WO2023140700A1 - Prevention or treatment of inflammatory bowel disease caused by western diets by inhibiting expression and activation of tas1r3 intestinal taste receptor - Google Patents

Prevention or treatment of inflammatory bowel disease caused by western diets by inhibiting expression and activation of tas1r3 intestinal taste receptor Download PDF

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WO2023140700A1
WO2023140700A1 PCT/KR2023/001053 KR2023001053W WO2023140700A1 WO 2023140700 A1 WO2023140700 A1 WO 2023140700A1 KR 2023001053 W KR2023001053 W KR 2023001053W WO 2023140700 A1 WO2023140700 A1 WO 2023140700A1
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tas1r3
bowel disease
inflammatory bowel
expression
intestinal
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French (fr)
Korean (ko)
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신동미
손우정
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서울대학교산학협력단
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    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
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    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/13—Nucleic acids or derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/32—Foods, ingredients or supplements having a functional effect on health having an effect on the health of the digestive tract
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/136—Screening for pharmacological compounds
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention has identified that inflammatory bowel disease caused by consumption of a western-style diet can be prevented or treated by suppressing the expression and activation of the intestinal taste receptor TAS1R3.
  • the present invention provides a method for screening a composition for treating inflammatory bowel disease or a drug for treating inflammatory bowel disease, which contains TAS1R3 gene expression deficiency and a substance inhibiting the corresponding protein activity as an active ingredient.
  • IBD Inflammatory bowel disease
  • US CDC Center for Disease Control and Prevention
  • IBD patients there are more than 4 million IBD patients worldwide, and 1.4 million people in the United States alone are estimated to have inflammatory bowel disease, and more than 70,000 new patients per year are reported to be constantly occurring.
  • the global inflammatory bowel disease treatment market reached $11.7 billion as of 2014, and is expected to grow to more than KRW 19 trillion by 2023.
  • Inflammatory bowel disease including occasional abdominal pain, is a 'disease directly related to menstrual phenomena', so the emotional burden on patients is very great.
  • biological cytokine inhibitors such as anti-TNF drugs, anti-integrin drugs, and IL-12/23 inhibitors are used as the dominant treatment for the treatment of inflammatory bowel disease.
  • anti-TNF drugs 50 to 60% of patients do not respond or the response disappears within 1 year. There is a high demand for treatment development.
  • the present inventors focused on the pathogenesis of inflammatory bowel disease caused by environmental factors such as Western-style dietary intake and the fact that there is no separate therapeutic agent or treatment method targeted thereto, and made diligent efforts to develop a specific therapeutic agent for inflammatory bowel disease, particularly occurring in Asia.
  • the intestinal taste receptor TAS1R3 can be an important mediator in mediating such intestinal inflammation, knocking out TAS1R3
  • the transcription factor PPAR ⁇ in enterocytes significantly increases, i) the mucosal defense system in the intestine is increased by increasing the expression of genes involved in the secretion of tight junction proteins and antimicrobial peptides, thereby strengthening the protective mechanism from western diet-induced intestinal inflammation, and the increased PPAR ⁇ ii) makes the condition of the intestinal lumen hypoxic, leading to the expansion of anaerobic butyrate bacteria and intestinal oil
  • the present invention was completed by verifying that intestinal inflammation can be treated by inducing proliferation of beneficial bacteria and significantly reducing intestinal inflammation indicators.
  • An object of the present invention is to provide a therapeutic agent for inflammatory bowel disease caused by a western diet and a method for screening the same.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
  • the present invention also provides a novel use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for preventing or treating inflammatory bowel disease.
  • the present invention also provides a novel use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for the preparation of a drug for preventing or treating inflammatory bowel disease.
  • the present invention also provides a method for preventing or treating inflammatory bowel disease comprising administering a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor to a subject in need thereof.
  • the present invention also provides a food composition for preventing or improving inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
  • the TAS1R3 expression inhibitor may be a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA.
  • the TAS1R3 protein activity inhibitor may be a TAS1R3-specific antibody, aptamer or antagonist.
  • the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease induced by a western diet.
  • composition in the present invention, the composition
  • an increase in antimicrobial peptide secretion gene expression may be characterized by preventing, improving or treating inflammatory bowel disease by inducing.
  • the change in the microbiome in the intestine may be characterized by expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine.
  • the present invention also provides a method for screening a therapeutic agent for inflammatory bowel disease comprising the following steps:
  • the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease caused by a western diet.
  • TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor according to the present invention, there is an advantage in that inflammatory bowel disease can be effectively prevented, improved, or treated by changing the intestinal microbiome, strengthening the tight junction between intestinal cells, and suppressing the secretion of inflammatory cytokines.
  • Figure 1a is a result of comparing the degree of intestinal inflammation through tissue staining in normal diet (ND) and western diet (WD) mice.
  • Figure 1b is the result of calculating the inflammation score (inflammation score) in mice fed a normal diet and a western diet. Levels of inflammation were extremely elevated in mice fed a western diet.
  • Figure 1c compares the results of RNA sequencing analysis of intestinal tissues extracted from normal and Western diet mice. Intestinal genome expression in Western-fed mice (red squares) was significantly different from that in normal-fed mice (black circles).
  • Figure 1d is a result of selecting (screening) genes whose expression was statistically significantly changed in the intestinal tissues of mice fed a normal diet and a western diet.
  • 1e is a result of functional classification of genes (Differentially expressed genes, DEGs) showing significant differences between mice fed a normal diet and a western diet. Significantly, changes in the signal transduction system related to taste receptor activity along with an inflammatory response are characteristic of the intestines of mice fed a western diet.
  • DEGs Differently expressed genes
  • FIG. 1f shows a result confirming that expression of the taste receptor Tas1r3 was significantly increased among various types of taste receptors in mice fed a western diet.
  • Figure 1g shows the result of confirming the increased protein expression of TAS1R3 in inflammatory intestinal tissues of mice fed a western diet.
  • FIG. 2a is a schematic diagram of an experiment in which the enteroendocrine cell line NCI-H716 was treated with various combinations of glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M), and the expression of the intestinal taste receptor TAS1R3 and its downstream molecule, GLP-1, was examined.
  • Figure 2b is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) in the combination shown in Figure 2a, and normalizing the mRNA expression level of TAS1R3 to the mRNA expression level of GAPDH after 12 hours.
  • Figure 2c is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) in the combination shown in Figure 2a, normalizing the mRNA expression level of GCG to the mRNA expression level of GAPDH, and observing for 24 hours.
  • Figure 2d is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) in the combination shown in Figure 2a, and observing the level of GLP-1 secretion for 24 hours.
  • Secretion of GLP-1 means the degree of activation of TAS1R3.
  • Figure 2e is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) in the combination shown in Figure 2a, normalizing the mRNA expression level of IL1B to the mRNA expression level of GAPDH, and observing for 24 hours.
  • Figure 2f is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) in the combination shown in Figure 2a, normalizing the mRNA expression level of IL6 to the mRNA expression level of GAPDH, and observing for 24 hours.
  • Figure 2g is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) in the combination shown in Figure 2a, normalizing the mRNA expression level of TNF to the mRNA expression level of GAPDH, and observing for 24 hours.
  • Figure 2h is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) in the combination shown in Figure 2a, normalizing the mRNA expression level of MCP-1 to the mRNA expression level of GAPDH, and observing for 24 hours.
  • Figure 3a is the result of confirming the amount of TAS1R3 transcript according to TAS1R3 siRNA treatment.
  • Figure 3b is the result of confirming the TAS1R3 expression inhibition effect according to TAS1R3 siRNA treatment at the protein level.
  • Figure 3c is the result of confirming the effect of suppressing GCG transcript expression according to TAS1R3 siRNA treatment.
  • Figure 3d is the result of confirming the active GLP-1 inhibitory effect according to TAS1R3 siRNA treatment.
  • Figure 3e is the result of confirming the effect of inhibiting IL1B transcript expression according to TAS1R3 siRNA treatment.
  • Figure 3f is the result of confirming the effect of inhibiting IL6 transcript expression according to TAS1R3 siRNA treatment.
  • 3g shows the result of confirming the effect of inhibiting IL8 transcript expression according to TAS1R3 siRNA treatment.
  • Figure 3h is the result of confirming the effect of inhibiting IL8 secretion according to TAS1R3 siRNA treatment.
  • Figure 3i is the result of confirming the effect of TNF transcript expression inhibition according to TAS1R3 siRNA treatment.
  • Figure 3j is the result of confirming the TNF- ⁇ secretion inhibitory effect according to TAS1R3 siRNA treatment.
  • 3k shows the result of confirming the effect of inhibiting GCG transcript expression by treatment with Lactisole, an antagonist for TAS1R3.
  • Figure 3l is the result of confirming the active GLP-1 inhibitory effect according to lactisole treatment.
  • 3m is a result confirming the effect of inhibiting IL1B transcript expression according to lactisole treatment.
  • 3n shows the result of confirming the effect of inhibiting IL6 transcript expression according to lactisole treatment.
  • Figure 3o shows the result of confirming the effect of inhibiting IL8 transcript expression according to lactisole treatment.
  • Figure 3p is the result of confirming the effect of inhibiting IL8 secretion according to lactisole treatment.
  • Figure 3q is the result of confirming the effect of inhibiting TNF transcript expression according to lactisole treatment.
  • Figure 3r is a result confirming the effect of inhibiting TNF- ⁇ secretion according to lactisole treatment.
  • Tas1r3 transcript expression is suppressed in the small intestine of Tas1r3 knockout mice fed a normal or western diet.
  • FIG. 5B is a result confirming that Tas1r3 protein expression is suppressed in the small intestine of Tas1r3 knockout mice fed a normal or western diet.
  • Figure 6a is a result of comparison of spleen weights in wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • Figure 6b is a result of comparing the length of the small intestine in wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • Figure 6c shows the results of comparison of colon lengths in wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • Figure 6d shows the results of comparison of inflammation in intestinal tissue through histological examination in wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • Figure 6e is a comparison of the intestinal inflammatory index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • Figure 6f compares the pattern of IL-1b transcript expression in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • 6g shows the comparison of Tnf transcript expression patterns in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • Figure 6h shows the comparison of IL6 transcript expression patterns in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • mTOR 7a is a comparison result of protein expression levels of phosphorylated-mTOR (mTOR), mTOR, and PPAR- ⁇ in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • 7b is a result of quantifying the protein expression levels of phosphorylated-mTOR (mTOR) protein and PPAR- ⁇ in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • mTOR phosphorylated-mTOR
  • FIGS. 7C and 7D each is a tight Junction gene (TJP1, OCLN1, CLDN7), a wild type or tas1r3 knockout mouse with normal or western diets, respectively. This is the result of the expression of the expression of the expression of antimicrobial peptides (Reg3g, Lyz1, Defa2, Defa3).
  • 8a is a comparison result of Shannon's diversity index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • 8b is a comparison result of Faith's phylogenetic index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
  • Figure 8c shows the results of comparison of ⁇ -dirversity of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet using the Bray-Curtis method.
  • 8d shows the results of comparison of ⁇ -diversity of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet using the weighted PCoA method.
  • FIG. 8E shows the results of comparing the differences in the gut microbiome of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet at the phylum level.
  • FIG. 8f shows the results of comparing the differences in gut microbial communities at the family (family) and genus (genus) levels in wild-type or Tas1r3 knockout mice fed a western-style diet. Bacteria showing significant differences between the two groups are shown through Cladogram.
  • Figure 8g shows the relative abundance of butyrate-producing strains (genus Butyrivibrio , Roseburia , Ruminococcus , Butyricicoccus , Faecalibacterium ) showing the most distinct characteristics in Tas1r3 knockout mice fed a western diet. Results were compared.
  • 8h is a result of comparison of the amount of butyrate in the feces of wild-type or Tas1r3 knockout mice fed a western-style diet.
  • 10a is a result confirming the effect of inhibiting MTOR transcript expression according to TAS1R3 siRNA treatment.
  • 10b is a result confirming the effect of enhancing PPAR ⁇ transcript expression according to TAS1R3 siRNA treatment.
  • 10c is a result confirming the effect of enhancing PPAR ⁇ protein expression according to TAS1R3 siRNA treatment.
  • 10d is a result confirming the effect of inhibiting MTOR transcript expression according to treatment with Lactisole, a TAS1R3 antagonist.
  • 10e is a result confirming the effect of enhancing PPAR ⁇ transcript expression according to lactisole treatment.
  • 10f is a result confirming the effect of enhancing PPAR ⁇ protein expression according to Lactisole treatment.
  • 11 is a result of analyzing differences in expression patterns of transcripts in intestinal biopsy samples of a normal control group and an experimental group of patients with inflammatory bowel disease (11a - 11c), and confirming the expression levels (11d - 11f) of TAS1R3 and downstream molecules (mTOR, PPAR ⁇ ) and association with related molecules (11g).
  • FIG. 12 is a schematic diagram showing a key mechanism by which the intestinal taste receptor TAS1R3 regulates intestinal inflammation.
  • TAS1R3 an intestinal taste receptor
  • the present invention relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient, and in another aspect, it relates to a food composition for preventing or improving inflammatory bowel disease comprising a TAS1R3 expression inhibitor or TAS1R3 protein activity inhibitor as an active ingredient.
  • the gene sequence encoding the human TAS1R3 may be represented by SEQ ID NO: 1
  • the human TAS1R3 protein sequence may be represented by SEQ ID NO: 3, but is not limited thereto.
  • TAS1R3 Homo sapiens taste 1 receptor member 3
  • Mus muscle taste receptor type 1, member 3 (Tas1r3), mRNA
  • Taste receptor type 1 member 3 precursor [Homo sapiens]
  • Taste receptor type 1 member 3 precursor [Mus musculus]
  • the TAS1R3 expression inhibitor may be a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA, but is not limited thereto.
  • the TAS1R3 expression inhibitor may be siRNA represented by the following sequence, but is not limited thereto.
  • the TAS1R3 protein activity inhibitor may be a TAS1R3-specific antibody aptamer or an antagonist, but is not limited thereto.
  • the TAS1R3 protein activity inhibitor may be lactisole, but is not limited thereto.
  • the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease induced by a western diet.
  • composition in the present invention, the composition
  • (iii) increase in antimicrobial peptide secretion gene expression may be characterized in that inflammatory bowel disease is prevented or treated by inducing, but the effect is not limited thereto.
  • the intestinal microbiome change may be characterized by expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine, but is not limited thereto.
  • the anaerobic butyrate bacteria may be characterized in that at least one selected from bacteria consisting of the genus Butyrivibrio , the genus Roseburia , the genus Ruminococcus , the genus Butyricicoccus and the genus Faecalibacterium , but is not limited thereto.
  • the harmful bacteria may be characterized in that at least one selected from bacteria consisting of the genus Prevotella , the family Paraprevotellaceae, the family Enterobacteriaceae, and the genus Enterobacteriales , but is not limited thereto.
  • Inhibition of TAS1R3 expression or activity according to the present invention significantly increases PPAR ⁇ in enterocytes, i) increases the expression of genes involved in the secretion of tight junction proteins and antimicrobial peptides, thereby increasing the defense system in the intestine, thereby strengthening the protective mechanism from western diet-induced intestinal inflammation, and the increased PPAR ⁇ ii) makes the intestinal lumen hypoxic and expands anaerobic butyrate bacteria While inducing, the proliferation of beneficial bacteria in the intestine is induced, and the intestinal inflammation index is significantly reduced, so that intestinal inflammation can be prevented or treated.
  • the present invention relates to a method for screening a therapeutic agent for inflammatory bowel disease comprising the following steps:
  • the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease caused by a western diet.
  • the therapeutic agent may be provided as a pharmaceutical composition, including tas1R3 specific antisense oligonucleotides, siRNA, shrna, miRNA, or TAS1R3 specific antibodies, aptamers or antagonists, or include a pharmaceutically acceptable carrier, excipient or diluent, and the composite is a disease and the complex. It may be included in the pharmaceutical composition in an appropriate pharmaceutically effective amount, depending on the severity, age, weight, health, gender, administration route, and treatment period of the patient.
  • pharmaceutically acceptable refers to a composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans.
  • composition according to the present invention may further include a pharmaceutically acceptable excipient.
  • Excipients that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; and the like.
  • composition of the present invention may be formulated into a dosage form for oral administration or a dosage form for parenteral administration by a conventional method, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, cryoprotectants, etc. It can be prepared using.
  • composition of the present invention when formulated as a solid preparation for oral administration, it includes tablets, pills, powders, granules, capsules, etc., and such solid preparations may contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, or gelatin in the active ingredient.
  • excipients such as starch, calcium carbonate, sucrose, lactose, or gelatin in the active ingredient.
  • magnesium stearate and lubricants such as talc may be included, but are not limited thereto.
  • composition of the present invention when formulated as a liquid formulation for oral administration, it includes suspensions, internal solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included, but are not limited thereto.
  • composition of the present invention when formulated into a formulation for parenteral administration, it may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, and a suppository.
  • Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate, but are not limited thereto.
  • a base for the suppository witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
  • the content of the novel lactic acid bacteria which is an active ingredient of the pharmaceutical composition of the present invention, may be adjusted in various ranges depending on the specific form, purpose or aspect of the composition.
  • the content of the active ingredient in the pharmaceutical composition according to the present invention is not particularly limited, and may be, for example, 0.01 to 99% by weight, specifically 0.1 to 75% by weight, more specifically 0.5 to 50% by weight based on the total weight of the composition.
  • composition for food according to the present invention may be characterized in that it is a composition for food or food additives, but is not limited thereto, and can be easily utilized as, for example, a main ingredient, a supplementary ingredient, food additive, health functional food or functional beverage of food.
  • the food means a natural product or processed product containing one or more nutrients, preferably means a product that can be directly eaten through a certain degree of processing, and in a conventional sense, includes food, food additives, health functional food and functional beverages.
  • Foods to which the food composition according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and functional foods.
  • food includes special nutritional foods (eg, formula milk, infant food, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasonings (eg, soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candy, chocolates, gum, ice cream, dairy products (eg , fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.).
  • the food, beverage or food additive may be prepared by a conventional manufacturing method.
  • the health functional food refers to food that is designed and processed to sufficiently express the body's regulatory functions related to biological defense rhythm control, disease prevention and recovery, etc. of a food group or food composition in which added value is added so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods.
  • the functional food may include food additives that are acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.
  • the functional beverage refers to a general term for drinking to quench thirst or enjoy taste, and there is no particular limitation on other ingredients other than including the composition as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates, etc. may be included as additional ingredients.
  • the food containing the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. can be used
  • the amount of the composition according to the present invention may be included in 0.001% to 100% by weight, preferably 1% to 99% by weight, based on the total weight of the food, and in the case of beverages, it may be included in a ratio of 0.001g to 10g, preferably 0.01g to 1g based on 100ml, but may be included for health and hygiene purposes or health control In the case of intended long-term intake, it may be below the above range, and since the active ingredient has no problem in terms of safety, it may be used in an amount above the above range, so it is not limited to the above range.
  • antisense oligonucleotides are useful for regulating the expression of nucleic acid molecules (eg, regulating the expression of TAS1R3) through an antisense mechanism.
  • modulation can be performed, for example, by providing an oligonucleotide that is complementary to and/or hybridizes to one or more target nucleic acid molecules, such as mRNA.
  • an oligonucleotide of the invention is complementary to a specific region of a target nucleic acid.
  • an oligonucleotide of the invention is capable of hybridizing to a specific region of a target nucleic acid.
  • the oligonucleotide compounds of the present invention are complementary to one or more target nucleic acids and interfere with the normal function of the targeted nucleic acids (eg, by an antisense mechanism of action). It interferes with or modulates the function of a target nucleic acid by means of an oligonucleotide of the invention that specifically hybridizes to what is commonly referred to as "antisense".
  • the functions of DNA that are disturbed can include replication and transcription.
  • Functions of the RNA that are hindered may include functions such as, for example, translocation of the RNA to a protein translation site, translation of a protein from the RNA, splicing of the RNA to provide one or more mRNA species, and catalytic activity that may be engaged or promoted by the RNA.
  • the overall effect of interfering with target nucleic acid function is regulation of expression of the product of such target nucleic acid.
  • siRNA small interfering RNA refers to a short double-stranded RNA capable of inducing RNA interference through cleavage of a specific mRNA.
  • siRNA is not limited to complete pairing of double-stranded RNA parts paired with each other, but may include unpaired parts due to mismatch (corresponding bases are not complementary), bulge (no base corresponding to one chain), etc. Paired bases are 15 to 30 bases in length.
  • the siRNA end structure either a blunt end or a protruding end may be used as long as the expression of the target gene can be suppressed by the RNA interference effect.
  • the sticky end structure can be both a 3' end protruding structure and a 5' end protruding structure.
  • the siRNA of the present invention includes variants having one or more substitutions, insertions, deletions, and combinations thereof that do not reduce its activity.
  • Such variants may have 80% or more sequence homology to the siRNA sequence described above, preferably 90% or more, and more preferably 95% or more sequence homology.
  • siRNA of the present invention can be synthesized by various methods known in the art, such as direct chemical synthesis (Sui G et al., Proc. Natl. Acad. Sci. USA (2002) 99:5515-5520) or synthesis using in vitro transcription (Brummelkamp TR et al., Science (2002) 296:550-553).
  • shRNA is called short hairpin RNA, and is an RNA molecule having a stem-loop structure so that a part of one strand forms a complementary strand with another region.
  • miRNA is a single-stranded RNA of about 21 to 25 bases that has an effect of suppressing gene expression.
  • miRNAs are generated from genes transcribed from single or clustered miRNA precursors. That is, pri-miRNA, the primary transcript from the gene, is transcribed, and subsequently, in step-by-step processing from pri-miRNA to mature miRNA, pre-miRNA of about 70 to 80 bases with a characteristic hairpin structure is generated from pri-mRNA, and then mature miRNA is generated from pre-miRNA by Dicer.
  • miRNAs are deeply involved in vital phenomena such as differentiation, cell proliferation, and apoptosis that are indispensable to living organisms.
  • antibody refers to a protein that binds to other molecules (antigens) through variable regions of light and heavy chains, and includes IgG, IgD, IgA, and IgE-derived proteins.
  • the antibodies herein include monoclonal antibodies having various types of structures, for example, intact antibodies comprising two full-length heavy chains and two full-length light chains, as well as fragments thereof with or without constant regions, chimeric antibodies, humanized antibodies, or other genetically modified antibodies having the characteristics according to the present application.
  • Antibodies of the present application may exist as multimers such as dimers, trimers, tetramers, pentamers, etc., including at least a portion of the monomer's antigen-binding ability. These multimers also include homomultimers or heteromultimers. Since antibody multimers contain multiple antigen-binding sites, they have superior antigen-binding ability compared to monomers. Multimers of antibodies are also easy to construct multifunctional (bifunctional, trifunctional, tetrafunctional) antibodies.
  • Antibodies that specifically bind to Tas1R3 described herein can be prepared by various methods known in the art (Scheving, L.A. et al., American Journal of Physiology, 306:G370-81, 2014; Kaufmann, A. et al., J. Cell. Biochem. 114:681-69, 2013; Herndon, CA. et al., Cell Res, 319: 718-730, 2013; Lacoste, B. et al., Journal of Neuroinflammation, 10:57, 2013; del Blanco, B. et al., J. Immunol, 188:3278-3293, 2012).
  • an aptamer refers to a small single-stranded oligonucleic acid capable of specifically recognizing a target material with high affinity.
  • SELEX Systematic Evolution of Ligand by Exponential Enrichment
  • an aptamer Craig et al., Science, 249: 505-510, 1990.
  • the aptamer specifically binding to Egr-1 of the present invention can be prepared by the SELEX standard method (Bock LC et al., Nature, 355:564-6, 1992), a technique known in the art (Mannironi et al., Biochemistry 36:9726, 1997; Ellington and Szostak et al., Nature, 346:818, 1990; Tuerk and Gold , et al., Science 249:505, WO 00/20040, WO 99/54506, WO 99/27133, WO 97/42317). Therefore, it will be apparent to those skilled in the art that an aptamer for Tas1R3 can be easily prepared using known techniques even if it is not described in the examples.
  • the step of measuring the binding degree of the aptamer that specifically binds to Tas1R3 may be performed using a DNA aptamer binding measurement technique commonly used in the related art.
  • a method of measuring fluorescence or radioactive intensity by binding biotin to an aptamer end, labeling a fluorescent or radioactive substance, or imaging and observing the aptamer end may be used, but is not limited thereto.
  • an antagonist means, in one aspect, a compound that inhibits or blocks intracellular signal transduction caused by TAS1R3, which can also be referred to as a compound that inhibits the TAS1R3 signal.
  • a compound may be a naturally occurring compound or an artificially synthesized compound. It may also be a low molecular weight compound or a high molecular weight compound such as protein.
  • gene expression is inhibited means that the gene itself is deleted or its expression is reduced, and enzymes or proteins produced by the genes are completely removed, as well as enzymes or proteins produced by the genes.
  • modified to be attenuated compared to the intrinsic activity means deletion of a gene showing activity, inactivation of a gene (eg, substitution with a mutant gene), attenuation of gene expression (eg, substitution with a weak promoter, introduction of siRNA, gRNA, sRNA, etc., substitution of the start codon from ATG to GTG, etc.), inhibition of the activity of an enzyme expressed by a gene (eg, addition of a non-competitive repressor or competitive repressor). This means that the activity after this is reduced.
  • intrinsic activity means the original active state of an enzyme or the like in an unmodified state, and "modified to be attenuated compared to the intrinsic activity” means that the activity disappears or is further reduced when compared to the enzymatic activity in the state before modification.
  • deletion is a concept encompassing preventing a corresponding gene from being expressed through a method of mutating, substituting, or deleting a part or all bases of a corresponding gene, or preventing a protein's inherent function from being expressed. It includes anything that blocks a biosynthetic pathway or signal transduction pathway involved in a protein expressed by a corresponding gene.
  • the present invention relates to the use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for preventing or treating inflammatory bowel disease.
  • the present invention relates to the use of the pharmaceutical composition for preventing or treating inflammatory bowel disease.
  • the present invention relates to the use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for the preparation of a drug for preventing or treating inflammatory bowel disease.
  • the present invention relates to a novel use of the pharmaceutical composition for the preparation of a drug for preventing or treating inflammatory bowel disease.
  • the present invention relates to a method for preventing or treating inflammatory bowel disease comprising administering a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor to a subject in need thereof.
  • the present invention relates to a method for preventing or treating inflammatory bowel disease comprising administering the pharmaceutical composition to a subject in need thereof.
  • mice 8-10 week old C57BL/6J mice were obtained from the Jackson Laboratory (West Grove, PA, USA), and 10 mice per experimental group were classified into either a normal diet group or a western diet group [sugar solution (30% w/v) + 60% high fat diet] and fed ad libitum for 10 weeks.
  • mice were sacrificed using 20% urethane (U2500, Sigma-Aldrich, St. Louis, MO, USA), and then terminal ileum specimens were obtained. The ileum tissue was fixed in 10% formalin and subjected to H&E staining. Histological analysis followed the previously reported histologic colitis scoring system (Severity of DSS-induced colitis is reduced in Ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks. Sci Rep , 2015, 5:17305). The degree of inflammation, the extent of inflammation, the extent of damage to the crypt, and the extent of damage to the entire small intestine were measured, and each variable was added to calculate the inflammation score.
  • cells of the lamina intestinal were analyzed by flow cytometry using Cell Quest software of FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA).
  • the antibodies used were CD4 (GK1.5; BioLegend, San Diego, CA, USA), CD8a (53-6.7; BioLegend), CD45 (30-F11; BioLegend), phycoerythrin (PE), allophycocyanin (APC), APC-CyChrome7 (APC-Cy7), Alexa Fluor 700 (AF700).
  • mice were sacrificed, and inflamed small intestine tissue was extracted and RNA sequencing was performed in the tissue.
  • RNA was quantified using a NanoDrop 2000/2000c Spectrophotometer (Thermo Scientific). Intact mRNA was captured with a total of 1 ⁇ g (1000ng) of total RNA (using Dynabeads mRNA DIRECT Micro Kit (Ambion)), and the captured mRNA was used for library construction through Ion Total RNA Seq Kit v2 (Life Technologies).
  • the prepared library sample was finally loaded into the Ion PI Chip Kit v3 (Life Technologies) and then put into the Ion Proton Sequencer (Life Technologies) and run. All experiments were performed according to the instructions of each kit manufacturer.
  • Enteroendocrine cell line NCI-H716 (CCL-251, American Type Culture Collection, Manassas, VA, USA) was treated with various combinations of glucose (10 mM), fructose (10 mM), and palmitate (10 ⁇ M) (all Sigma-Aldrich, St. Louis, MO, USA), and the expression of the intestinal taste receptor TAS1R3 and its downstream molecule, GLP-1, was analyzed at the mRNA and protein levels ( Fig. 2a).
  • RNAqueous (AM1914, Ambion) kit total RNA in intestinal tissue was extracted using RNAqueous (AM1914, Ambion) kit.
  • the RNA extracted with the kit was synthesized into cDNA using the MML-V reverse transcriptase protocol (11917010, Invitrogen), and the quantitative polymerase reaction was measured to quantitatively express mRNA expression.
  • TAS1R3 As a result, compared to the case of processing glucose, fructose, or palmitate alone, TAS1R3 It was confirmed that the amount of mRNA expression significantly increased (Fig. 2b).
  • the NCI-H716 cell line an enteroendocrine cell
  • glucose (10 mM) glucose (10 mM)
  • fructose (10 mM) fructose
  • palmitate 10 ⁇ M
  • the secretion of GLP-1 means the degree of activation of TAS1R3.
  • GLP-1 secreted from cells centrifugation was performed at 4°C and 3000 g for 10 minutes, and only supernatants were collected and stored at -80°C.
  • a GLP-1 (Active) ELISA kit (EGLP-35k, Millipore, St. Charles, MI, USA) was used and the experiment was performed according to the manufacturer's protocol.
  • GLP-1 mRNA expression level
  • peptide level activate GLP-1
  • the NCI-H716 cell line was treated with (i) glucose (10 mM), fructose (10 mM), palmitate (10 ⁇ M) (control group), (ii) glucose (10 mM), fructose (10 mM), palmitate (10 ⁇ M), TAS1R3 siRNA treatment (siRNA treatment group), (iii) glucose (10 mM), fructose (10 mM), palmitate (1 0 ⁇ M), TAS1R3 antagonist (lactisole) treatment (antagonist treatment group), followed by additional incubation.
  • Human enteroendocrine NCI-H716 cells (CCL-251; American Type Culture Collection, Manassas, VA, USA) were maintained in suspension culture at 37° C. and 5% CO 2 according to the supplier's protocol.
  • the culture medium was RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin and 100 ⁇ g/mL streptomycin.
  • 1 ⁇ 10 6 cells were seeded in 24-well culture plates pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). On the day of the experiment, the supernatant was replaced with medium containing 10 mM glucose, fructose, glucose + fructose and/or 10 ⁇ M palmitate.
  • siRNA duplex against TAS1R3 was synthesized by Bioneer (Daejeon, South Korea). Scrambled negative control siRNA was also purchased from Bioneer.
  • 5 x 10 5 endocrine differentiated NCI-H716 cells were plated in 6-well culture plates and cultured for 48 hours.
  • TAS1R3 (10 nM) or control (10 nM) siRNA was transfected into cells using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA). 48 hours after transfection, cells were incubated for 12 hours with medium containing 10 mM glucose, 10 mM fructose and 10 ⁇ M palmitate.
  • NCI-H716 cells were pretreated with the TAS1R3 antagonist lactisole (2.5 mM) for 30 min and stimulated with a medium containing fructose (10 mM), glucose (10 mM) and palmitate (10 ⁇ M) for 12 h.
  • Glucose, fructose, palmitate and lactisol were all purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). After incubation, media was collected and centrifuged at 1,000 x g for 10 min at 4°C to remove floating cells and frozen at -20°C for subsequent biochemical analysis.
  • qPCR and ELISA were performed in the same manner as in Example 2, and the amount of IL-8 protein was analyzed using the IL-8 ELISA kit (Cat# ab46032, Abcam, Cambridge, UK), and the amount of TNF protein was analyzed using the TNF- ⁇ ELISA kit (Cat# ab181421, Abcam, Cambridge, UK).
  • TAS1R3 expressed in the intestine can directly regulate the secretion of inflammatory cytokines induced by diet, and that inhibiting TAS1R3 in enterocytes can inhibit the inflammatory mechanism of enterocytes.
  • Example 4 Functional study of taste receptor TAS1R3 in intestinal inflammation induced by western diet using Tas1r3 knockout mouse model
  • Tas1r3 tm1Csz (JAX013066) breeding pair (purchased frozen sperm) was purchased from The Jackson Lab, and Tas1r3 gene-deficient (knock-out, KO) mice were constructed. Backcrossing with C57BL/6 was performed for at least 7 generations, and the genotype of Tas1r3 knockout mice was confirmed through PCR genotyping. Experiments were conducted according to the Jax protocol, primer information used for genotyping is shown in Table 2, Reaction A information is shown in Table 3, and PCR conditions are shown in Table 4.
  • Wild -type controls (TAS1R3 WT ) (West Grove, PA, USA), where age and weight match, and the lust -out mouse (TAS1R3 KO ) in the SPF (SpeciFic Pathogen Free) animal breeding room and immunochemical chemical dyeing method (Immunohi) Stochemistry, IHC) confirmed whether the expression of tas1R3 was actually deficient in the small intestine and the large intestine of each mouse at the protein level.
  • SPF SeiFic Pathogen Free
  • sample tissues cut to a thickness of 4-6 ⁇ m were fixed with formalin, the tissues were deparaffinized, rehydrated through xylene and alcohol series, and placed in citrate buffer (pH6.0, Sigma, C9999) and heated at 100 ° C. for 20 minutes. After washing with phosphate-buffered saline, the slides were incubated with 1% bovine serum albumin (Sigma-Aldrich) and Phosphate-buffered saline-tween for 1 hour, and an anti-TAS1R3 antibody (1:100, OSR00184W, Invitrogen, Camarillo, CA, USA) was attached. Mouse and Rabbit-Specific HRP/DAB Detection IHC kit (ab64264, Abcam) was used as a secondary antibody.
  • Tas1r3 WT wild-type control
  • Tas1r3 KO Tas1r3 knockout mice
  • mice were sacrificed, and the spleen, small intestine, and colon were removed and histological examination was performed to compare their sizes.
  • IF immunofluorescence staining
  • pan-leukocytes (CD45) (1:100, ab10558, Abcam, Cambridge, UK), anti-CD4 (14-0041-86; eBioscience, San Diego, CA, USA), and rat anti-CD8 (ab22378, Abcam), and mouse anti-CD11b (14-0112-82, eBioscience)
  • Alexafluor 488 anti-mouse/human CD11b (101219, BioLegend, San Diego, CA, USA), donkey anti-rabbit Alexafluor 594 (A21207, Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-mouse Alexafluor 488 (A21202, Thermo Fisher Scientific), and donkey anti-rat Alexafluor 594 (A21209, Thermo Fisher Scientific).
  • transcriptome analysis in the small intestine was performed using the mice of Example 4-2 to investigate the mechanism.
  • the transcriptome experiment is the same as the RNA sequence analysis method of Example 1.
  • the PPAR ⁇ gene in the intestine is known as a transcription factor that regulates the expression of tight junction proteins and antimicrobial peptides that strengthen the intestinal barrier. Accordingly, as a result of further confirming changes in the expression of tight junction proteins and antimicrobial peptides, it was confirmed that the expressions of tight junction proteins and antimicrobial peptides were also significantly changed in Tas1r3 knockout mice (Fig. 7c).
  • the intestinal taste receptor TAS1R3 functions as an intrinsic regulator of the transcription factor PPAR ⁇ in enterocytes and can strengthen the barrier by increasing the tight junctions between enterocytes from intestinal inflammation induced by a western diet.
  • Tas1r3 knockout mice are protected from intestinal inflammation induced by a western diet, and the intestinal commensal is known to play an important role in intestinal inflammation, the intestinal commensal profile of Tas1r3 knockout mice was analyzed by 16S rRNA sequencing (microbiome).
  • a 16S rRNA sequencing library was constructed according to the 16S metagenomics sequencing library preparation protocol (Illumina, San Diego, CA, USA) targeting the V3 and V4 hypervariable regions of the 16S rRNA gene.
  • a KAPA HiFi HotStart ReadyMix KAPA Biosystems, Wilmington, MA, USA
  • Agencourt AMPure XP system Beckman Coulter Genomics, Brea, CA, USA
  • Final amplicons were sequenced in paired-end mode (PE275) using the MiSeq system (Illumina).
  • the PPAR ⁇ signaling pathway increased by Tas1r3 knockout showed a very significant positive correlation with anaerobic butyrate-producing bacteria. It is interpreted that when Tas1r3 deficiency increases the intestinal transcription factor PPAR ⁇ , the intestinal lumen becomes hypoxic, induces the expansion of anaerobic butyrate bacteria living in an anaerobic environment, and consequently exerts an effect of protecting against intestinal inflammation.
  • Example 5 Regulation of the mTOR/PPAR ⁇ axis through inhibition of the intestinal taste receptor TAS1R3
  • Treatment with siRNA and TAS1R3 antagonist lactisole was performed in the same manner as in Example 3, and the primary antibodies for mTOR, phospho-mTOR, and PPAR ⁇ were monoclonal rabbit anti-mTOR (1:1,000) (Cat# 2972S, RRID: AB_330978), rabbit anti-phospho-mTOR (1:1,000) (Cat# 297 1S, RRID: AB_330970) and rabbit anti-PPARgamma (1:1,000) (Cat# 2443S, RRID: AB_823598) were purchased from Cell Signaling Technology (Danvers, MA, USA) and analyzed.
  • mouse anti- ⁇ -tubulin (1:10,000) (Cat# T5168, RRID: AB_477579) was used.
  • Secondary antibodies were Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000) (Cat# 7074S, RRID: AB_2099233) from Cell Signaling Technology (Danvers, MA, USA) and goat anti-mouse secondary antibodies (1:10,000) (Cat# G21040, RRID: AB_2536527) from Invitrogen (Carls bad, CA, USA) and used.
  • Example 6 Expression analysis of TAS1R3 and related molecules (mTOR/PPAR ⁇ ) in intestinal tissues of patients with inflammatory bowel disease (IBD)
  • TAS1R3 and related molecules TAS1R3 and related molecules (mTOR/PPAR ⁇ ) in the intestinal tissue of patients with inflammatory bowel disease (IBD)
  • IBD inflammatory bowel disease
  • biopsy mRNA microarray data sets of inflammatory bowel disease patients and healthy controls were downloaded from the GEO database (public database, available online: http://www.ncbi.nlm.nih.gov/geo ), and the expression levels of each molecule were analyzed.
  • the raw microarray data was downloaded from the GEO database and then preprocessed using Partek® version 6.6 (Partek®) with a robust multi-chip analysis (RMA) algorithm to perform background correction, quantile normalization and probe summarization.
  • Partek® version 6.6 Partek® version 6.6
  • RMA multi-chip analysis
  • Differentially expressed genes were analyzed using R/Bioconductor (available online: http://www.bioconductor.org /), and an FDR P ⁇ 0.001 was considered as the cut off value when selecting DEGs.

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Abstract

The present invention provides a composition for treating inflammatory bowel disease or a method for the screening of drugs for treating inflammatory bowel disease, the composition comprising, as an active ingredient, a material for reducing the expression of TAS1R3 gene or inhibiting the protein activity thereof. According to the present invention, when a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor is used, the microbial community in the intestine is changed, tight junctions between intestinal cells are enhanced, and inflammatory cytokine secretion is inhibited, thus inflammatory bowel disease can be effectively prevented, alleviated, or treated, and this benefit can be effectively used for treating inflammatory bowel disease in Asian people, which is rapidly increasing due to western diets and for which appropriate treatment has not been developed to date.

Description

장 미각 수용체 TAS1R3 발현 및 활성화 억제를 통한 서구식 식이 유발 염증성 장 질환의 예방 또는 치료Prevention or treatment of western diet-induced inflammatory bowel disease by inhibiting the expression and activation of the intestinal taste receptor TAS1R3
본 발명은 장 미각 수용체 TAS1R3 발현 및 활성화 억제를 통해 서구식 식이 섭취로 인해 유발되는 염증성 장 질환을 예방 또는 치료할 수 있음을 규명한 것으로, 본 발명에서는 TAS1R3 유전자 발현 결핍 및 해당 단백질 활성 억제 물질을 유효성분으로 하는 염증성 장 질환 치료용 조성물 또는 염증성 장 질환 치료용 약물을 스크리닝 하는 방법을 제공한다. The present invention has identified that inflammatory bowel disease caused by consumption of a western-style diet can be prevented or treated by suppressing the expression and activation of the intestinal taste receptor TAS1R3. The present invention provides a method for screening a composition for treating inflammatory bowel disease or a drug for treating inflammatory bowel disease, which contains TAS1R3 gene expression deficiency and a substance inhibiting the corresponding protein activity as an active ingredient.
염증성 장질환 (IBD)은 장관 내 원인 불명의 만성적인 염증이 호전과 재발을 반복하는 질환으로, 궤양성 대장염과 크론병이 대표적이다. 미국 CDC(질병관리예방센터)에 의하면, 전 세계적으로 IBD 환자는 약 400만명 이상에 달하며, 미국에서만 140만명이 염증성 장질환 환자로 추정, 연간 7만여명 이상의 신규 환자가 끊임없이 발생되는 것으로 보고되고 있다. 이에 따라 전세계 염증성 장질환 치료제 시장은 2014년 기준 117억 달러에 달하며, 오는 2023년에는 19조원이 넘는 시장으로 성장할 것으로 예측되고 있다.Inflammatory bowel disease (IBD) is a disease in which chronic inflammation of unknown cause in the intestinal tract repeatedly improves and recurs, and ulcerative colitis and Crohn's disease are representative examples. According to the US CDC (Center for Disease Control and Prevention), there are more than 4 million IBD patients worldwide, and 1.4 million people in the United States alone are estimated to have inflammatory bowel disease, and more than 70,000 new patients per year are reported to be constantly occurring. Accordingly, the global inflammatory bowel disease treatment market reached $11.7 billion as of 2014, and is expected to grow to more than KRW 19 trillion by 2023.
주목할 만한 점은, 아시아 국가의 염증성 장질환 발병률은 낮았으나 (실제로 과거에는 매우 희귀질환으로 분류됨), 최근 10여년 간 염증성 장질환 국내 환자의 수가 급격하게 증가하고 있다는 점이다. 국내 건강보험심사평가원 자료를 분석한 결과, 우리나라(한국)의 2015년 한해동안 크론병 진료 인원은 1만 8천명, 총 진료비는 473억원을 넘어섰으며, 신규 환자 또한 2010년 대비 2019년 10년 새 약 2배 이상 증가했을 뿐만 아니라, 관해와 재발이 반복되는 질환 특성상 국내 환자 수는 앞으로 꾸준히 증가할 것으로 평가되고 있다.It is noteworthy that the incidence of inflammatory bowel disease in Asian countries was low (in fact, it was classified as a very rare disease in the past), but the number of domestic patients with inflammatory bowel disease has increased rapidly over the past decade. As a result of analyzing data from the National Health Insurance Review and Assessment Service, in Korea (Korea), in 2015 alone, 18,000 people were treated for Crohn's disease, and the total treatment cost exceeded 47.3 billion won.
이처럼 아시아에서의 갑작스러운 발병 증가의 원인으로는 환경 요인 변화를 들 수 있는데, 특히 서구화(Westernized)된 식생활 패턴의 변화가 질병 발생과 매우 유의한 것으로 보고되었다 (The role of diet in the aetiopathogenesis of inflammatory bowel disease. Nature Review Gastroenterology Hepatology, 2018, 15:525-535).As such, the cause of the sudden increase in incidence in Asia can be attributed to changes in environmental factors, and in particular, changes in Westernized dietary patterns have been reported to be very significant in the occurrence of the disease (The role of diet in the aetiopathogenesis of inflammatory bowel disease. Nature Review Gastroenterology Hepatology , 2018, 15:525-535).
염증성 장질환은 시시때때로 나타나는 복통을 포함하여 '생리현상과 직접적으로 관계된 질환'의 특성상 환자들의 정서적 부담감이 매우 크다. 현재 염증성 장질환의 치료를 위해 항-TNF 치료제, 항-인테그린 제제, IL-12/23 억제제 등 생물학적 사이토카인 억제제가 지배적인 치료제로 사용되고 있으나, 항-TNF 제제의 경우 환자의 50~60%는 반응하지 않거나, 1년 이내에 반응이 사라지는 등 치료환자 중 기존 표준 약물치료 (5-Aminosalicylic acid(5-ASA) 제제, 스테로이드, 면역조절제 등)에 실패하거나 치료제 부작용으로 수술을 받게 되는 경우가 많아, 새로운 치료제 개발에 대한 요구도가 높은 상황이다. Inflammatory bowel disease, including occasional abdominal pain, is a 'disease directly related to menstrual phenomena', so the emotional burden on patients is very great. Currently, biological cytokine inhibitors such as anti-TNF drugs, anti-integrin drugs, and IL-12/23 inhibitors are used as the dominant treatment for the treatment of inflammatory bowel disease. However, in the case of anti-TNF drugs, 50 to 60% of patients do not respond or the response disappears within 1 year. There is a high demand for treatment development.
뿐만 아니라, 현재 시판되고 있는 치료제 및 치료방법은 보통 미국·유럽 등 서양인에 타겟된 치료이므로, 서구화된 식습관으로 발생이 급격히 증가되어 있는 아시아인의 염증성 장 질환에는 실제로 그 효과가 현저히 떨어진다는 연구결과도 보고된 바 있다 (Treatment of inflammatory bowel disease in Aisa. Intest Res, 2016, 14(3):231-239; Best practices on immunomodulators and biologic agents for ulcerative colitis and Crohn's disease in Asia, Intest Res, 2019, 17(3):285-310). In addition, since currently marketed treatments and treatment methods are usually targeted at Westerners, such as those in the US and Europe, a study has also reported that their effectiveness is significantly less effective for inflammatory bowel disease in Asians, whose incidence is rapidly increasing due to westernized eating habits (Treatment of inflammatory bowel disease in Aisa. Intest Res , 2016, 14(3):231-239; Best practices on immunomodulators and biologic agents for ulcerative colitis and Crohn's disease in Asia, Intest Res , 2019, 17(3):285-310).
이에, 본 발명자들은 서구식 식이 섭취와 같은 환경적 요인으로 유발된 염증성 장 질환의 발병 기전과 이에 타겟된 치료제 또는 치료 방법이 별도로 마련되어 있지 않다는 점에 착안하여, 특히 아시아 지역에서 발생되는 염증성 장 질환 특이적 치료제를 개발하고자 예의 노력한 결과, 장기간의 서구식 식이 섭취는 실제로 장 내 심각한 염증을 유도하고, 특히 장 미각 수용체 TAS1R3가 이러한 장 염증을 매개하는데 중요한 매개체가 될 수 있음을 확인하였으며, TAS1R3를 넉아웃 시키면 장세포 내 전사인자(Transcription factor) PPARγ이 유의하게 증가하면서, i) 밀착 연접 강화 (Tight junction protein) 및 항균 펩타이드 (Antimicrobial peptide) 분비에 관여하는 유전자들의 발현을 증가시켜 장 내 점막 방어 시스템(defense system)이 증가되어 서구식 식이 유도 장 염증으로부터 보호 기전이 강화될 뿐만 아니라, 증가된 PPARγ는 ii) 장 내강의 상태를 저산소 상태로 만들어, 혐기성 부티레이트균의 확장을 유도시키면서 장내유익균(Beneficial bacteria)들의 증식을 유발, 장 염증 지표가 유의적으로 감소되어 장 염증이 치료될 수 있음을 검증함으로써 본 발명을 완성하였다. Accordingly, the present inventors focused on the pathogenesis of inflammatory bowel disease caused by environmental factors such as Western-style dietary intake and the fact that there is no separate therapeutic agent or treatment method targeted thereto, and made diligent efforts to develop a specific therapeutic agent for inflammatory bowel disease, particularly occurring in Asia. As a result, long-term Western-style dietary intake actually induces severe inflammation in the intestine, and in particular, it was confirmed that the intestinal taste receptor TAS1R3 can be an important mediator in mediating such intestinal inflammation, knocking out TAS1R3 When activated, the transcription factor PPARγ in enterocytes significantly increases, i) the mucosal defense system in the intestine is increased by increasing the expression of genes involved in the secretion of tight junction proteins and antimicrobial peptides, thereby strengthening the protective mechanism from western diet-induced intestinal inflammation, and the increased PPARγ ii) makes the condition of the intestinal lumen hypoxic, leading to the expansion of anaerobic butyrate bacteria and intestinal oil The present invention was completed by verifying that intestinal inflammation can be treated by inducing proliferation of beneficial bacteria and significantly reducing intestinal inflammation indicators.
본 발명의 목적은 서구식 식이로 발병되는 염증성 장 질환의 치료제 및 이를 스크리닝 하는 방법을 제공하는 것이다.An object of the present invention is to provide a therapeutic agent for inflammatory bowel disease caused by a western diet and a method for screening the same.
상기 목적을 달성하기 위하여, 본 발명은 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 유효성분으로 포함하는 염증성 장 질환 예방 또는 치료용 약학적 조성물을 제공한다. In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
본 발명은 또한, 염증성 장 질환 예방 또는 치료를 위한 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제의 신규한 용도를 제공한다.The present invention also provides a novel use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for preventing or treating inflammatory bowel disease.
본 발명은 또한, 염증성 장 질환 예방 또는 치료를 위한 약물의 제조를 위한 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제의 신규한 용도를 제공한다.The present invention also provides a novel use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for the preparation of a drug for preventing or treating inflammatory bowel disease.
본 발명은 또한, TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 염증성 장 질환 예방 또는 치료방법을 제공한다. The present invention also provides a method for preventing or treating inflammatory bowel disease comprising administering a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor to a subject in need thereof.
본 발명은 또한, TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 유효성분으로 포함하는 염증성 장 질환 예방 또는 개선용 식품용 조성물을 제공한다.The present invention also provides a food composition for preventing or improving inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
본 발명에 있어서, 상기 TAS1R3 발현 억제제는 TAS1R3 특이적인 안티센스올리고뉴클레오티드, siRNA, shRNA 또는 miRNA인 것을 특징으로 할 수 있다.In the present invention, the TAS1R3 expression inhibitor may be a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA.
본 발명에 있어서, 상기 TAS1R3 단백질 활성 억제제는 TAS1R3 특이적인 항체, 압타머 또는 안타고니스트(antagonist)인 것을 특징으로 할 수 있다.In the present invention, the TAS1R3 protein activity inhibitor may be a TAS1R3-specific antibody, aptamer or antagonist.
본 발명에 있어서, 상기 염증성 장 질환은 서구식 식이에 의해 유도되는 염증성 장 질환인 것을 특징으로 할 수 있다.In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease induced by a western diet.
본 발명에 있어서, 상기 조성물은 In the present invention, the composition
(i) 장 내 미생물군집(microbiome) 변화; (i) changes in the gut microbiome;
(ii) 장 내 세포간 밀착 연접 강화; 및/또는(ii) strengthening the tight junctions between cells in the intestine; and/or
(iii) 항균 펩타이드 분비 유전자 발현의 증가;를 유도하여 염증성 장 질환을 예방, 개선 또는 치료하는 것을 특징으로 할 수 있다. (iii) an increase in antimicrobial peptide secretion gene expression; may be characterized by preventing, improving or treating inflammatory bowel disease by inducing.
본 발명에 있어서, 상기 장 내 미생물군집(microbiome) 변화는 장 내 혐기성 부티레이트 균의 확장 및/또는 장 내 유해균의 억제인 것을 특징으로 할 수 있다.In the present invention, the change in the microbiome in the intestine may be characterized by expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine.
본 발명은 또한, 다음 단계를 포함하는 염증성 장 질환 치료제 스크리닝 방법을 제공한다: The present invention also provides a method for screening a therapeutic agent for inflammatory bowel disease comprising the following steps:
(a) TAS1R3가 발현되거나 TAS1R3 단백질이 활성을 가지는 세포 또는 조직에 후보 물질을 처리하는 단계; (a) treating cells or tissues in which TAS1R3 is expressed or the TAS1R3 protein is active with a candidate substance;
(b) TAS1R3 발현이 억제되거나 TAS1R3 단백질 활성이 억제되는 경우 상기 후보 물질을 염증성 장 질환 치료제로 선별하는 단계. (b) selecting the candidate substance as a therapeutic agent for inflammatory bowel disease when TAS1R3 expression is inhibited or TAS1R3 protein activity is inhibited.
본 발명에 있어서, 상기 염증성 장 질환은 서구식 식이에 의해 유발되는 염증성 장 질환인 것을 특징으로 할 수 있다.In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease caused by a western diet.
본 발명에 따라 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 사용하는 경우, 장 내 미생물군집을 변화시키고, 장 세포 간의 밀착 연접을 강화시키며, 염증성 사이토카인 분비를 억제하여, 염증성 장 질환을 효과적으로 예방, 개선 또는 치료할 수 있는 장점이 있고, 이는 현재까지 적절한 치료제가 개발되지 않은 서구식 식이에 의해 급증하는 아시아인들의 염증성 장 질환 치료 용도로 유용하게 사용될 수 있을 것이다.In the case of using a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor according to the present invention, there is an advantage in that inflammatory bowel disease can be effectively prevented, improved, or treated by changing the intestinal microbiome, strengthening the tight junction between intestinal cells, and suppressing the secretion of inflammatory cytokines.
도 1a는 정상 식이(ND)와 서구식 식이(WD) 마우스에서 조직 염색을 통해 장 염증 정도를 비교한 결과이다.Figure 1a is a result of comparing the degree of intestinal inflammation through tissue staining in normal diet (ND) and western diet (WD) mice.
도 1b는 정상 식이와 서구식 식이 마우스에서 염증 지수(inflammation score)를 계산한 결과이다. 서구식 식이 마우스에서 염증의 수치가 극도로 높아졌다. Figure 1b is the result of calculating the inflammation score (inflammation score) in mice fed a normal diet and a western diet. Levels of inflammation were extremely elevated in mice fed a western diet.
도 1c는 정상 식이와 서구식 식이 마우스에서 장 조직을 적출하여 해당 조직에서의 RNA 시퀀싱 분석 결과를 비교한 것이다. 서구식 식이 마우스(빨간 사각형)의 장 유전체 발현은 정상 식이 마우스(검정 원)의 장 유전체 발현과 확연히 구분되었다.Figure 1c compares the results of RNA sequencing analysis of intestinal tissues extracted from normal and Western diet mice. Intestinal genome expression in Western-fed mice (red squares) was significantly different from that in normal-fed mice (black circles).
도 1d는 정상 식이와 서구식 식이 마우스의 장 조직에서 통계적으로 발현이 유의하게 변한 유전자들을 선별(스크리닝) 한 결과이다.Figure 1d is a result of selecting (screening) genes whose expression was statistically significantly changed in the intestinal tissues of mice fed a normal diet and a western diet.
도 1e는 정상 식이와 서구식 식이 마우스에서 유의한 차이를 보이는 유전자 (Differentially expressed genes, DEGs)들의 기능적 분류 (Functional classification)를 진행한 결과이다. 크게 염증 반응 (Inflammatory response)과 함께 미각 수용체 활성(Taste receptor activity) 관련 신호전달체계의 변화가 서구식 식이 마우스의 장에서 나타나는 특징이다.1e is a result of functional classification of genes (Differentially expressed genes, DEGs) showing significant differences between mice fed a normal diet and a western diet. Significantly, changes in the signal transduction system related to taste receptor activity along with an inflammatory response are characteristic of the intestines of mice fed a western diet.
도 1f는 서구식 식이 마우스에서 여러 미각 수용체의 종류 중에서도 미각 수용체 Tas1r3 발현이 매우 유의하게 증가되었음을 확인한 결과이다. FIG. 1f shows a result confirming that expression of the taste receptor Tas1r3 was significantly increased among various types of taste receptors in mice fed a western diet.
도 1g는 서구식 식이 마우스의 염증성 장 조직에서 TAS1R3의 단백질 발현 증가를 확인한 결과이다.Figure 1g shows the result of confirming the increased protein expression of TAS1R3 in inflammatory intestinal tissues of mice fed a western diet.
도 1h는 TAS1R3와 여러 염증 지표 간의 유의한 양의 상관관계를 확인한 결과이다. 서구식 식이 마우스에서 증가한 장 내 TAS1R3 발현은 여러 염증지표들과 유의한 양의 상관관계를 나타낸다. 1h is a result confirming a significant positive correlation between TAS1R3 and various inflammatory markers. Increased intestinal TAS1R3 expression in mice fed a western diet showed significant positive correlations with several inflammatory markers.
도 2a는 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 다양한 조합으로 처리하고, 장 미각수용체 TAS1R3 및 그 다운스트림 분자인 GLP-1의 발현 여부를 확인하는 실험의 모식도이다.FIG. 2a is a schematic diagram of an experiment in which the enteroendocrine cell line NCI-H716 was treated with various combinations of glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the expression of the intestinal taste receptor TAS1R3 and its downstream molecule, GLP-1, was examined.
도 2b는 도 2a에 도식된 조합으로 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, 12시간 후 TAS1R3의 mRNA 발현량을 GAPDH의 mRNA 발현량으로 정상화하여 표현한 결과이다.Figure 2b is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 µM) in the combination shown in Figure 2a, and normalizing the mRNA expression level of TAS1R3 to the mRNA expression level of GAPDH after 12 hours.
도 2c는 도 2a에 도식된 조합으로 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, GCG의 mRNA 발현량을 GAPDH의 mRNA 발현량으로 정상화하여 24시간 동안 관찰한 결과이다.Figure 2c is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) in the combination shown in Figure 2a, normalizing the mRNA expression level of GCG to the mRNA expression level of GAPDH, and observing for 24 hours.
도 2d는 도 2a에 도식된 조합으로 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, GLP-1의 분비 정도를 24시간 동안 관찰한 결과이다. GLP-1의 분비는 TAS1R3의 활성화 정도를 의미한다. Figure 2d is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) in the combination shown in Figure 2a, and observing the level of GLP-1 secretion for 24 hours. Secretion of GLP-1 means the degree of activation of TAS1R3.
도 2e는 도 2a에 도식된 조합으로 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, IL1B의 mRNA 발현량을 GAPDH의 mRNA 발현량으로 정상화하여 24시간 동안 관찰한 결과이다.Figure 2e is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) in the combination shown in Figure 2a, normalizing the mRNA expression level of IL1B to the mRNA expression level of GAPDH, and observing for 24 hours.
도 2f는 도 2a에 도식된 조합으로 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, IL6의 mRNA 발현량을 GAPDH의 mRNA 발현량으로 정상화하여 24시간 동안 관찰한 결과이다.Figure 2f is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) in the combination shown in Figure 2a, normalizing the mRNA expression level of IL6 to the mRNA expression level of GAPDH, and observing for 24 hours.
도 2g는 도 2a에 도식된 조합으로 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, TNF의 mRNA 발현량을 GAPDH의 mRNA 발현량으로 정상화하여 24시간 동안 관찰한 결과이다.Figure 2g is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) in the combination shown in Figure 2a, normalizing the mRNA expression level of TNF to the mRNA expression level of GAPDH, and observing for 24 hours.
도 2h는 도 2a에 도식된 조합으로 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, MCP-1의 mRNA 발현량을 GAPDH의 mRNA 발현량으로 정상화하여 24시간 동안 관찰한 결과이다.Figure 2h is the result of treating the NCI-H716 cell line, which is an enteroendocrine cell, with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) in the combination shown in Figure 2a, normalizing the mRNA expression level of MCP-1 to the mRNA expression level of GAPDH, and observing for 24 hours.
도 3a는 TAS1R3 siRNA 처리에 따른 TAS1R3 전사체 양을 확인한 결과이다.Figure 3a is the result of confirming the amount of TAS1R3 transcript according to TAS1R3 siRNA treatment.
도 3b는 TAS1R3 siRNA 처리에 따른 TAS1R3 발현 억제 효과를 단백질 수준에서 확인한 결과이다.Figure 3b is the result of confirming the TAS1R3 expression inhibition effect according to TAS1R3 siRNA treatment at the protein level.
도 3c는 TAS1R3 siRNA 처리에 따른 GCG 전사체 발현 억제 효과를 확인한 결과이다. Figure 3c is the result of confirming the effect of suppressing GCG transcript expression according to TAS1R3 siRNA treatment.
도 3d는 TAS1R3 siRNA 처리에 따른 활성 GLP-1 억제 효과를 확인한 결과이다.Figure 3d is the result of confirming the active GLP-1 inhibitory effect according to TAS1R3 siRNA treatment.
도 3e는 TAS1R3 siRNA 처리에 따른 IL1B 전사체 발현 억제 효과를 확인한 결과이다. Figure 3e is the result of confirming the effect of inhibiting IL1B transcript expression according to TAS1R3 siRNA treatment.
도 3f는 TAS1R3 siRNA 처리에 따른 IL6 전사체 발현 억제 효과를 확인한 결과이다. Figure 3f is the result of confirming the effect of inhibiting IL6 transcript expression according to TAS1R3 siRNA treatment.
도 3g는 TAS1R3 siRNA 처리에 따른 IL8 전사체 발현 억제 효과를 확인한 결과이다. 3g shows the result of confirming the effect of inhibiting IL8 transcript expression according to TAS1R3 siRNA treatment.
도 3h는 TAS1R3 siRNA 처리에 따른 IL8 분비 억제 효과를 확인한 결과이다.Figure 3h is the result of confirming the effect of inhibiting IL8 secretion according to TAS1R3 siRNA treatment.
도 3i는 TAS1R3 siRNA 처리에 따른 TNF 전사체 발현 억제 효과를 확인한 결과이다. Figure 3i is the result of confirming the effect of TNF transcript expression inhibition according to TAS1R3 siRNA treatment.
도 3j는 TAS1R3 siRNA 처리에 따른 TNF-α 분비 억제 효과를 확인한 결과이다.Figure 3j is the result of confirming the TNF-α secretion inhibitory effect according to TAS1R3 siRNA treatment.
도 3k는 TAS1R3 안타고니스트인 Lactisole 처리에 따른 GCG 전사체 발현 억제 효과를 확인한 결과이다. 3k shows the result of confirming the effect of inhibiting GCG transcript expression by treatment with Lactisole, an antagonist for TAS1R3.
도 3l은 Lactisole 처리에 따른 활성 GLP-1 억제 효과를 확인한 결과이다.Figure 3l is the result of confirming the active GLP-1 inhibitory effect according to lactisole treatment.
도 3m은 Lactisole 처리에 따른 IL1B 전사체 발현 억제 효과를 확인한 결과이다. 3m is a result confirming the effect of inhibiting IL1B transcript expression according to lactisole treatment.
도 3n 은 Lactisole 처리에 따른 IL6 전사체 발현 억제 효과를 확인한 결과이다. 3n shows the result of confirming the effect of inhibiting IL6 transcript expression according to lactisole treatment.
도 3o는 Lactisole 처리에 따른 IL8 전사체 발현 억제 효과를 확인한 결과이다. Figure 3o shows the result of confirming the effect of inhibiting IL8 transcript expression according to lactisole treatment.
도 3p는 Lactisole 처리에 따른 IL8 분비 억제 효과를 확인한 결과이다.Figure 3p is the result of confirming the effect of inhibiting IL8 secretion according to lactisole treatment.
도 3q는 Lactisole 처리에 따른 TNF 전사체 발현 억제 효과를 확인한 결과이다. Figure 3q is the result of confirming the effect of inhibiting TNF transcript expression according to lactisole treatment.
도 3r은 Lactisole 처리에 따른 TNF-α 분비 억제 효과를 확인한 결과이다.Figure 3r is a result confirming the effect of inhibiting TNF-α secretion according to lactisole treatment.
도 4는 본 발명에서 제작된 미각 수용체 Tas1r3 넉아웃 마우스의 소장(Small intestine) 및 대장(Large intestine) 내에서 TAS1R3 미발현을 확인한 결과이다. 4 is a result of confirming non-expression of TAS1R3 in the small intestine and large intestine of the taste receptor Tas1r3 knockout mouse prepared in the present invention.
도 5a는 정상 또는 서구식 식이가 공급된 Tas1r3 넉아웃 마우스의 소장에서 Tas1r3 전사체 발현이 억제됨을 확인한 결과이다. 5a is a result confirming that Tas1r3 transcript expression is suppressed in the small intestine of Tas1r3 knockout mice fed a normal or western diet.
도 5b는 정상 또는 서구식 식이가 공급된 Tas1r3 넉아웃 마우스의 소장에서 Tas1r3 단백질 발현이 억제됨을 확인한 결과이다. FIG. 5B is a result confirming that Tas1r3 protein expression is suppressed in the small intestine of Tas1r3 knockout mice fed a normal or western diet.
도 6a는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 비장(Spleen)의 무게를 비교한 결과이다.Figure 6a is a result of comparison of spleen weights in wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 6b는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 소장(small intestine)의 길이를 비교한 결과이다.Figure 6b is a result of comparing the length of the small intestine in wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 6c는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 결장(colon)의 길이를 비교한 결과이다.Figure 6c shows the results of comparison of colon lengths in wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 6d는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 조직학적 검사를 통해 장 조직 내 염증 발생을 비교한 결과이다.Figure 6d shows the results of comparison of inflammation in intestinal tissue through histological examination in wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 6e는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 장 염증 지수를 비교한 결과이다.Figure 6e is a comparison of the intestinal inflammatory index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 6f는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 소장에서 IL-1b 전사체 발현 양상을 비교한 결과이다.Figure 6f compares the pattern of IL-1b transcript expression in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 6g는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 소장에서 Tnf 전사체 발현 양상을 비교한 결과이다.6g shows the comparison of Tnf transcript expression patterns in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 6h는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 소장에서 IL6 전사체 발현 양상을 비교한 결과이다.Figure 6h shows the comparison of IL6 transcript expression patterns in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 7a는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 소장에서 인산화 된 mTOR(phosphorylated-mTOR), mTOR, PPAR-γ의 단백질 발현 정도를 비교한 결과이다.7a is a comparison result of protein expression levels of phosphorylated-mTOR (mTOR), mTOR, and PPAR-γ in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 7b는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 소장에서 인산화된 mTOR(phosphorylated-mTOR) 단백질 및 PPAR-γ의 단백질 발현 정도를 정량화한 결과이다.7b is a result of quantifying the protein expression levels of phosphorylated-mTOR (mTOR) protein and PPAR-γ in the small intestine of wild-type or Tas1r3 knockout mice fed a normal or western diet.
[규칙 제91조에 의한 보정 14.02.2023]
도 7c 및 도 7d는 각각 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 소장에서 전사인자(Transcription factor) PPAR-γ의 조절 유전자인 밀착 연접(Tight junction) 유전자(Tjp1, Ocln, Cldn1, Cldn7)들의 발현 정도와 항균 펩타이드(Antimicrobial peptide) 유전자(Reg3g, Lyz1, Defa2, Defa3)들의 발현 정도를 비교한 결과이다.[Amendment 14.02.2023 under Rule 91]
FIGS. 7C and 7D each is a tight Junction gene (TJP1, OCLN1, CLDN7), a wild type or tas1r3 knockout mouse with normal or western diets, respectively. This is the result of the expression of the expression of the expression of antimicrobial peptides (Reg3g, Lyz1, Defa2, Defa3).
도 8a는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 Shannon's diversity index를 비교한 결과이다.8a is a comparison result of Shannon's diversity index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 8b는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 Faith's phylogenetic index를 비교한 결과이다.8b is a comparison result of Faith's phylogenetic index in wild-type or Tas1r3 knockout mice fed a normal or western diet.
도 8c는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 β-dirversity를 Bray-Curtis 방식을 통해 비교한 결과이다. Figure 8c shows the results of comparison of β-dirversity of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet using the Bray-Curtis method.
도 8d는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 β-diversity를 Weighted PCoA 방식을 통해 비교한 결과이다.8d shows the results of comparison of β-diversity of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet using the weighted PCoA method.
도 8e는 정상 또는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 장내 미생물 군집의 차이를 Phylum(문) 수준에서 비교한 결과이다.FIG. 8E shows the results of comparing the differences in the gut microbiome of wild-type or Tas1r3 knockout mice fed a normal or Western-style diet at the phylum level.
도 8f는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스에서 차이를 보이는 장내 미생물 군집의 차이를 Family(과) 및 Genus(속) 수준에서 비교한 결과이다. 두 그룹 간 유의하게 차이를 보이는 박테리아를 클라도그램(Cladogram)을 통해 보여주고 있다. FIG. 8f shows the results of comparing the differences in gut microbial communities at the family (family) and genus (genus) levels in wild-type or Tas1r3 knockout mice fed a western-style diet. Bacteria showing significant differences between the two groups are shown through Cladogram.
도 8g는 서구식 식이가 공급된 Tas1r3 넉아웃 마우스에서 가장 뚜렷한 특징을 보이는 부티레이트 생성균주(Butyrivibrio 속, Roseburia 속, Ruminococcus 속, Butyricicoccus 속, Faecalibacterium 속)의 상대적 풍부도(Relative abundance)를 비교한 결과이다. Figure 8g shows the relative abundance of butyrate-producing strains (genus Butyrivibrio , Roseburia , Ruminococcus , Butyricicoccus , Faecalibacterium ) showing the most distinct characteristics in Tas1r3 knockout mice fed a western diet. Results were compared.
도 8h는 서구식 식이가 공급된 야생형 또는 Tas1r3 넉아웃 마우스의 대변 내 부티레이트의 양을 비교한 결과이다. 8h is a result of comparison of the amount of butyrate in the feces of wild-type or Tas1r3 knockout mice fed a western-style diet.
[규칙 제91조에 의한 보정 14.02.2023]
도 9a 내지 도 9e는 Tas1r3 결핍에 의해 증가된 PPARγ 신호전달 경로와 혐기성 부티레이트 생성 박테리아가 유의한 상관관계가 있음을 보여주는 결과이다.[Amendment 14.02.2023 under Rule 91]
9a to 9e are results showing that there is a significant correlation between the PPARγ signaling pathway increased by Tas1r3 deficiency and anaerobic butyrate-producing bacteria.
도 10a는 TAS1R3 siRNA 처리에 따른 MTOR 전사체 발현 억제 효과를 확인한 결과이다. 10a is a result confirming the effect of inhibiting MTOR transcript expression according to TAS1R3 siRNA treatment.
도 10b는 TAS1R3 siRNA 처리에 따른 PPARγ 전사체 발현 증진 효과를 확인한 결과이다. 10b is a result confirming the effect of enhancing PPARγ transcript expression according to TAS1R3 siRNA treatment.
도 10c는 TAS1R3 siRNA 처리에 따른 PPARγ 단백질 발현 증진 효과를 확인한 결과이다. 10c is a result confirming the effect of enhancing PPARγ protein expression according to TAS1R3 siRNA treatment.
도 10d는 TAS1R3 antagonist인 Lactisole 처리에 따른 MTOR 전사체 발현 억제 효과를 확인한 결과이다. 10d is a result confirming the effect of inhibiting MTOR transcript expression according to treatment with Lactisole, a TAS1R3 antagonist.
도 10e는 Lactisole 처리에 따른 PPARγ 전사체 발현 증진 효과를 확인한 결과이다. 10e is a result confirming the effect of enhancing PPARγ transcript expression according to lactisole treatment.
도 10f는 Lactisole 처리에 따른 PPARγ 단백질 발현 증진 효과를 확인한 결과이다. 10f is a result confirming the effect of enhancing PPARγ protein expression according to Lactisole treatment.
도 11은 정상 대조군과 염증성 장 질환 환자 실험군의 장 생검 샘플에서 전사체들의 발현 양상의 차이를 분석하고(11a - 11c), TAS1R3 및 다운스트림 분자(mTOR, PPARγ)의 발현정도(11d - 11f) 및 관련 분자(molecules)들과의 연관성(11g)을 확인한 결과이다.11 is a result of analyzing differences in expression patterns of transcripts in intestinal biopsy samples of a normal control group and an experimental group of patients with inflammatory bowel disease (11a - 11c), and confirming the expression levels (11d - 11f) of TAS1R3 and downstream molecules (mTOR, PPARγ) and association with related molecules (11g).
도 12는 장 미각 수용체 TAS1R3가 장 염증을 조절하는 핵심 메커니즘을 보여주는 모식도이다. 12 is a schematic diagram showing a key mechanism by which the intestinal taste receptor TAS1R3 regulates intestinal inflammation.
다른 식으로 정의되지 않는 한, 본 명세서에서 사용된 모든 기술적 및 과학적 용어들은 본 발명이 속하는 기술분야에서 숙련된 전문가에 의해서 통상적으로 이해되는 것과 동일한 의미를 갖는다. 일반적으로 본 명세서에서 사용된 명명법은 본 기술 분야에서 잘 알려져 있고 통상적으로 사용되는 것이다.Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is one well known and commonly used in the art.
본 발명에서는 서구식 식이에 의해 유도되는 염증성 장 질환 개체에서 장 내 미각수용체인 TAS1R3 발현이 증가되어 있음을 확인하였고, TAS1R3 발현을 억제하는 경우 서구식 식이에 의해 유도되는 염증성 장 질환을 치료할 수 있을 것이라는 가설 아래, TAS1R3 넉아웃 동물 모델을 제작하여 실험을 진행한 결과, TAS1R3 발현이 억제되면 염증성 사이토카인 및 케모카인의 발현이 억제되고, mTOR/PPARγ를 축으로 하는 신호전달 경로가 조절되어, 장 내 밀착 연접이 강화되고 장 내 미생물군집이 장 건강에 유리하게 변화되어, 염증성 장 질환이 효과적으로 치료될 수 있음을 확인하였다. In the present invention, it was confirmed that the expression of TAS1R3, an intestinal taste receptor, was increased in subjects with inflammatory bowel disease induced by a western-style diet. Under the hypothesis that suppressing TAS1R3 expression would be able to treat inflammatory bowel disease induced by a western-style diet, a TAS1R3 knockout animal model was constructed and tested. It was confirmed that inflammatory bowel disease can be effectively treated by regulating the signal transduction pathway, strengthening the intestinal tight junction and changing the intestinal microbial community in favor of intestinal health.
따라서, 본 발명은 일 관점에서 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 유효성분으로 포함하는 염증성 장 질환 예방 또는 치료용 약학적 조성물에 관한 것이고, 다른 관점에서 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 유효성분으로 포함하는 염증성 장 질환 예방 또는 개선용 식품용 조성물에 관한 것이다.Accordingly, in one aspect, the present invention relates to a pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient, and in another aspect, it relates to a food composition for preventing or improving inflammatory bowel disease comprising a TAS1R3 expression inhibitor or TAS1R3 protein activity inhibitor as an active ingredient.
본 발명에 있어서, 상기 인간 TAS1R3를 인코딩하는 유전자 서열은 서열번호 1로 표시될 수 있고, 인간 TAS1R3 단백질 서열은 서열번호 3으로 표시될 수 있으나, 이에 한정되지는 않는다. In the present invention, the gene sequence encoding the human TAS1R3 may be represented by SEQ ID NO: 1, and the human TAS1R3 protein sequence may be represented by SEQ ID NO: 3, but is not limited thereto.
TAS1R3 유전자 서열 (Human) TAS1R3 gene sequence (Human)
Homo sapiens taste 1 receptor member 3 (TAS1R3), mRNAHomo sapiens taste 1 receptor member 3 (TAS1R3), mRNA
> NM_152228.3 Homo sapiens taste 1 receptor member 3 (TAS1R3), mRNA> NM_152228.3 Homo sapiens taste 1 receptor member 3 (TAS1R3), mRNA
Figure PCTKR2023001053-appb-img-000001
Figure PCTKR2023001053-appb-img-000001
Tas1r3 유전자 서열 (Mouse) Tas1r3 gene sequence (Mouse)
Mus musculus taste receptor, type 1, member 3 (Tas1r3), mRNAMus muscle taste receptor, type 1, member 3 (Tas1r3), mRNA
> NM_031872.2 Mus musculus taste receptor, type 1, member 3 (Tas1r3), mRNA> NM_031872.2 Mus muscle taste receptor, type 1, member 3 (Tas1r3), mRNA
Figure PCTKR2023001053-appb-img-000002
Figure PCTKR2023001053-appb-img-000002
TAS1R3 단백질 서열 (Human)TAS1R3 protein sequence (Human)
Taste receptor type 1 member 3 precursor [Homo sapiens] Taste receptor type 1 member 3 precursor [Homo sapiens]
NCBI Reference Sequence: NP_689414.2NCBI Reference Sequence: NP_689414.2
mlgpavlgls lwallhpgtg aplclsqqlr mkgdyvlggl fplgeaeeag lrsrtrpsspmlgpavlgls lwallhpgtg aplclsqqlr mkgdyvlggl fplgeaeeag lrsrtrpssp
vctrfssngl lwalamkmav eeinnksdll pglrlgydlf dtcsepvvam kpslmflakavctrfssngl lwalamkmav eeinnksdll pglrlgydlf dtcsepvvam kpslmflaka
gsrdiaaycn ytqyqprvla vigphssela mvtgkffsff lmpqvsygas mellsaretfgsrdiaaycn ytqyqprvla vigphssela mvtgkffsff lmpqvsygas mellsaretf
psffrtvpsd rvqltaaael lqefgwnwva algsddeygr qglsifsala aargiciahepsffrtvpsd rvqltaaael lqefgwnwva algsddeygr qglsifsala aargiciahe
glvplpradd srlgkvqdvl hqvnqssvqv vllfasvhaa halfnysiss rlspkvwvasglvplpradd srlgkvqdvl hqvnqssvqv vllfasvhaa halfnysiss rlspkvwvas
eawltsdlvm glpgmaqmgt vlgflqrgaq lhefpqyvkt hlalatdpaf csalgereqgeawltsdlvm glpgmaqmgt vlgflqrgaq lhefpqyvkt hlalatdpaf csalgereqg
leedvvgqrc pqcdcitlqn vsaglnhhqt fsvyaavysv aqalhntlqc nasgcpaqdpleedvvgqrc pqcdcitlqn vsaglnhhqt fsvyaavysv aqalhntlqc nasgcpaqdp
vkpwqllenm ynltfhvggl plrfdssgnv dmeydlklwv wqgsvprlhd vgrfngslrtvkpwqllenm ynltfhvggl plrfdssgnv dmeydlklwv wqgsvprlhd vgrfngslrt
erlkirwhts dnqkpvsrcs rqcqegqvrr vkgfhsccyd cvdceagsyr qnpddiactferlkirwhts dnqkpvsrcs rqcqegqvrr vkgfhsccyd cvdceagsyr qnpddiactf
cgqdewsper strcfrrrsr flawgepavl llllllslal glvlaalglf vhhrdsplvqcgqdewsper strcfrrrsr flawgepavl llllllslal glvlaalglf vhhrdsplvq
asggplacfg lvclglvcls vllfpgqpsp arclaqqpls hlpltgclst lflqaaeifvasggplacfg lvclglvcls vllfpgqpsp arclaqqpls hlpltgclst lflqaaeifv
eselplswad rlsgclrgpw awlvvllaml vevalctwyl vafppevvtd whmlptealveselplswad rlsgclrgpw awlvvllaml vevalctwyl vafppevvtd whmlptealv
hcrtrswvsf glahatnatl aflcflgtfl vrsqpgcynr argltfamla yfitwvsfvphcrtrswvsf glahatnatl aflcflgtfl vrsqpgcynr argltfamla yfitwvsfvp
llanvqvvlr pavqmgalll cvlgilaafh lprcyllmrq pglntpeffl gggpgdaqgqllanvqvvlr pavqmgalll cvlgilaafh lprcyllmrq pglntpeffl gggpgdaqgq
ndgntgnqgk hendgntgnqgk he
TAS1R3 단백질 서열 (Mouse)TAS1R3 protein sequence (Mouse)
Taste receptor type 1 member 3 precursor [Mus musculus] Taste receptor type 1 member 3 precursor [Mus musculus]
NCBI Reference Sequence: NP_114078.1NCBI Reference Sequence: NP_114078.1
mpalaimgls laaflelgmg aslclsqqfk aqgdyilggl fplgsteeat lnqrtqpnsimpalaimgls laaflelgmg aslclsqqfk aqgdyilggl fplgsteeat lnqrtqpnsi
pcnrfsplgl flamamkmav eeinngsall pglrlgydlf dtcsepvvtm ksslmflakvpcnrfsplgl flamamkmav eeinngsall pglrlgydlf dtcsepvvtm ksslmflakv
gsqsiaaycn ytqyqprvla vigphssela litgkffsff lmpqvsysas mdrlsdretfgsqsiaaycn ytqyqprvla vigphssela litgkffsff lmpqvsysas mdrlsdretf
psffrtvpsd rvqlqavvtl lqnfswnwva algsdddygr eglsifssla nargiciahepsffrtvpsd rvqlqavvtl lqnfswnwva algsdddygr eglsifssla nargiciahe
glvpqhdtsg qqlgkvldvl rqvnqskvqv vvlfasarav yslfsysihh glspkvwvasglvpqhdtsg qqlgkvldvl rqvnqskvqv vvlfasarav yslfsysihh glspkvwvas
eswltsdlvm tlpniarvgt vlgflqrgal lpefshyvet hlalaadpaf caslnaeldleswltsdlvm tlpniarvgt vlgflqrgal lpefshyvet hlalaadpaf caslnaeldl
eehvmgqrcp rcddimlqnl ssgllqnlsa gqlhhqifat yaavysvaqa lhntlqcnvseehvmgqrcp rcddimlqnl ssgllqnlsa gqlhhqifat yaavysvaqa lhntlqcnvs
hchvsehvlp wqllenmynm sfhardltlq fdaegnvdme ydlkmwvwqs ptpvlhtvgthchvsehvlp wqllenmynm sfhardltlq fdaegnvdme ydlkmwvwqs ptpvlhtvgt
fngtlqlqqs kmywpgnqvp vsqcsrqckd gqvrrvkgfh sccydcvdck agsyrkhpddfngtlqlqqs kmywpgnqvp vsqcsrqckd gqvrrvkgfh sccydcvdck agsyrkhpdd
ftctpcnqdq wspekstacl prrpkflawg epvvlsllll lclvlglala alglsvhhwdftctpcnqdq wspekstacl prrpkflawg epvvlsllll lclvlglala alglsvhhwd
splvqasggs qfcfgliclg lfclsvllfp grpssascla qqpmahlplt gclstlflqasplvqasggs qfcfgliclg lfclsvllfp grpssascla qqpmahlplt gclstlflqa
aetfveselp lswanwlcsy lrglwawlvv llatfveaal cawyliafpp evvtdwsvlpaetfveselp lswanwlcsy lrglwawlvv llatfveaal cawyliafpp evvtdwsvlp
tevlehchvr swvslglvhi tnamlaflcf lgtflvqsqp grynrarglt famlayfitwtevlehchvr swvslglvhi tnamlaflcf lgtflvqsqp grynrarglt famlayfitw
vsfvpllanv qvayqpavqm gailvcalgi lvtfhlpkcy vllwlpklnt qefflgrnakvsfvpllanv qvayqpavqm gailvcalgi lvtfhlpkcy vllwlpklnt qefflgrnak
kaadensggg eaaqghnekaadensggg eaaqghne
본 발명에 있어서, 상기 TAS1R3 발현 억제제는 TAS1R3 특이적인 안티센스올리고뉴클레오티드, siRNA, shRNA 또는 miRNA인 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다.In the present invention, the TAS1R3 expression inhibitor may be a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA, but is not limited thereto.
예컨대, 상기 TAS1R3 발현 억제제는 하기 서열로 표시되는 siRNA일 수 있으나, 이에 한정되지는 않는다.For example, the TAS1R3 expression inhibitor may be siRNA represented by the following sequence, but is not limited thereto.
TAS1R3 siRNA 시퀀스 정보TAS1R3 siRNA sequence information
Forward: CUGUCUACGCAGCUGUGUA (dTdT) Forward: CUGUCUACGCAGCUGUGUA (dTdT)
Reverse: UACACAGCUGCGUAGACAG (dTdT)Reverse: UACACAGCUGCGUAGACAG (dTdT)
본 발명에 있어서, 상기 TAS1R3 단백질 활성 억제제는 TAS1R3 특이적인 항체 압타머, 또는 안타고니스트인 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다.In the present invention, the TAS1R3 protein activity inhibitor may be a TAS1R3-specific antibody aptamer or an antagonist, but is not limited thereto.
본 발명에 있어서, 상기 TAS1R3 단백질 활성 억제제는 lactisole일 수 있으나, 이에 한정되지는 않는다. In the present invention, the TAS1R3 protein activity inhibitor may be lactisole, but is not limited thereto.
본 발명에 있어서, 상기 염증성 장 질환은 서구식 식이에 의해 유도되는 염증성 장 질환인 것을 특징으로 할 수 있다.In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease induced by a western diet.
본 발명에 있어서, 상기 조성물은 In the present invention, the composition
(i) 장 내 미생물군집(microbiome) 변화; (i) changes in the gut microbiome;
(ii) 장 내 세포간 밀착 연접 강화; 및/또는(ii) strengthening the tight junctions between cells in the intestine; and/or
(iii) 항균 펩타이드 분비 유전자 발현의 증가;를 유도하여 염증성 장 질환을 예방 또는 치료하는 것을 특징으로 할 수 있으나, 그 효과가 이에 한정되지는 않는다.(iii) increase in antimicrobial peptide secretion gene expression; may be characterized in that inflammatory bowel disease is prevented or treated by inducing, but the effect is not limited thereto.
본 발명에 있어서, 상기 장 내 미생물군집(microbiome) 변화는 장 내 혐기성 부티레이트 균의 확장 및/또는 장 내 유해균의 억제인 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다.In the present invention, the intestinal microbiome change may be characterized by expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine, but is not limited thereto.
본 발명에 있어서, 상기 혐기성 부티레이트 균은 Butyrivibrio 속, Roseburia 속, Ruminococcus 속, Butyricicoccus 속 및 Faecalibacterium 속 균으로 구성된 균에서 선택되는 1종 이상인 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다.In the present invention, the anaerobic butyrate bacteria may be characterized in that at least one selected from bacteria consisting of the genus Butyrivibrio , the genus Roseburia , the genus Ruminococcus , the genus Butyricicoccus and the genus Faecalibacterium , but is not limited thereto.
본 발명에 있어서, 상기 유해균은 Prevotella 속, Paraprevotellaceae 과, Enterobacteriaceae 과 및 Enterobacteriales 속 균으로 구성된 균에서 선택되는 1종 이상인 것을 특징으로 할 수 있으나, 이에 한정되지는 않는다. In the present invention, the harmful bacteria may be characterized in that at least one selected from bacteria consisting of the genus Prevotella , the family Paraprevotellaceae, the family Enterobacteriaceae, and the genus Enterobacteriales , but is not limited thereto.
본 발명에 따라 TAS1R3 발현 또는 활성을 억제시키면 장세포 내 PPARγ이 유의하게 증가하면서, i) 밀착 연접 강화 (Tight junction protein) 및 항균 펩타이드 (Antimicrobial peptide) 분비에 관여하는 유전자들의 발현을 증가시켜 장 내 점막 방어 시스템(defense system)이 증가되어 서구식 식이 유도 장 염증으로부터 보호 기전이 강화될 뿐만 아니라, 증가된 PPARγ는 ii) 장 내강의 상태를 저산소 상태로 만들어, 혐기성 부티레이트균의 확장을 유도시키면서 장내 유익균(Beneficial bacteria)들의 증식을 유발, 장 염증 지표가 유의적으로 감소되는바, 장 염증이 예방 또는 치료될 수 있다.Inhibition of TAS1R3 expression or activity according to the present invention significantly increases PPARγ in enterocytes, i) increases the expression of genes involved in the secretion of tight junction proteins and antimicrobial peptides, thereby increasing the defense system in the intestine, thereby strengthening the protective mechanism from western diet-induced intestinal inflammation, and the increased PPARγ ii) makes the intestinal lumen hypoxic and expands anaerobic butyrate bacteria While inducing, the proliferation of beneficial bacteria in the intestine is induced, and the intestinal inflammation index is significantly reduced, so that intestinal inflammation can be prevented or treated.
본 발명은 또 다른 관점에서, 다음 단계를 포함하는 염증성 장 질환 치료제 스크리닝 방법에 관한 것이다: In another aspect, the present invention relates to a method for screening a therapeutic agent for inflammatory bowel disease comprising the following steps:
(a) TAS1R3가 발현되거나 TAS1R3 단백질이 활성을 가지는 세포 또는 조직에 후보 물질을 처리하는 단계; (a) treating cells or tissues in which TAS1R3 is expressed or the TAS1R3 protein is active with a candidate substance;
(b) TAS1R3 발현이 억제되거나 TAS1R3 단백질 활성이 억제되는 경우 상기 후보 물질을 염증성 장 질환 치료제로 선별하는 단계. (b) selecting the candidate substance as a therapeutic agent for inflammatory bowel disease when TAS1R3 expression is inhibited or TAS1R3 protein activity is inhibited.
본 발명에 있어서, 상기 염증성 장 질환은 서구식 식이에 의해 유발되는 염증성 장 질환인 것을 특징으로 할 수 있다.In the present invention, the inflammatory bowel disease may be characterized in that it is an inflammatory bowel disease caused by a western diet.
상기 치료제는 TAS1R3 특이적인 안티센스올리고뉴클레오티드, siRNA, shRNA, miRNA 또는 TAS1R3 특이적인 항체, 압타머 또는 안타고니스트를 단독으로 포함하거나, 하나 이상의 약학적으로 허용되는 담체, 부형제 또는 희석제를 포함하여 약학 조성물로 제공될 수 있으며, 상기 복합체는 질환 및 이의 중증정도, 환자의 연령, 체중, 건강상태, 성별, 투여 경로 및 치료 기간 등에 따라 적절한 약학적으로 유효한 양으로 약학 조성물에 포함될 수 있다.The therapeutic agent may be provided as a pharmaceutical composition, including tas1R3 specific antisense oligonucleotides, siRNA, shrna, miRNA, or TAS1R3 specific antibodies, aptamers or antagonists, or include a pharmaceutically acceptable carrier, excipient or diluent, and the composite is a disease and the complex. It may be included in the pharmaceutical composition in an appropriate pharmaceutically effective amount, depending on the severity, age, weight, health, gender, administration route, and treatment period of the patient.
상기에서 “약학적으로 허용되는”이란 생리학적으로 허용되고 인간에게 투여될 때, 통상적으로 위장 장애, 현기증과 같은 알레르기 반응 또는 이와 유사한 반응을 일으키지 않는 조성물을 말한다. As used herein, "pharmaceutically acceptable" refers to a composition that is physiologically acceptable and does not cause allergic reactions such as gastrointestinal disorders and dizziness or similar reactions when administered to humans.
본 발명에 따른 조성물은 약학적으로 허용가능한 부형제를 더 포함할 수 있다. 본 발명의 조성물에 포함될 수 있는 부형제로는 락토즈, 덱스트로즈, 수크로스, 솔비톨, 만니톨, 자일리톨, 에리스리톨, 말티톨, 전분, 아카시아 고무, 알지네이트, 젤라틴, 칼슘 포스페이트, 칼슘 실리케이트, 셀룰로즈, 메틸 셀룰로즈, 미정질 셀룰로스, 폴리비닐 피롤리돈, 물, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 탈크, 마그네슘 스테아레이트 및 광물유 등을 포함하나 이에 국한되지 않는다.The composition according to the present invention may further include a pharmaceutically acceptable excipient. Excipients that may be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil; and the like.
본 발명의 조성물은 통상의 방법에 의해 경구 투여를 위한 제형 또는 비경구 투여를 위한 제형으로 제제화될 수 있고, 제제화할 경우 보통 사용하는 충진제, 증량제, 결합제, 습윤제, 붕해제, 계면활성제, 동결보호제 등을 사용하여 조제될 수 있다. The composition of the present invention may be formulated into a dosage form for oral administration or a dosage form for parenteral administration by a conventional method, and when formulated, commonly used fillers, extenders, binders, wetting agents, disintegrants, surfactants, cryoprotectants, etc. It can be prepared using.
본 발명의 조성물이 경구 투여를 위한 고형 제제로 제제화된 경우 정제, 환제, 산제, 과립제, 캡슐제 등을 포함하며, 이러한 고형 제제는 유효성분에 적어도 하나 이상의 부형제 예를 들면, 전분, 칼슘카보네이트(Calcium carbonate), 수크로스(Sucrose), 락토스(Lactose) 또는 젤라틴 등을 포함할 수 있다. 또한, 단순한 부형제 이외에 마그네슘 스테아레이트, 탈크 같은 윤활제 등을 포함할 수 있으나 이에 국한되지 않는다. When the composition of the present invention is formulated as a solid preparation for oral administration, it includes tablets, pills, powders, granules, capsules, etc., and such solid preparations may contain at least one or more excipients such as starch, calcium carbonate, sucrose, lactose, or gelatin in the active ingredient. In addition, in addition to simple excipients, magnesium stearate and lubricants such as talc may be included, but are not limited thereto.
본 발명의 조성물이 경구 투여를 위한 액상 제제로 제제화된 경우 현탁제, 내용액제, 유제 및 시럽제 등을 포함하며, 흔히 사용되는 단순희석제인 물, 리퀴드 파라핀 이외에 여러 가지 부형제, 예를 들면 습윤제, 감미제, 방향제, 보존제, 등을 포함할 수 있으나 이에 국한되지 않는다.When the composition of the present invention is formulated as a liquid formulation for oral administration, it includes suspensions, internal solutions, emulsions, syrups, etc., and in addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, aromatics, preservatives, etc. may be included, but are not limited thereto.
본 발명의 조성물이 비경구 투여를 위한 제제로 제제화된 경우 멸균된 수용액, 비수성 용제, 현탁제, 유제, 좌제를 포함할 수 있다. 비수성용제, 현탁용제로는 프로필렌글리콜(Propylene glycol), 폴리에틸렌 글리콜, 올리브 오일과 같은 식물성 기름, 에틸올레이트와 같은 주사 가능한 에스테르 등을 포함할 수 있으나, 이에 국한되지 않는다. 좌제의 기제로는 위텝솔(witepsol), 마크로골, 트윈(tween) 61, 카카오지, 라우린지, 글리세로제라틴 등이 사용될 수 있다.When the composition of the present invention is formulated into a formulation for parenteral administration, it may include a sterilized aqueous solution, a non-aqueous solvent, a suspension, an emulsion, and a suppository. Non-aqueous solvents and suspending agents may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate, but are not limited thereto. As a base for the suppository, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogeratin and the like may be used.
본 발명의 약학 조성물의 유효성분인 신규한 유산균 등의 함량은 조성물의 구체적인 형태, 사용 목적 내지 양상에 따라 다양한 범위에서 조정될 수 있다. 본 발명에 따른 약학 조성물에서 유효성분의 함량은 크게 제한되지 않으며, 예를 들어 조성물 총 중량을 기준으로 0.01 내지 99 중량%, 구체적으로 0.1 내지 75 중량%, 보다 구체적으로 0.5 내지 50 중량%일 수 있다.The content of the novel lactic acid bacteria, which is an active ingredient of the pharmaceutical composition of the present invention, may be adjusted in various ranges depending on the specific form, purpose or aspect of the composition. The content of the active ingredient in the pharmaceutical composition according to the present invention is not particularly limited, and may be, for example, 0.01 to 99% by weight, specifically 0.1 to 75% by weight, more specifically 0.5 to 50% by weight based on the total weight of the composition.
본 발명에 따른 식품용 조성물은 식품 또는 식품 첨가용 조성물인 것을 특징으로 할 수 있으나, 이에 한정되지는 않으며, 예컨대, 식품의 주원료, 부원료, 식품 첨가제, 건강기능식품 또는 기능성 음료로 용이하게 활용할 수 있다.The composition for food according to the present invention may be characterized in that it is a composition for food or food additives, but is not limited thereto, and can be easily utilized as, for example, a main ingredient, a supplementary ingredient, food additive, health functional food or functional beverage of food.
상기 식품이란, 영양소를 한 가지 또는 그 이상 함유하고 있는 천연물 또는 가공품을 의미하며, 바람직하게는 어느 정도의 가공 공정을 거쳐 직접 먹을 수 있는 상태가 된 것을 의미하며, 통상적인 의미로서, 식품, 식품 첨가제, 건강기능식품 및 기능성 음료를 모두 포함하는 것을 말한다.The food means a natural product or processed product containing one or more nutrients, preferably means a product that can be directly eaten through a certain degree of processing, and in a conventional sense, includes food, food additives, health functional food and functional beverages.
본 발명에 따른 상기 식품용 조성물을 첨가할 수 있는 식품으로는 예를 들어, 각종 식품류, 음료, 껌, 차, 비타민 복합제, 기능성 식품 등이 있다. 추가로, 본원발명에서 식품에는 특수영양식품(예, 조제유류, 영, 유아식 등), 식육가공품, 어육제품, 두부류, 묵류, 면류(예, 라면류, 국수류 등), 빵류, 건강보조식품, 조미식품(예, 간장, 된장, 고추장, 혼합장 등), 소스류, 과자류(예, 스넥류), 캔디류, 쵸코렛류, 껌류, 아이스크림류, 유가공품(예, 발효유, 치즈 등), 기타 가공식품, 김치, 절임식품(각종 김치류, 장아찌 등), 음료(예, 과실 음료, 채소류 음료, 두유류, 발효음료류 등), 천연조미료(예, 라면 스프 등)을 포함하나 이에 한정되지 않는다. 상기 식품, 음료 또는 식품첨가제는 통상의 제조방법으로 제조될 수 있다.Foods to which the food composition according to the present invention can be added include, for example, various foods, beverages, gum, tea, vitamin complexes, and functional foods. In addition, in the present invention, food includes special nutritional foods (eg, formula milk, infant food, baby food, etc.), processed meat products, fish meat products, tofu, jelly, noodles (eg, ramen, noodles, etc.), breads, health supplements, seasonings (eg, soy sauce, soybean paste, gochujang, mixed paste, etc.), sauces, confectionery (eg, snacks), candy, chocolates, gum, ice cream, dairy products (eg , fermented milk, cheese, etc.), other processed foods, kimchi, pickled foods (various types of kimchi, pickles, etc.), beverages (e.g., fruit drinks, vegetable drinks, soy milk, fermented beverages, etc.), natural seasonings (e.g., ramen soup, etc.). The food, beverage or food additive may be prepared by a conventional manufacturing method.
상기 건강기능식품이란 식품에 물리적, 생화학적, 생물공학적 수법 등을 이용하여 해당 식품의 기능을 특정 목적에 작용, 발현하도록 부가가치를 부여한 식품군이나 식품 조성이 갖는 생체방어리듬조절, 질병방지와 회복 등에 관한 체내조절기능을 생체에 대하여 충분히 발현하도록 설계하여 가공한 식품을 의미한다. 상기 기능성 식품에는 식품학적으로 허용 가능한 식품 보조 첨가제를 포함할 수 있으며, 기능성 식품의 제조에 통상적으로 사용되는 적절한 담체, 부형제 및 희석제를 더욱 포함할 수 있다.The health functional food refers to food that is designed and processed to sufficiently express the body's regulatory functions related to biological defense rhythm control, disease prevention and recovery, etc. of a food group or food composition in which added value is added so that the function of the food acts for a specific purpose by using physical, biochemical, or bioengineering methods. The functional food may include food additives that are acceptable in food science, and may further include appropriate carriers, excipients, and diluents commonly used in the manufacture of functional foods.
본 발명에서 상기 기능성 음료란 갈증을 해소하거나 맛을 즐기기 위하여 마시는 것의 총칭을 의미하며, 지시된 비율로 필수 성분으로서 상기 조성물을 포함하는 것 외에 다른 성분에는 특별한 제한이 없으며 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다.In the present invention, the functional beverage refers to a general term for drinking to quench thirst or enjoy taste, and there is no particular limitation on other ingredients other than including the composition as an essential ingredient in the indicated ratio, and various flavors or natural carbohydrates, etc. may be included as additional ingredients.
나아가 상기 기술한 것 이외에 본 발명의 식품용 조성물을 함유하는 식품은 여러 가지 영양제, 비타민, 광물(전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 충진제(치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산 음료에 사용되는 탄산화제 등을 함유할 수 있으며, 상기 성분은 독립적으로 또는 조합하여 사용할 수 있다.In addition to the above, the food containing the food composition of the present invention may contain various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, coloring agents and fillers (cheese, chocolate, etc.), pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc. can be used
본 발명의 식품용 조성물을 함유하는 식품에 있어서, 상기 본 발명에 따른 조성물의 양은 전체 식품 중량의 0.001중량% 내지 100중량%로 포함할 수 있으며, 바람직하게는 1중량% 내지 99중량%로 포함할 수 있고, 음료의 경우, 100ml를 기준으로 0.001g 내지 10g, 바람직하게는 0.01g 내지 1g의 비율로 포함할 수 있으나, 건강 및 위생을 목적으로 하거나 건강 조절을 목적으로 하는 장기간 섭취의 경우에는 상기 범위 이하일 수 있으며, 유효성분은 안전성 면에서 아무런 문제가 없기 때문에 상기 범위 이상의 양으로 사용될 수 있으므로 상기 범위에 한정되는 것은 아니다.In the food containing the composition for food of the present invention, the amount of the composition according to the present invention may be included in 0.001% to 100% by weight, preferably 1% to 99% by weight, based on the total weight of the food, and in the case of beverages, it may be included in a ratio of 0.001g to 10g, preferably 0.01g to 1g based on 100ml, but may be included for health and hygiene purposes or health control In the case of intended long-term intake, it may be below the above range, and since the active ingredient has no problem in terms of safety, it may be used in an amount above the above range, so it is not limited to the above range.
본 발명에 있어서, 안티센스 올리고뉴클레오티드란 안티센스 작용 기전을 통해 핵산 분자의 발현을 조절(예를 들어, TAS1R3의 발현을 조절)하는데 유용하다. 상기 조절은, 예를 들어 하나 이상의 표적 핵산 분자, 예를 들어 mRNA에 상보성이고/이거나 상기 분자에 하이브리드화하는 올리고뉴클레오티드를 제공함으로써 수행될 수 있다. 일부 양태에서, 본 발명의 올리고뉴클레오티드는 표적 핵산의 특정한 영역에 상보성이다. 일부 양태에서, 본 발명의 올리고뉴클레오티드는 표적 핵산의 특정한 영역에 하이브리드화 할 수 있다. 본 발명의 올리고뉴클레오티드 화합물은 하나 이상의 표적 핵산에 상보성이고 상기 표적화된 핵산의 통상적인 기능을 (예를 들어, 안티센스 작용 기전에 의해) 방해한다. 이는 일반적으로 "안티센스"라 지칭되는 것에 특이적으로 하이브리드화하는 본 발명의 올리고뉴클레오티드에 의해 표적 핵산의 기능을 방해하거나 조절한다. 방해받는 DNA의 기능은 복제 및 전사를 포함할 수 있다. 방해받는 RNA의 기능은, 예를 들어 상기 RNA의 단백질 번역 부위로의 전좌, 상기 RNA로부터의 단백질의 번역, 하나 이상의 mRNA 종을 제공하기 위한 상기 RNA의 연접, 및 상기 RNA에 의해 참여되거나 촉진될 수 있는 촉매 활성과 같은 기능들을 포함할 수 있다. 일부 양태에서, 표적 핵산 기능을 방해하는 전반적인 효과는 상기와 같은 표적 핵산의 산물의 발현 조절이다.In the present invention, antisense oligonucleotides are useful for regulating the expression of nucleic acid molecules (eg, regulating the expression of TAS1R3) through an antisense mechanism. Such modulation can be performed, for example, by providing an oligonucleotide that is complementary to and/or hybridizes to one or more target nucleic acid molecules, such as mRNA. In some embodiments, an oligonucleotide of the invention is complementary to a specific region of a target nucleic acid. In some embodiments, an oligonucleotide of the invention is capable of hybridizing to a specific region of a target nucleic acid. The oligonucleotide compounds of the present invention are complementary to one or more target nucleic acids and interfere with the normal function of the targeted nucleic acids (eg, by an antisense mechanism of action). It interferes with or modulates the function of a target nucleic acid by means of an oligonucleotide of the invention that specifically hybridizes to what is commonly referred to as "antisense". The functions of DNA that are disturbed can include replication and transcription. Functions of the RNA that are hindered may include functions such as, for example, translocation of the RNA to a protein translation site, translation of a protein from the RNA, splicing of the RNA to provide one or more mRNA species, and catalytic activity that may be engaged or promoted by the RNA. In some embodiments, the overall effect of interfering with target nucleic acid function is regulation of expression of the product of such target nucleic acid.
본 발명에 있어서 siRNA(small interfering RNA)란 특정 mRNA의 절단을 통하여 RNA 간섭을 유도할 수 있는 짧은 이중가닥 RNA를 말한다. siRNA는 RNA끼리 짝을 이루는 이중가닥 RNA 부분이 완전히 쌍을 이루는 것에 한정되지 않고 미스매치(대응하는 염기가 상보적이지 않음), 벌지(일방의 사슬에 대응하는 염기가 없음) 등에 의하여 쌍을 이루지 않는 부분이 포함될 수 있다. 쌍을 이루는 염기 길이는 15 내지 30 염기이다. siRNA 말단 구조는 표적 유전자의 발현을 RNA 간섭 효과에 의하여 억제할 수 있는 것이면 평활 말단 또는 돌출 말단 모두 가능하다. 점착 말단 구조는 3' 말단 돌출 구조와 5' 말단 돌출 구조 모두 가능하다.In the present invention, siRNA (small interfering RNA) refers to a short double-stranded RNA capable of inducing RNA interference through cleavage of a specific mRNA. siRNA is not limited to complete pairing of double-stranded RNA parts paired with each other, but may include unpaired parts due to mismatch (corresponding bases are not complementary), bulge (no base corresponding to one chain), etc. Paired bases are 15 to 30 bases in length. As the siRNA end structure, either a blunt end or a protruding end may be used as long as the expression of the target gene can be suppressed by the RNA interference effect. The sticky end structure can be both a 3' end protruding structure and a 5' end protruding structure.
본 발명의 siRNA는 그 활성을 저하시키지 않는 하나 이상의 치환, 삽입, 결실 및 그 조합을 갖는 변형체를 포함한다. 이러한 변형체는 상기한 siRNA의 서열과 80% 이상의 서열 상동성을 가질 수 있으며, 바람직하게는 90%, 더욱 바람직하게는 95% 이상의 서열 상동성을 갖는다.The siRNA of the present invention includes variants having one or more substitutions, insertions, deletions, and combinations thereof that do not reduce its activity. Such variants may have 80% or more sequence homology to the siRNA sequence described above, preferably 90% or more, and more preferably 95% or more sequence homology.
본 발명의 siRNA는 직접 화학적으로 합성하거나(Sui G et al., Proc. Natl. Acad. Sci. USA (2002) 99:5515-5520), 인비트로 전사를 이용하여 합성하는 방법(Brummelkamp TR et al., Science (2002) 296:550-553) 등 당업계에 공지된 다양한 방법에 의해 합성할 수 있다.The siRNA of the present invention can be synthesized by various methods known in the art, such as direct chemical synthesis (Sui G et al., Proc. Natl. Acad. Sci. USA (2002) 99:5515-5520) or synthesis using in vitro transcription (Brummelkamp TR et al., Science (2002) 296:550-553).
본 발명에 있어서, shRNA란 쇼트 헤어핀 RNA(short hairpin RNA)이라고 불리고, 1개 쇄의 일부의 영역이 다른 영역과 상보쇄를 형성하기 위하여 스템루프(stem-loop) 구조를 갖는 RNA 분자이다. In the present invention, shRNA is called short hairpin RNA, and is an RNA molecule having a stem-loop structure so that a part of one strand forms a complementary strand with another region.
본 발명에 있어서, miRNA란, 유전자 발현을 억제하는 작용을 갖는 21 내지 25 염기 정도의 1개 쇄 RNA이다. 게놈 위에 코딩되어 있는 비코딩 영역에서 유래되는 small RNA로, 일반적으로 miRNA는, 단일 또는 클러스터화된 miRNA 전구체로부터 전사되는 유전자에서 생성된다. 즉, 유전자로부터의 1차 전사물인 pri-miRNA가 전사되고, 계속해서 pri-miRNA로부터 성숙형 miRNA로의 단계적 프로세싱에서, pri-mRNA로부터 특징적인 머리핀 구조를 갖는 약 70 내지 80 염기의 pre-miRNA가 생성되며, 이어서 Dicer에 의해 pre-miRNA로부터 성숙형 miRNA가 생성된다. miRNA는 분화, 세포 증식, 아폽토시스 등의 생물에게 있어 없어서는 안되는 생명 현상에 깊이 관여하고 있다.In the present invention, miRNA is a single-stranded RNA of about 21 to 25 bases that has an effect of suppressing gene expression. Small RNAs derived from non-coding regions encoded on the genome. Generally, miRNAs are generated from genes transcribed from single or clustered miRNA precursors. That is, pri-miRNA, the primary transcript from the gene, is transcribed, and subsequently, in step-by-step processing from pri-miRNA to mature miRNA, pre-miRNA of about 70 to 80 bases with a characteristic hairpin structure is generated from pri-mRNA, and then mature miRNA is generated from pre-miRNA by Dicer. miRNAs are deeply involved in vital phenomena such as differentiation, cell proliferation, and apoptosis that are indispensable to living organisms.
본 발명에 있어서, 항체란 경쇄 및 중쇄의 가변영역을 통해 다른 분자(항원)에 결합하는 단백질을 일컫는 것으로, IgG, IgD, IgA 및 IgE 유래를 포함한다. 또한 본원의 항체는 다양한 형태의 구조를 갖는 단클론 항체를 포함하는 것으로, 예를 들면, 두 개의 전장 중쇄 및 두 개의 전장 경쇄를 포함하는 온전한 항체(intact Ab)는 물론 불변영역을 포함하거나 또는 포함하지 않는 이의 단편, 키메라 항체, 인간화항체, 또는 본원에 따른 특징을 갖는 기타 유전공학적으로 변형된 항체를 일컫는 것이다. 온전한 항체 또는 그 단편을 포함하는 본원의 항체는 단량체의 항원 결합능의 적어도 일부를 포함하는 이량체, 삼량체, 사량체, 오량체 등의 다량체로 존재할 수 있다. 이러한 다량체는 또한 동종다량체, 또는 이종다량체를 포함하는 것이다. 항체 다량체는 다수의 항원 결합 부위를 포함하기 때문에 단량체와 비교하여 항원에 대한 결합능이 우수하다. 항체의 다량체는 또한 다기능성(bifunctional, trifunctional, tetrafunctional) 항체 제작에도 용이하다.In the present invention, antibody refers to a protein that binds to other molecules (antigens) through variable regions of light and heavy chains, and includes IgG, IgD, IgA, and IgE-derived proteins. In addition, the antibodies herein include monoclonal antibodies having various types of structures, for example, intact antibodies comprising two full-length heavy chains and two full-length light chains, as well as fragments thereof with or without constant regions, chimeric antibodies, humanized antibodies, or other genetically modified antibodies having the characteristics according to the present application. Antibodies of the present application, including intact antibodies or fragments thereof, may exist as multimers such as dimers, trimers, tetramers, pentamers, etc., including at least a portion of the monomer's antigen-binding ability. These multimers also include homomultimers or heteromultimers. Since antibody multimers contain multiple antigen-binding sites, they have superior antigen-binding ability compared to monomers. Multimers of antibodies are also easy to construct multifunctional (bifunctional, trifunctional, tetrafunctional) antibodies.
본원에 기재된 Tas1R3에 특이적으로 결합하는 항체는 당업계에 공지된 다양한 방법에 의해 제조될 수 있다(Scheving, L.A. et al., American Journal of Physiology, 306:G370-81, 2014; Kaufmann, A. et al., J. Cell. Biochem. 114:681-69, 2013; Herndon, CA. et al., Cell Res, 319:718-730, 2013; Lacoste, B. et al., Journal of Neuroinflammation, 10:57, 2013; del Blanco, B. et al., J. Immunol, 188:3278-3293, 2012).Antibodies that specifically bind to Tas1R3 described herein can be prepared by various methods known in the art (Scheving, L.A. et al., American Journal of Physiology, 306:G370-81, 2014; Kaufmann, A. et al., J. Cell. Biochem. 114:681-69, 2013; Herndon, CA. et al., Cell Res, 319: 718-730, 2013; Lacoste, B. et al., Journal of Neuroinflammation, 10:57, 2013; del Blanco, B. et al., J. Immunol, 188:3278-3293, 2012).
본 발명에서 압타머란, 높은 친화성으로 타겟물질을 특이적으로 인지할 수 있는 작은 단일가닥 올리고핵산을 말한다.In the present invention, an aptamer refers to a small single-stranded oligonucleic acid capable of specifically recognizing a target material with high affinity.
구체적으로, 특정물질과 결합하는 핵산을 선별하는 방법을 셀렉스(SELEX, Systematic Evolution of Ligand by Exponential enrichment)라 하고, 이렇게 선별된 산물인 핵산을 일반적으로 압타머(aptamer)라고 한다(Craig et al., Science, 249:505-510, 1990). 상기 SELEX법을 통하여 자연 상태에서 핵산과 결합할 수 있는 단백질뿐만 아니라 핵산과 결합하고 있지 않은 단백질을 비롯한 여러 생체분자와도 매우 높은 친화력으로 결합할 수 있는 분자들이 선별되고 있다. 본 발명의 Egr-1에 특이적으로 결합하는 압타머는 당해 기술 분야에 공지된 기술인 SELEX 표준방법(Bock LC et al., Nature, 355:564-6, 1992)에 의해 제조될 수 있다(Mannironi et al., Biochemistry 36:9726, 1997; Ellington and Szostak et al., Nature, 346:818, 1990; Tuerk and Gold, et al., Science 249:505, WO 00/20040, WO 99/54506, WO 99/27133, WO 97/42317). 따라서 실시예에 기재되지 않아도 공지기술을 이용하여 Tas1R3에 대한 압타머를 용이하게 제조할 수 있음은 당업자에게 있어서 자명한 사실이라고 할 것이다.Specifically, a method for selecting a nucleic acid that binds to a specific substance is called SELEX (Systematic Evolution of Ligand by Exponential Enrichment), and the nucleic acid that is the product of this selection is generally called an aptamer (Craig et al., Science, 249: 505-510, 1990). Through the SELEX method, molecules capable of binding with very high affinity to various biomolecules including proteins not bound to nucleic acids as well as proteins capable of binding to nucleic acids in nature are selected. The aptamer specifically binding to Egr-1 of the present invention can be prepared by the SELEX standard method (Bock LC et al., Nature, 355:564-6, 1992), a technique known in the art (Mannironi et al., Biochemistry 36:9726, 1997; Ellington and Szostak et al., Nature, 346:818, 1990; Tuerk and Gold , et al., Science 249:505, WO 00/20040, WO 99/54506, WO 99/27133, WO 97/42317). Therefore, it will be apparent to those skilled in the art that an aptamer for Tas1R3 can be easily prepared using known techniques even if it is not described in the examples.
상기 Tas1R3에 특이적으로 결합하는 압타머의 결합 정도를 측정하는 단계는 관련 기술 분야에서 통상적으로 사용되는 DNA 압타머 결합 측정 기술을 이용하여 수행될 수 있으며, 예컨대, 압타머 말단에 형광 또는 방사성 물질 표지하거나 비오틴을 결합시켜 형광 또는 방사성 세기를 측정하거나, 이미지화하여 관찰하는 방법 등을 이용할 수 있으나, 이에 제한되는 것은 아니다.The step of measuring the binding degree of the aptamer that specifically binds to Tas1R3 may be performed using a DNA aptamer binding measurement technique commonly used in the related art. For example, a method of measuring fluorescence or radioactive intensity by binding biotin to an aptamer end, labeling a fluorescent or radioactive substance, or imaging and observing the aptamer end may be used, but is not limited thereto.
본 발명에 있어서, 안타고니스트란, 일 태양에 있어서, TAS1R3에 의해 야기되는 세포내 시그널 전달을 억제 혹은 차단하는 화합물을 의미하고, 이것은 TAS1R3 시그널을 저해하는 화합물이라고 바꿔 말할 수도 있다. 그와 같은 화합물은 천연에 존재하는 화합물이어도 되고, 인공적으로 합성된 화합물이어도 된다. 또한, 저분자 화합물이어도 되고, 단백질과 같은 고분자 화합물이어도 된다. In the present invention, an antagonist means, in one aspect, a compound that inhibits or blocks intracellular signal transduction caused by TAS1R3, which can also be referred to as a compound that inhibits the TAS1R3 signal. Such a compound may be a naturally occurring compound or an artificially synthesized compound. It may also be a low molecular weight compound or a high molecular weight compound such as protein.
본 발명에서 유전자 발현이 억제되어 있다는 것은 유전자 자체를 결실시키거나 그 발현을 감소시키는 것으로, 유전자에 의해 생성되는 효소 또는 단백질이 완전히 제거하는 것뿐만 아니라, 상기 유전자에 의해 생성되는 효소 또는 단백질이 내재적 활성에 비하여 약화되도록 변형된 것을 포함한다.In the present invention, gene expression is inhibited means that the gene itself is deleted or its expression is reduced, and enzymes or proteins produced by the genes are completely removed, as well as enzymes or proteins produced by the genes.
본 발명에서 "내재적 활성에 비하여 약화되도록 변형"되었다는 것은, 활성을 나타내는 유전자의 결실이나 유전자의 불활성화(예를 들어, 돌연변이 유전자로의 치환), 유전자 발현의 약화(예를 들어, 약한 프로모터로의 치환, siRNA, gRNA, sRNA 등의 도입, 시작 코돈을 ATG에서 GTG 등으로의 치환), 유전자에 의해 발현된 효소의 활성 억제(예를 들어, 비경쟁적 억제자 또는 경쟁적 억제자 첨가) 등과 같은 조작이 이루어지기 전과 비교하여 조작이 이루어진 이후의 활성이 감소된 상태를 의미한다.In the present invention, "modified to be attenuated compared to the intrinsic activity" means deletion of a gene showing activity, inactivation of a gene (eg, substitution with a mutant gene), attenuation of gene expression (eg, substitution with a weak promoter, introduction of siRNA, gRNA, sRNA, etc., substitution of the start codon from ATG to GTG, etc.), inhibition of the activity of an enzyme expressed by a gene (eg, addition of a non-competitive repressor or competitive repressor). This means that the activity after this is reduced.
본 발명에서 사용되는 용어 "내재적 활성"이란, 변형되지 않은 상태에서 가지고 있는 효소 등의 본래의 활성 상태를 의미하고, "내재적 활성에 비해 약화되도록 변형"된다는 의미는 변형 전 상태의 효소 활성과 비교할 때 당해 활성이 사라지거나 더 감소된 것을 의미한다.As used herein, the term "intrinsic activity" means the original active state of an enzyme or the like in an unmodified state, and "modified to be attenuated compared to the intrinsic activity" means that the activity disappears or is further reduced when compared to the enzymatic activity in the state before modification.
본 발명에서 "결실"이란 해당 유전자의 일부 또는 전체염기를 변이, 치환 또는 삭제시키는 방법을 통해 해당 유전자가 발현되지 않도록 하거나 발현되더라도 단백질 고유의 기능을 나타내지 못하도록 하는 것을 포괄하는 개념으로 해당 유전자에 의해 발현되는 단백질이 관여하는 생합성 경로 또는 신호 전달 경로를 차단하는 모든 것을 포함한다.In the present invention, "deletion" is a concept encompassing preventing a corresponding gene from being expressed through a method of mutating, substituting, or deleting a part or all bases of a corresponding gene, or preventing a protein's inherent function from being expressed. It includes anything that blocks a biosynthetic pathway or signal transduction pathway involved in a protein expressed by a corresponding gene.
본 발명은 또 다른 관점에서, 염증성 장 질환 예방 또는 치료를 위한 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제의 용도에 관한 것이다.In another aspect, the present invention relates to the use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for preventing or treating inflammatory bowel disease.
본 발명은 또 다른 관점에서, 염증성 장 질환 예방 또는 치료를 위한 상기 약학적 조성물의 용도에 관한 것이다.In another aspect, the present invention relates to the use of the pharmaceutical composition for preventing or treating inflammatory bowel disease.
본 발명은 또 다른 관점에서, 염증성 장 질환 예방 또는 치료를 위한 약물의 제조를 위한 TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제의 용도에 관한 것이다.In another aspect, the present invention relates to the use of a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor for the preparation of a drug for preventing or treating inflammatory bowel disease.
본 발명은 또 다른 관점에서, 염증성 장 질환 예방 또는 치료를 위한 약물의 제조를 위한 상기 약학적 조성물의 신규한 용도에 관한 것이다.In another aspect, the present invention relates to a novel use of the pharmaceutical composition for the preparation of a drug for preventing or treating inflammatory bowel disease.
본 발명은 또 다른 관점에서, TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 이를 필요로 하는 개체에 투여하는 단계를 포함하는 염증성 장 질환 예방 또는 치료방법에 관한 것이다. In another aspect, the present invention relates to a method for preventing or treating inflammatory bowel disease comprising administering a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor to a subject in need thereof.
본 발명은 또 다른 관점에서, 상기 약학적 조성물을 이를 필요로 하는 개체에 투여하는 단계를 포함하는 염증성 장 질환 예방 또는 치료방법에 관한 것이다. In another aspect, the present invention relates to a method for preventing or treating inflammatory bowel disease comprising administering the pharmaceutical composition to a subject in need thereof.
실시예Example
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로서, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for exemplifying the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.
실시예 1. 서구식 식이 (Western diet)가 장 염증에 미치는 영향Example 1. Effect of Western diet on intestinal inflammation
8-10주령 C57BL/6J 마우스를 Jackson Laboratory (West Grove, PA, USA)에서 입수하여, 각 실험군 당 10 마리씩 정상 식이군 또는 서구식 식이군 [설탕용액(30% w/v) + 60% 고지방식이]으로 분류하고, 자유 급식으로 10주간 공급하였다. 실험에 사용된 고지방식이(60%, D12492) 및 지방의 함량 이외에 모든 조성이 동일한 정상 식이(D12450J)는 모두 Research Diets(New Brunswick, NJ, USA)로부터 구입하였다. 8-10 week old C57BL/6J mice were obtained from the Jackson Laboratory (West Grove, PA, USA), and 10 mice per experimental group were classified into either a normal diet group or a western diet group [sugar solution (30% w/v) + 60% high fat diet] and fed ad libitum for 10 weeks. The high-fat diet (60%, D12492) and the normal diet (D12450J), which had the same composition except for the fat content, were all purchased from Research Diets (New Brunswick, NJ, USA).
장 염증 정도를 분석하기 위하여, 조직학적 검사(Pathological examination) 및 유세포 분석 (FACS profiling)을 진행하였다. 10주간의 다이어트 유도 후 마우스는 20% 우레탄(U2500, Sigma-Aldrich, St. Louis, MO, USA)을 이용하여 희생시킨 후 회장 (Terminal ileum) 검체를 얻었다. 회장 조직은 10% 포르말린에 고정시키고, H&E 염색을 시행하였다. 조직학적인 분석은 이전에 보고된 histologic colitis scoring system을 따랐다 (Severity of DSS-induced colitis is reduced in Ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks. Sci Rep, 2015, 5:17305). 각 검체의 염증정도, 염증의 범위, 음와(Crypt) 손상정도, 전체 소장의 손상 범위를 측정하여 각 변수를 합산하여 염증지수(Inflammation score)를 산정하였다. In order to analyze the degree of intestinal inflammation, histological examination (Pathological examination) and flow cytometry analysis (FACS profiling) were performed. After 10 weeks of diet induction, mice were sacrificed using 20% urethane (U2500, Sigma-Aldrich, St. Louis, MO, USA), and then terminal ileum specimens were obtained. The ileum tissue was fixed in 10% formalin and subjected to H&E staining. Histological analysis followed the previously reported histologic colitis scoring system (Severity of DSS-induced colitis is reduced in Ido1-deficient mice with down-regulation of TLR-MyD88-NF-kB transcriptional networks. Sci Rep , 2015, 5:17305). The degree of inflammation, the extent of inflammation, the extent of damage to the crypt, and the extent of damage to the entire small intestine were measured, and each variable was added to calculate the inflammation score.
또한 소유고유층의 세포를 FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA)의 Cell Quest software를 사용하여 유세포 분석하였다. 사용된 항체는 CD4 (GK1.5; BioLegend, San Diego, CA, USA), CD8a (53-6.7; BioLegend), CD45 (30-F11; BioLegend), phycoerythrin (PE), allophycocyanin (APC), APC-CyChrome7 (APC-Cy7), Alexa Fluor 700 (AF700) 이다. In addition, cells of the lamina propria were analyzed by flow cytometry using Cell Quest software of FACSCalibur (Becton Dickinson, Franklin Lakes, NJ, USA). The antibodies used were CD4 (GK1.5; BioLegend, San Diego, CA, USA), CD8a (53-6.7; BioLegend), CD45 (30-F11; BioLegend), phycoerythrin (PE), allophycocyanin (APC), APC-CyChrome7 (APC-Cy7), Alexa Fluor 700 (AF700).
조직학적 검사 및 유세포 분석 결과, 10주간의 장기간 서구식 식이에 의해 마우스 장 염증이 크게 증가되는 것으로 확인되었다 (도 1a, 도 1b)As a result of histological examination and flow cytometry, it was confirmed that intestinal inflammation was significantly increased in mice by a long-term western diet for 10 weeks (FIGS. 1a and 1b).
서구식 식이 섭취 마우스에서 장 염증이 증가된 원인을 분자생물학적 수준에서 확인하고자, 마우스를 희생시키고 염증이 발생한 소장 조직을 적출하여 해당 조직에서의 RNA 서열 분석을 진행하였다. 적출된 장 조직에서 Total RNA를 추출한 후(RNAqueous kit (AM1914, Ambion) 사용), NanoDrop 2000/2000c Spectrophotometer(Thermo Scientific) 기기를 활용하여 RNA를 정량하였다. 총 1μg (1000ng)의 Total RNA를 가지고 Intact mRNA를 capture하였고(Dynabeads mRNA DIRECT Micro Kit(Ambion) 사용), captured mRNA는 Ion Total RNA Seq Kit v2(Life Technologies)를 통해 라이브러리 제작에 사용되었다. 제작된 라이브러리 샘플은 최종적으로 Ion PI Chip Kit v3(Life Technologies)에 로딩 후, Ion Proton Sequencer (Life Technologies)에 넣고 running하였다. 모든 실험은 각 키트 제조업체의 지침에 따라 진행하였다. In order to confirm the cause of increased intestinal inflammation in mice fed a western diet at the molecular level, mice were sacrificed, and inflamed small intestine tissue was extracted and RNA sequencing was performed in the tissue. After total RNA was extracted from the extracted intestinal tissue (using RNAqueous kit (AM1914, Ambion)), RNA was quantified using a NanoDrop 2000/2000c Spectrophotometer (Thermo Scientific). Intact mRNA was captured with a total of 1μg (1000ng) of total RNA (using Dynabeads mRNA DIRECT Micro Kit (Ambion)), and the captured mRNA was used for library construction through Ion Total RNA Seq Kit v2 (Life Technologies). The prepared library sample was finally loaded into the Ion PI Chip Kit v3 (Life Technologies) and then put into the Ion Proton Sequencer (Life Technologies) and run. All experiments were performed according to the instructions of each kit manufacturer.
그 결과, 염증 발생의 지표인 염증성 사이토카인 및 케모카인 관련 신호가 증가하였으며, 이와 동시에 미각 수용체 활성(Taste receptor activity) 관련 신호전달체계가 유의적으로 증가한 것으로 확인되었다 (도 1c-1e). 미각 수용체 활성에 관여하는 여러 유전자들 중에서도 특히, 미각 수용체 Tas1r3의 발현이 서구식 식이로 인한 염증성 장 조직에서 유의하게 증가하는 것으로 확인되었으며 (도 1f), 면역조직화학염색 결과에서도, 염증성 장 조직에서 TAS1R3의 단백질 발현 정도가 매우 높은 것으로 나타났다 (도 1g). As a result, it was confirmed that inflammatory cytokine and chemokine-related signals, which are indicators of inflammation, increased, and at the same time, signal transduction systems related to taste receptor activity significantly increased (FIG. 1c-1e). Among several genes involved in taste receptor activity, expression of the taste receptor Tas1r3 in particular was confirmed to be significantly increased in inflammatory intestinal tissues induced by the Western-style diet (Fig. 1f). Immunohistochemical staining also showed that the protein expression level of TAS1R3 was very high in inflammatory intestinal tissues (Fig. 1g).
한편, TAS1R3와 여러 염증을 나타내는 지표들(Inflammation score, lymph node에 침윤된 CD45+ leukocyte, CD4+ T cell, CD8+ T cell의 frequency, Il1b mRNA, and Tnf mRNA level)간 유의한 양의 상관관계가 확인되었다 (도 1h).On the other hand, significant positive correlations were confirmed between TAS1R3 and various markers of inflammation (inflammation score, frequency of CD45+ leukocytes, CD4+ T cells, and CD8+ T cells infiltrating lymph nodes, Il1b mRNA, and Tnf mRNA level) (Fig. 1h).
이와 같은 결과는, 장기간의 서구식 식이 섭취가 심각한 조직 손상 및 염증성 세포들의 침윤을 유도하고 이로써 장 내 심각한 염증을 유발하며, 장 미각 수용체 TAS1R3가 이러한 장 염증을 매개하는데 중요한 매개체가 될 수 있음을 시사한다.These results suggest that long-term consumption of a western-style diet induces severe tissue damage and infiltration of inflammatory cells, thereby causing severe inflammation in the intestine, and that the intestinal taste receptor TAS1R3 may be an important mediator in mediating such intestinal inflammation.
실시예 2. 장 미각수용체 TAS1R3가 서구식 유도 장 염증에 미치는 영향Example 2. Effect of intestinal taste receptor TAS1R3 on western diet-induced intestinal inflammation
장 내분비 세포인 NCI-H716 세포주 (CCL-251, American Type Culture Collection, Manassas, VA, USA)에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM) (모두 Sigma-Aldrich, St. Louis, MO, USA)를 다양한 조합으로 처리하고, 장 미각수용체 TAS1R3 및 그 다운스트림 분자인 GLP-1의 발현 여부를 mRNA 및 단백질 수준에서 분석하였다 (도 2a).Enteroendocrine cell line NCI-H716 (CCL-251, American Type Culture Collection, Manassas, VA, USA) was treated with various combinations of glucose (10 mM), fructose (10 mM), and palmitate (10 μM) (all Sigma-Aldrich, St. Louis, MO, USA), and the expression of the intestinal taste receptor TAS1R3 and its downstream molecule, GLP-1, was analyzed at the mRNA and protein levels ( Fig. 2a).
qPCR 수행을 위해, 먼저 장 조직 내 Total RNA를 RNAqueous (AM1914, Ambion) 키트를 이용하여 추출하였다. 해당 키트로 추출한 RNA를 MML-V reverse transcriptase의 protocol(11917010, Invitrogen)을 사용하여 cDNA로 합성하였고, mRNA의 발현을 정량적으로 표현하기 위해 정량 중합효소 반응을 측정하였다. 합성된 cDNA 2μl(Total volume 10 ng/μl)에 각각에 해당되는 유전자의 프라이머 (Forward+Reverse) 5 μl, SYBRGreen® (4367659, Applied Biosystems) 10 μl를 넣고 non-DEPC water로 최종 볼륨을 20μl로 맞춘 다음 StepOnePlus (Real-time PCR System, Applied Biosystems, Foster City, CA, USA) 장비를 이용하여 확인하였다. PCR은 92 ℃에서 30초, 60 ℃에서 45초, 그 후에 72 ℃에서 30초를 40 cycle로 진행하였다. qPCR에 사용된 프라이머 정보는 다음과 같다 (표 1).For qPCR, first, total RNA in intestinal tissue was extracted using RNAqueous (AM1914, Ambion) kit. The RNA extracted with the kit was synthesized into cDNA using the MML-V reverse transcriptase protocol (11917010, Invitrogen), and the quantitative polymerase reaction was measured to quantitatively express mRNA expression. 5 μl of primers (Forward+Reverse) for each gene and 10 μl of SYBRGreen ® (4367659, Applied Biosystems) were added to 2 μl of synthesized cDNA (total volume 10 ng/μl), and the final volume was adjusted to 20 μl with non-DEPC water, and then confirmed using StepOnePlus (Real-time PCR System, Applied Biosystems, Foster City, CA, USA) equipment. PCR was performed at 92 ° C for 30 seconds, 60 ° C for 45 seconds, and then 72 ° C for 30 seconds in 40 cycles. Primer information used for qPCR is as follows (Table 1).
GeneGene Forward primer sequences(5'-3')Forward primer sequences (5'-3') Reverse primer sequences(5'-3')Reverse primer sequences (5'-3')
Human GAPDHHuman GAPDH CCACTCCTCCACCTTTGACGCCACTCCTCCACCTTTGACG CCACCACCCTGTTGCTGTAGCCACCACCCTGTTGCTGTAG
Human TAS1R3Human TAS1R3 CACCAGACGTTCTCTGTCTACGCACCAGACGTTCTCTGTCTACG CTGAGGCGTTGCACTGAAGACTGAGGCGTTGCACTGAAGA
Human GCGHuman GCG CTGAAGGGACCTTTACCAGTGACTGAAGGGACCTTTACCAGTGA CCTGGCGGCAAGATTATCAAGCCTGGCGGCAAGATTATCAAG
Human IL1BHuman IL1B ATGATGGCTTATTACAGTGGCAAATGATGGCTTATTACAGTGGCAA GTCGGAGATTCGTAGCTGGAGTCGGAGATTCGTAGCTGGA
Human IL6Human IL6 CCTGAACCTTCCAAAGATGGCCCTGAACCTTCCAAAGATGGC TTCACCAGGCAAGTCTCCTCATTCACCAGGCAAGTCTCCTCA
Human IL8Human IL8 ATGACTTCCAAGCTGGCCGTGGCTATGACTTCCAAGCTGGCCGTGGCT TCTCAGCCCTCTTCAAAAACTTCTCTCTCAGCCCTCTTCAAAAACTTCTC
Human TNFHuman TNF GAGGCCAAGCCCTGGTATGGAGGCCAAGCCCTGGTATG CGGGCCGATTGATCTCAGCCGGGCCGATTGATCTCAGC
Human CCL2(MCP-1)Human CCL2 (MCP-1) GCTCAGCCAGATGCAATCAGCTCAGCCAGATGCAATCA TTTGCTTGTCCAGGTGGTCTTTGCTTGTCCAGGTGGTC
그 결과, 포도당이나 과당, 팔미테이트를 단독으로 처리하는 경우에 비하여 포도당/과당/팔미테이트를 조합하여 처리하는 경우 TAS1R3 mRNA 발현량이 현저히 증가하는 것으로 확인되었다 (도 2b). As a result, compared to the case of processing glucose, fructose, or palmitate alone, TAS1R3 It was confirmed that the amount of mRNA expression significantly increased (Fig. 2b).
실제로 발현량이 높아진 TAS1R3가 기능의 활성화까지도 유발되었는지 확인하고자, 장 내분비 세포인 NCI-H716 세포주에 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 처리하고, GLP-1의 분비 정도를 24시간 동안 관찰하였다. 이 때에 GLP-1의 분비는 TAS1R3의 활성화 정도를 의미한다. In order to confirm whether TAS1R3, whose expression level was actually increased, even activated the function, the NCI-H716 cell line, an enteroendocrine cell, was treated with glucose (10 mM), fructose (10 mM), and palmitate (10 μM), and the level of GLP-1 secretion was observed for 24 hours. At this time, the secretion of GLP-1 means the degree of activation of TAS1R3.
세포에서 분비되는 GLP-1을 측정하기 위하여 4℃, 3000g로 10분간 원심분리하여 세포의 상층액(Supernatants)만을 채취하여 -80℃에 보관하였다. GLP-1 (Active) ELISA kit (EGLP-35k, Millipore, St. Charles, MI, USA)를 사용하였고, 제조업체의 프로토콜에 따라 실험을 수행하였다. To measure GLP-1 secreted from cells, centrifugation was performed at 4°C and 3000 g for 10 minutes, and only supernatants were collected and stored at -80°C. A GLP-1 (Active) ELISA kit (EGLP-35k, Millipore, St. Charles, MI, USA) was used and the experiment was performed according to the manufacturer's protocol.
그 결과, 포도당이나 과당, 팔미테이트를 단독으로 처리하는 경우에 비하여 포도당/과당/팔미테이트를 조합하여 처리하는 경우 GLP-1의 mRNA 발현량(GCG) 및 펩타이드 수준(Active GLP-1)이 현저히 증가하는 것으로 확인되었다 (도 2c-2d).As a result, compared to the case of processing glucose, fructose, or palmitate alone, when glucose/fructose/palmitate was treated in combination, the level of GLP-1 was increased. It was confirmed that mRNA expression level (GCG) and peptide level (Active GLP-1) were significantly increased (Fig. 2c-2d).
이와 같이 식이 리간드 자극에 의해 활성화된 장 미각수용체 TAS1R3가 직접적으로 염증성 사이토카인 및 케모카인의 분비를 유도하는지 여부를 확인하고자, 염증성 장 질환에서 주요한 염증성 사이토카인인 IL1B, IL6, TNF 및 케모카인 MCP-1의 mRNA 수준을 표 1에 기재된 프라이머를 사용하여 상기한 qPCR 방법으로 확인하였다. In order to confirm whether the intestinal taste receptor TAS1R3 activated by dietary ligand stimulation directly induces the secretion of inflammatory cytokines and chemokines, mRNA levels of IL1B, IL6, TNF, and chemokine MCP-1, which are major inflammatory cytokines in inflammatory bowel disease, were confirmed by the qPCR method described above using the primers shown in Table 1.
그 결과, 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM)를 단독 또는 조합하여 처리한지 12시간 경과한 시점에서 IL1B, IL6, TNF 및 케모카인 MCP-1의 mRNA 수준은 특히 포도당/과당/팔미테이트를 조합하여 처리한 실험군에서 현저하게 증가하는 것을 확인할 수 있었다 (도 2e - 도 2h)As a result, at 12 hours after glucose (10mM), fructose (10mM), and palmitate (10μM) alone or in combination, the mRNA levels of IL1B, IL6, TNF, and the chemokine MCP-1 increased significantly in the experimental group treated with the combination of glucose/fructose/palmitate (Fig. 2e - Fig. 2h)
이와 같은 결과로부터, 서구식 식이 섭취로 장 염증이 악화되고, 이 때 미각수용체 TAS1R3가 염증 반응을 유도하는 것으로 확인된 실시예 1의 동물 실험 결과가 in vitro 실험에서도 그대로 검증되는 것을 알 수 있었다. From these results, it can be seen that the results of the animal experiment in Example 1, which were confirmed to exacerbate intestinal inflammation with the intake of a western-style diet and that the taste receptor TAS1R3 induces an inflammatory response at this time, were also verified in an in vitro experiment.
실시예 3. 장 미각수용체 TAS1R3 억제를 통한 장 세포 염증 기전 억제Example 3. Inhibition of Intestinal Cell Inflammatory Mechanism through Inhibition of Intestinal Taste Receptor TAS1R3
장 미각수용체 TAS1R3 억제를 위하여 siRNA 또는 수용체 안타고니스트인 락티솔(lactisole)을 처리하고 그 다운스트림 분자인 GLP-1의 발현과 장 내 사이토카인(IL1B, IL6, TNF) 및 케모카인(MCP-1) 발현을 qPCR 및 ELISA로 확인하였다. In order to inhibit the intestinal taste receptor TAS1R3, siRNA or receptor antagonist lactisole was treated, and the expression of GLP-1, a downstream molecule, and the expression of cytokines (IL1B, IL6, TNF) and chemokines (MCP-1) in the intestine were confirmed by qPCR and ELISA.
이를 위하여 NCI-H716 세포주에 (i) 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM) 처리 (대조군), (ii) 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM), TAS1R3 siRNA 처리 (siRNA 처리군), (iii) 포도당 (10mM), 과당 (10mM), 팔미테이트 (10μM), TAS1R3 antagonist (lactisole) 처리 (안타고니스트 처리군) 후 추가 인큐베이션하였다. To this end, the NCI-H716 cell line was treated with (i) glucose (10 mM), fructose (10 mM), palmitate (10 μM) (control group), (ii) glucose (10 mM), fructose (10 mM), palmitate (10 μM), TAS1R3 siRNA treatment (siRNA treatment group), (iii) glucose (10 mM), fructose (10 mM), palmitate (1 0 μM), TAS1R3 antagonist (lactisole) treatment (antagonist treatment group), followed by additional incubation.
공급업체의 프로토콜에 따라 인간 장내분비 NCI-H716 세포(CCL-251; American Type Culture Collection, Manassas, VA, USA)를 37℃ 및 5% CO2에서 현탁 배양으로 유지하였다. 배양 배지는 10% 태아 소 혈청, 2mM L-글루타민, 100IU/mL 페니실린 및 100㎍/mL 스트렙토마이신이 보충된 RPMI-1640(Invitrogen)를 사용하였다. 실험 2일 전에 Matrigel(BD Biosciences, Franklin Lakes, NJ, USA)로 사전 코팅된 24-웰 배양 플레이트에 1 x 106 세포를 분주하였다. 실험 당일, 상등액(supernatant)을 10 mM 포도당, 과당, 포도당 + 과당 및/또는 10 μM 팔미테이트를 포함하는 배지로 교체하였다.Human enteroendocrine NCI-H716 cells (CCL-251; American Type Culture Collection, Manassas, VA, USA) were maintained in suspension culture at 37° C. and 5% CO 2 according to the supplier's protocol. The culture medium was RPMI-1640 (Invitrogen) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 IU/mL penicillin and 100 μg/mL streptomycin. Two days before the experiment, 1×10 6 cells were seeded in 24-well culture plates pre-coated with Matrigel (BD Biosciences, Franklin Lakes, NJ, USA). On the day of the experiment, the supernatant was replaced with medium containing 10 mM glucose, fructose, glucose + fructose and/or 10 μM palmitate.
siRNA 녹다운siRNA knockdown
TAS1R3에 대한 siRNA duplex는 Bioneer (Daejeon, South Korea)에서 합성하였다. Scrambled negative control siRNA도 Bioneer에서 구입하였다. 녹다운 실험을 위해 5 x 105 내분비 분화 NCI-H716 세포를 6-웰 배양 플레이트에 플레이팅하고 48시간 동안 배양하였다. TAS1R3 (10nM) 또는 대조군 (10nM) siRNA를 Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA)를 사용하여 세포에 형질감염시켰다. 형질감염 48시간 후, 세포를 10mM 포도당, 10mM 과당 및 10μM 팔미테이트를 함유하는 배지로 12시간 동안 인큐베이션 하였다. siRNA duplex against TAS1R3 was synthesized by Bioneer (Daejeon, South Korea). Scrambled negative control siRNA was also purchased from Bioneer. For knockdown experiments, 5 x 10 5 endocrine differentiated NCI-H716 cells were plated in 6-well culture plates and cultured for 48 hours. TAS1R3 (10 nM) or control (10 nM) siRNA was transfected into cells using Lipofectamine RNAiMAX Reagent (Invitrogen, Carlsbad, CA, USA). 48 hours after transfection, cells were incubated for 12 hours with medium containing 10 mM glucose, 10 mM fructose and 10 μM palmitate.
TAS1R3 siRNA 시퀀스 정보TAS1R3 siRNA sequence information
Forward: CUGUCUACGCAGCUGUGUA (dTdT) Forward: CUGUCUACGCAGCUGUGUA (dTdT)
Reverse: UACACAGCUGCGUAGACAG (dTdT)Reverse: UACACAGCUGCGUAGACAG (dTdT)
TAS1R3 길항제(antagonist) lactisole 처리Treatment with TAS1R3 antagonist lactisole
TAS1R3 길항제 실험에서 NCI-H716 세포는 TAS1R3 길항제인 lactisole(2.5mM)로 30분 동안 전처리되었고, 과당(10mM), 포도당(10mM) 및 팔미테이트(10μM)를 포함하는 배지로 12시간 동안 자극되었다. 포도당, 과당, 팔미테이트 및 락티솔은 모두 Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA)에서 구입하였다. 배양 후 배지를 수집하고 1,000 x g에서 10분 동안 4℃에서 원심분리하여 부유 세포를 제거하고 후속 생화학 분석을 위해 -20℃에서 동결하였다.In the TAS1R3 antagonist experiment, NCI-H716 cells were pretreated with the TAS1R3 antagonist lactisole (2.5 mM) for 30 min and stimulated with a medium containing fructose (10 mM), glucose (10 mM) and palmitate (10 μM) for 12 h. Glucose, fructose, palmitate and lactisol were all purchased from Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO, USA). After incubation, media was collected and centrifuged at 1,000 x g for 10 min at 4°C to remove floating cells and frozen at -20°C for subsequent biochemical analysis.
qPCR과 ELISA는 실시예 2와 동일한 방법으로 진행하였으며, IL-8의 단백질량은 IL-8 ELISA kit (Cat# ab46032, Abcam, Cambridge, UK)를 사용하고, TNF 단백질량은 TNF-α ELISA kit (Cat# ab181421, Abcam, Cambridge, UK)를 사용하여 분석하였다. qPCR and ELISA were performed in the same manner as in Example 2, and the amount of IL-8 protein was analyzed using the IL-8 ELISA kit (Cat# ab46032, Abcam, Cambridge, UK), and the amount of TNF protein was analyzed using the TNF-α ELISA kit (Cat# ab181421, Abcam, Cambridge, UK).
그 결과, 대조군에서는 포도당(10mM), 과당(10mM), 팔미테이트(10μM)를 처리함에 따라 TAS1R3가 활성화(activation)되며 GLP-1의 mRNA 발현 및 분비가 증가한 반면, siRNA 처리군에서는 TAS1R3의 mRNA 및 단백질 발현이 약 70% 억제된 결과 GLP-1의 분비 또한 억제(inhibition)되었음을 확인할 수 있었다 (도 3a-3d). As a result, in the control group, treatment with glucose (10 mM), fructose (10 mM), and palmitate (10 μM) activated TAS1R3 and increased the mRNA expression and secretion of GLP-1, whereas in the siRNA-treated group, TAS1R3 mRNA and protein expression were suppressed by about 70%, confirming that GLP-1 secretion was also inhibited (FIGS. 3a-3d).
또한 대조군에서 TAS1R3 활성화에 따라 염증성 사이토카인인 IL1B, IL6, IL8 및 TNF의 mRNA 수준 및 IL8, TNF의 단백질 수준이 유의하게 증가하였으나, siRNA 처리군에서는 TAS1R3 발현이 억제되며 염증성 사이토카인의 수준을 낮게 유지시켰다 (도 3e-3j). In addition, in the control group, the mRNA levels of IL1B, IL6, IL8, and TNF, which are inflammatory cytokines, and the protein levels of IL8 and TNF were significantly increased according to TAS1R3 activation, but in the siRNA-treated group, TAS1R3 expression was suppressed and the level of inflammatory cytokines was kept low (Fig. 3e-3j).
유사하게, TAS1R3 수용체에 안타고니스트인 lactisole의 처리한 결과, 대조군과 비교하여 GLP-1의 mRNA 발현 및 분비를 유의적으로 억제시킬 수 있었고(도 3k-3l), IL1B, IL6, IL8, TNF의 mRNA 발현 및 IL-8, TNF의 분비 단백질 수준까지도 유의하게 억제시킬 수 있음을 확인하였다 (도 3m-3r).Similarly, as a result of treatment with lactisole, an antagonist to the TAS1R3 receptor, compared to the control group, the mRNA expression and secretion of GLP-1 could be significantly inhibited (Figs. 3k-3l), and the mRNA expression of IL1B, IL6, IL8, TNF, and secreted protein levels of IL-8 and TNF were also significantly inhibited (Figs. 3m-3r).
상기와 같은 결과를 종합하면, 장에서 발현되는 TAS1R3는 식이로 유도되는 염증성 사이토카인의 분비를 직접적으로 조절할 수 있으며, 장 세포에서 TAS1R3을 억제하는 경우, 장 세포의 염증 기전을 억제시킬 수 있음을 세포 수준에서 확인할 수 있었다. Summarizing the above results, it was confirmed at the cellular level that TAS1R3 expressed in the intestine can directly regulate the secretion of inflammatory cytokines induced by diet, and that inhibiting TAS1R3 in enterocytes can inhibit the inflammatory mechanism of enterocytes.
실시예 4. Tas1r3 넉아웃 마우스 모델을 이용한 서구식 식이 유도된 장 염증에서 미각 수용체 TAS1R3의 기능 연구Example 4. Functional study of taste receptor TAS1R3 in intestinal inflammation induced by western diet using Tas1r3 knockout mouse model
4-1. Tas1r3 넉아웃 마우스 제작4-1. Construction of Tas1r3 knockout mice
Tas1r3tm1Csz(JAX013066)의 Breeding pair(동결된 sperm 구매)를 The Jackson Lab에서 구매하여, Tas1r3 유전자 결핍(knock-out, KO) 마우스를 제작하였다. 최소 7세대 동안 C57BL/6와 백크로싱(backcrossing)을 진행하였으며, Tas1r3 넉아웃 마우스의 유전자형은 PCR 지노타이핑(Genotyping)을 통해 확인하였다. Jax protocol에 따라 실험을 진행하였으며, 지노타이핑에 사용된 프라이머 정보는 표 2, Reaction A 정보는 표 3, PCR 조건은 표 4와 같다.Tas1r3 tm1Csz (JAX013066) breeding pair (purchased frozen sperm) was purchased from The Jackson Lab, and Tas1r3 gene-deficient (knock-out, KO) mice were constructed. Backcrossing with C57BL/6 was performed for at least 7 generations, and the genotype of Tas1r3 knockout mice was confirmed through PCR genotyping. Experiments were conducted according to the Jax protocol, primer information used for genotyping is shown in Table 2, Reaction A information is shown in Table 3, and PCR conditions are shown in Table 4.
PRIMERPRIMER Sequence (5'->3')Sequence (5'->3') Reaction reaction
1182911829 CCA CAG TCA ACA GAA ACCCCA CAG TCA ACA GAA ACC AA
1183011830 CAT CCC AAG CTC CAG GAACAT CCC AAG CTC CAG GAA AA
1183111831 TAA GGG CCA GCT CAT TCCTAA GGG CCA GCT CAT TCC AA
ComponentComponent Final concentrationFinal concentration
ddH2OddH 2 O
Kapa 2G HS Buffer Kapa 2G HS Buffer 1.30Ⅹ1.30Ⅹ
MgCl2 MgCl 2 2.60mM2.60 mM
dNTP KAPAdNTPs KAPA 0.26mM0.26 mM
1182911829 0.50uM0.50 uM
1183011830 0.50uM0.50 uM
1183111831 0.50uM0.50 uM
)Glycerol)Glycerol 6.50%6.50%
DyeDye 1.00Ⅹ1.00Ⅹ
Kapa 2G HS taq polymeraseKapa 2G HS taq polymerase 0.03U/ul0.03U/ul
DNADNA
STEPSTEP TEMP (℃TEMP (℃ NOTENOTE
1One 94.094.0
22 94.094.0
33 65.065.0 -0.5℃ per cycle decrease-0.5℃ per cycle decrease
44 68.068.0
55 Repeat steps 2-4 for 10 cycles (Touchdown)Repeat steps 2-4 for 10 cycles (Touchdown)
66 94.094.0
77 60.060.0
88 72.072.0
99 Repeat steps 6-8 for 28 cyclesRepeat steps 6-8 for 28 cycles
1010 72.072.0
1111 10.010.0 holdhold
연령 및 체중이 일치하는 야생형 대조군(Tas1r3WT) (Jackson Laboratory (West Grove, PA, USA)과 녹아웃 마우스(Tas1r3KO)를 SPF(Specific Pathogen Free) 동물 사육실에서 사육하며, 면역조직화학염색법(Immunohistochemistry, IHC)을 통해, 각 마우스의 소장(Small intestine) 및 대장(Large intestine)에서 실제로 TAS1R3의 발현이 결핍되었는지를 단백질 수준에서 확인하였다. Wild -type controls (TAS1R3 WT ) (West Grove, PA, USA), where age and weight match, and the lust -out mouse (TAS1R3 KO ) in the SPF (SpeciFic Pathogen Free) animal breeding room and immunochemical chemical dyeing method (Immunohi) Stochemistry, IHC) confirmed whether the expression of tas1R3 was actually deficient in the small intestine and the large intestine of each mouse at the protein level.
면역조직화학염색법(IHC)을 위해, 4-6 μm 두께로 자른 샘플조직을 포르말린으로 고정한 후, 조직을 탈파라핀화 한 후, 자일렌 및 알코올 시리즈를 통해 재수화하고, 시트레이트 완충용액 (pH6.0, Sigma, C9999)에 넣은 후 100℃에서 20분간 가열하였다. 인산염 완충 식염수로 세척한 후 슬라이드를 1% bovine serum albumin(Sigma-Aldrich), Phosphate-buffered saline-tween과 함께 1시간동안 incubation하고 Anti-TAS1R3 항체 (TAS1R3 antibody (1:100, OSR00184W, Invitrogen, Camarillo, CA, USA)를 붙어주었다. Mouse and Rabbit-Specific HRP/DAB Detection IHC kit (ab64264, Abcam)을 2차 항체로 사용하였다. For immunohistochemical staining (IHC), sample tissues cut to a thickness of 4-6 μm were fixed with formalin, the tissues were deparaffinized, rehydrated through xylene and alcohol series, and placed in citrate buffer (pH6.0, Sigma, C9999) and heated at 100 ° C. for 20 minutes. After washing with phosphate-buffered saline, the slides were incubated with 1% bovine serum albumin (Sigma-Aldrich) and Phosphate-buffered saline-tween for 1 hour, and an anti-TAS1R3 antibody (1:100, OSR00184W, Invitrogen, Camarillo, CA, USA) was attached. Mouse and Rabbit-Specific HRP/DAB Detection IHC kit (ab64264, Abcam) was used as a secondary antibody.
그 결과, 대조군인 야생형 (Tas1r3WT) 마우스와 비교하여, 본 발명에서 제작된 넉아웃 마우스(Tas1r3KO)의 장조직에서는 TAS1R3의 발현이 결핍되었음을 확인하였다(도 4, 도 5a, 도 5b). As a result, compared to wild-type (Tas1r3 WT ) mice as a control group, it was confirmed that the intestinal tissue of knockout mice (Tas1r3 KO ) prepared in the present invention lacked expression of TAS1R3 (FIGS. 4, 5a, and 5b).
4-2. 야생형 및 Tas1r3 넉아웃 마우스에서 정상 식이와 서구식 식이에 의한 장 염증 유도 여부 확인4-2. Induction of Intestinal Inflammation by Normal Diet and Western Diet in Wild-Type and Tas1r3 Knockout Mice
체중이 유사한 8-10 주령의 야생형 대조군(Tas1r3WT)과 Tas1r3 녹아웃 마우스(Tas1r3KO)를 SPF(Specific Pathogen Free) 동물 사육실에서 사육하며, 각 실험군 당 10 마리씩 정상 식이군 또는 서구식 식이군 [설탕용액(30% w/v) + 60% 고지방식이]으로 분류하고, 자유 급식으로 10주간 공급하였다. 8-10-week-old wild-type control (Tas1r3 WT ) and Tas1r3 knockout mice (Tas1r3 KO ) of similar body weight were bred in SPF (Specific Pathogen Free) animal breeding rooms, and 10 animals per experimental group were classified as normal diet or western diet [sugar solution (30% w/v) + 60% high-fat diet] and fed free-feed for 10 weeks.
장 염증 정도를 분석하기 위하여, 마우스를 희생시키고, 비장, 소장, 결장을 적출하여 그 크기를 비교하는 조직학적 검사를 수행하였다. In order to analyze the degree of intestinal inflammation, the mice were sacrificed, and the spleen, small intestine, and colon were removed and histological examination was performed to compare their sizes.
그 결과, 야생형 대조군에서 서구식 식이에 의해 급격하게 증가된 비장의 크기가, Tas1r3 녹아웃 마우스에서는 서구식 식이를 공급하여도 크게 증가되지 않는 것으로 확인되었고 (도 6a), 야생형 대조군에서는 서구식 식이에 의해 소장과 대장의 크기가 현저하게 감소한 반면, 서구식 식이를 공급한 Tas1r3 녹아웃 마우스는 정상 식이를 공급한 마우스와 유사한 수준의 소장과 대장 크기를 유지하였다 (도 6b, 6c). As a result, it was confirmed that the size of the spleen, which was rapidly increased by the Western-style diet in the wild-type control group, was not significantly increased in the Tas1r3 knockout mice even when fed the Western-style diet (Fig. 6a). In the wild-type control group, the size of the small and large intestines was significantly reduced by the Western-style diet, whereas in the Tas1r3 knockout mice fed the Western-style diet, the small and large intestines were maintained at similar levels to those fed the normal diet (Figs. 6b, 6c).
장 내 침윤된 염증세포를 분석하고자, 적출된 조직을 이용하여 및 면역형광염색법(Immunofluorescence, IF)으로 분석하였다. 면역형광염색법(IF)의 실험방법은 면역조직화학염색법(IHC)과 동일하며, 사용된 항체의 정보는 다음과 같다.In order to analyze the inflammatory cells infiltrating the intestine, the extracted tissues were analyzed using immunofluorescence (IF). The experimental method of immunofluorescence staining (IF) is the same as that of immunohistochemical staining (IHC), and the information of the antibody used is as follows.
Primary antibodies: pan-leukocytes (CD45) (1:100, ab10558, Abcam, Cambridge, UK), anti-CD4 (14-0041-86; eBioscience, San Diego, CA, USA), and rat anti-CD8 (ab22378, Abcam), and mouse anti-CD11b (14-0112-82, eBioscience)Primary antibodies: pan-leukocytes (CD45) (1:100, ab10558, Abcam, Cambridge, UK), anti-CD4 (14-0041-86; eBioscience, San Diego, CA, USA), and rat anti-CD8 (ab22378, Abcam), and mouse anti-CD11b (14-0112-82, eBioscience)
Secondary and secondary-conjugated primary antibodies: Alexafluor 488 anti-mouse/human CD11b (101219, BioLegend, San Diego, CA, USA), donkey anti-rabbit Alexafluor 594 (A21207, Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-mouse Alexafluor 488 (A21202, Thermo Fisher Scientific), and donkey anti-rat Alexafluor 594 (A21209, Thermo Fisher Scientific). Secondary and secondary-conjugated primary antibodies: Alexafluor 488 anti-mouse/human CD11b (101219, BioLegend, San Diego, CA, USA), donkey anti-rabbit Alexafluor 594 (A21207, Thermo Fisher Scientific, Waltham, MA, USA), donkey anti-mouse Alexafluor 488 (A21202, Thermo Fisher Scientific), and donkey anti-rat Alexafluor 594 (A21209, Thermo Fisher Scientific).
면역반응성은 공초점 현미경 시스템(LSM 510; Carl Zeiss, Oberkochen, Germany)을 사용하여 시각화(Visualization) 하였다.Immunoreactivity was visualized using a confocal microscope system (LSM 510; Carl Zeiss, Oberkochen, Germany).
그 결과, Tas1r3 녹아웃 마우스에서 장 염증이 감소하고, 야생형 대조군에서 서구식 식이에 의해 급격하게 증가된 염증 수치가 서구식 식이를 공급한 Tas1r3 녹아웃 마우스에서는 정상 식이를 공급하는 수준으로 유지되었으며, 염증성 사이토카인인 IL1B, Tnfα, IL6의 mRNA 발현량도 정상 식이를 공급하는 수준으로 유지되어(도 6d-6h), Tas1r3 넉아웃 마우스는 서구식 식이 유도 장 염증으로부터 보호되고 있음을 확인하였다. As a result, intestinal inflammation was reduced in Tas1r3 knockout mice, and the inflammation level rapidly increased by western diet in wild-type control mice was maintained at the normal diet level in Tas1r3 knockout mice fed a western diet, and mRNA expression levels of inflammatory cytokines IL1B, Tnfα, and IL6 were also maintained at normal diet levels (FIG. 6d-6h). It was confirmed that it was protected from inflammation.
4-3. 야생형 및 Tas1r3 넉아웃 마우스에서 정상 식이와 서구식 식이에 의한 장 내 전사체 분석4-3. Intestinal transcriptome analysis in wild-type and Tas1r3 knockout mice fed a normal and western diet
Tas1r3 넉아웃 마우스는 서구식 식이 유도 장 염증으로부터 보호되고 있는바, 그 메커니즘을 규명하기 위하여 실시예 4-2의 마우스를 이용하여 소장(ileum)에서의 전사체(transcriptome) 분석을 진행하였다. 전사체 실험은, 실시예 1의 RNA 서열 분석 방법과 동일하다.Since Tas1r3 knockout mice are protected from intestinal inflammation induced by a western diet, transcriptome analysis in the small intestine was performed using the mice of Example 4-2 to investigate the mechanism. The transcriptome experiment is the same as the RNA sequence analysis method of Example 1.
그 결과, Tas1r3 넉아웃 마우스에서 장 내 mTOR/PPARγ 축(axis)이 야생형 마우스와 비교하여 크게 변화되어 있음이 확인되었는데, 구체적으로 mTOR의 인산화가 억제되고, PPARγ의 발현이 증가되는 것으로 확인되었다 (도 7a, 도 7b). As a result, it was confirmed that the intestinal mTOR/PPARγ axis was significantly changed in Tas1r3 knockout mice compared to wild-type mice. Specifically, mTOR phosphorylation was inhibited and PPARγ expression was increased (FIGS. 7a and 7b).
장 내 PPARγ 유전자는 장벽을 강화시키는 밀착 연접 단백질 (tight junction protein) 및 항균 펩타이드(antimicrobial peptide)의 발현을 조절하는 전사인자로 알려져 있다. 이에, 밀착 연접 단백질 (tight junction protein) 및 항균 펩타이드 (antimicrobial peptide)의 발현 변화를 추가로 확인해 본 결과, 밀착연접 단백질 (tight junction protein) 및 항균 펩타이드(antimicrobial peptide)의 발현 역시 Tas1r3 넉아웃 마우스에서 크게 변화되어 있음을 확인할 수 있었다 (도 7c).The PPARγ gene in the intestine is known as a transcription factor that regulates the expression of tight junction proteins and antimicrobial peptides that strengthen the intestinal barrier. Accordingly, as a result of further confirming changes in the expression of tight junction proteins and antimicrobial peptides, it was confirmed that the expressions of tight junction proteins and antimicrobial peptides were also significantly changed in Tas1r3 knockout mice (Fig. 7c).
이와 같은 결과로부터, 장 미각수용체 TAS1R3가 장세포 내 전사인자 PPARγ의 내재적 조절자로 기능하여 서구식 식이로부터 유도된 장 염증으로부터 장 세포간 밀착 연접을 증가시켜 장벽을 강화시킬 수 있음을 확인하였다.From these results, it was confirmed that the intestinal taste receptor TAS1R3 functions as an intrinsic regulator of the transcription factor PPARγ in enterocytes and can strengthen the barrier by increasing the tight junctions between enterocytes from intestinal inflammation induced by a western diet.
4-4. 야생형 및 Tas1r3 넉아웃 마우스에서 정상 식이와 서구식 식이에 의한 장 내 공생물체 프로파일 변화 분석4-4. Analysis of intestinal commensal profiles in wild-type and Tas1r3 knockout mice induced by normal and western-style diets
Tas1r3 넉아웃 마우스는 서구식 식이 유도 장 염증으로부터 보호되고 있고, 장 염증에는 장 내 공생물체의 역할이 중요한 것으로 알려져 있는바, Tas1r3 넉아웃 마우스의 장 내 공생물체 프로파일을 16S rRNA 시퀀싱(microbiome)으로 분석하였다. Since Tas1r3 knockout mice are protected from intestinal inflammation induced by a western diet, and the intestinal commensal is known to play an important role in intestinal inflammation, the intestinal commensal profile of Tas1r3 knockout mice was analyzed by 16S rRNA sequencing (microbiome).
16S rRNA 시퀀싱(microbiome) 분석을 위한 대변 샘플은 수집 직후 냉동되어 -80℃에 보관되었다. QIAamp®Stool Mini Kit(Qiagen, Hilden, Germany)를 사용하여 제조업체의 프로토콜에 따라 대변 샘플에서 총 박테리아 DNA를 분리하였다. Stool samples for 16S rRNA sequencing (microbiome) analysis were frozen immediately after collection and stored at -80°C. Total bacterial DNA was isolated from stool samples using the QIAamp®Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol.
16S rRNA 시퀀싱 라이브러리는 16S rRNA 유전자의 V3 및 V4 초가변 영역을 표적으로 하는 16S metagenomics sequencing library preparation protocol (Illumina, San Diego, CA, USA)에 따라 구성되었다. KAPA HiFi HotStart ReadyMix(KAPA Biosystems, Wilmington, MA, USA) 및 Agencourt AMPure XP 시스템(Beckman Coulter Genomics, Brea, CA, USA)은 각각 PCR 산물을 증폭 및 정제하는 데 사용되었으며. 최종 Amplicon은 MiSeq system(Illumina)을 사용하여 paired-end 모드(PE275)에서 시퀀싱되었다. A 16S rRNA sequencing library was constructed according to the 16S metagenomics sequencing library preparation protocol (Illumina, San Diego, CA, USA) targeting the V3 and V4 hypervariable regions of the 16S rRNA gene. A KAPA HiFi HotStart ReadyMix (KAPA Biosystems, Wilmington, MA, USA) and Agencourt AMPure XP system (Beckman Coulter Genomics, Brea, CA, USA) were used to amplify and purify PCR products, respectively. Final amplicons were sequenced in paired-end mode (PE275) using the MiSeq system (Illumina).
그 결과, 서구식 식이가 공급된 야생형 마우스와 비교하여 서구식 식이가 공급된 Tas1r3 넉아웃 마우스에서 장 내 유용세균(절대 혐기성균), 특히 부티르산 생성 박테리아(Faecalibacterium 및 Roseburia 등)의 증식이 매우 유의하게 증가되었고, 병원성 유해균(Enterobacteriaceae)의 풍부도는 유의적으로 낮은 것으로 확인되었다 (도 8a -8h).As a result, the proliferation of useful bacteria (absolutely anaerobic bacteria) in the intestine, particularly butyric acid-producing bacteria (Faecalibacterium and Roseburia, etc.), was significantly increased in Tas1r3 knockout mice fed a western diet compared to wild-type mice fed a western diet, and the abundance of pathogenic harmful bacteria (Enterobacteriaceae) was confirmed to be significantly low (FIGS. 8a - 8h).
4-5. Tas1r3 넉아웃 마우스에서 부티르산 생성 박테리아균의 확장 원인 규명4-5. Identification of the cause of expansion of butyric acid-producing bacteria in Tas1r3 knockout mice
최근 연구에서는 전사인자 PPARγ가 장세포 내 β-oxidation을 증가시켜, 산소분압이 낮은 장 내강의 상태(hypoxia)를 유지시키는 역할을 하는 것으로 밝혀졌다(Microbiota-activated PPAR-γ dysbiotic Enterobacteriaceae expansion, Science, 2017, 357:570-575).In a recent study, it was found that the transcription factor PPARγ increases β-oxidation in enterocytes and plays a role in maintaining hypoxia in the intestinal lumen with low oxygen partial pressure (Microbiota-activated PPAR-γ dysbiotic Enterobacteriaceae expansion, Science , 2017, 357:570-575).
서구식 식이가 공급된 Tas1r3 넉아웃 마우스에서 장 내 유용세균인 부티르산 생성 박테리아의 증식이 증가되는 이유가 장세포 내 전사인자 PPARγ 발현이 증가되는 것에 기인할 수 있는 것으로 판단하여, 이에 대해 분석하였다. 부티레이트 생성 박테리아의 상대적 풍부함과 PPAR 관련 분자의 전사체 발현 사이의 상관 관계는 Spearman의 상관 관계 테스트를 사용하여 분석되었다. It was determined that the reason why the proliferation of butyric acid-producing bacteria, a useful bacterium in the intestine, was increased in Tas1r3 knockout mice fed a Western-style diet could be due to increased expression of the transcription factor PPARγ in enterocytes, and this was analyzed. The correlation between the relative abundance of butyrate-producing bacteria and the transcript expression of PPAR-related molecules was analyzed using Spearman's correlation test.
그 결과, 도 9에서와 같이 Tas1r3 넉아웃에 의해 증가된 PPARγ 신호전달 경로가 혐기성 부티레이트 생성 박테리아와 매우 유의적인 양(positive)의 상관관계를 나타내었다. 이는 Tas1r3 결핍이 장 내 전사인자 PPARγ를 증가시키면, 장 내강이 저산소의 상태(hypoxia)가 되며, 혐기적인 환경에서 사는 혐기성 부티레이트균의 확장을 유도하여 결과적으로 장 염증으로부터 보호되는 효과가 발휘되는 것으로 해석된다.As a result, as shown in FIG. 9, the PPARγ signaling pathway increased by Tas1r3 knockout showed a very significant positive correlation with anaerobic butyrate-producing bacteria. It is interpreted that when Tas1r3 deficiency increases the intestinal transcription factor PPARγ, the intestinal lumen becomes hypoxic, induces the expansion of anaerobic butyrate bacteria living in an anaerobic environment, and consequently exerts an effect of protecting against intestinal inflammation.
실시예 5. 장 미각수용체 TAS1R3 억제를 통한 mTOR/PPARγ axis의 조절Example 5. Regulation of the mTOR/PPARγ axis through inhibition of the intestinal taste receptor TAS1R3
장 미각수용체 TAS1R3를 억제하기 위하여 siRNA 또는 장 미각수용체 TAS1R3 안타고니스트인 락티솔(lactisole)을 처리하는 경우 mTOR/PPARγ axis가 조절되는지 확인하고자 하였다. In order to suppress the intestinal taste receptor TAS1R3, we tried to confirm whether the mTOR/PPARγ axis is regulated when treated with siRNA or lactisole, an antagonist of the intestinal taste receptor TAS1R3.
siRNA와 TAS1R3 안타고니스트인 락티솔(lactisole)처리는 실시예 3와 동일한 방법으로 진행하였으며, mTOR, phospho-mTOR, 및 PPARγ의 1차 항체는 monoclonal rabbit anti-mTOR (1:1,000) (Cat# 2972S, RRID: AB_330978), rabbit anti-phospho-mTOR (1:1,000) (Cat# 2971S, RRID: AB_330970), rabbit anti-PPARgamma (1:1,000) (Cat# 2443S, RRID: AB_823598)를 Cell Signaling Technology (Danvers, MA, USA)로부터 구입하여 분석하였다. 대조군으로 mouse anti-α-tubulin (1:10,000)(Cat# T5168, RRID: AB_477579)을 사용하였다. 2차 항체는 Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000) (Cat# 7074S, RRID: AB_2099233)를 Cell Signaling Technology (Danvers, MA, USA)로부터, goat anti-mouse secondary antibodies (1:10,000) (Cat# G21040, RRID: AB_2536527)를 Invitrogen (Carlsbad, CA, USA)으로부터 구입하여 사용하였다. Treatment with siRNA and TAS1R3 antagonist lactisole was performed in the same manner as in Example 3, and the primary antibodies for mTOR, phospho-mTOR, and PPARγ were monoclonal rabbit anti-mTOR (1:1,000) (Cat# 2972S, RRID: AB_330978), rabbit anti-phospho-mTOR (1:1,000) (Cat# 297 1S, RRID: AB_330970) and rabbit anti-PPARgamma (1:1,000) (Cat# 2443S, RRID: AB_823598) were purchased from Cell Signaling Technology (Danvers, MA, USA) and analyzed. As a control, mouse anti-α-tubulin (1:10,000) (Cat# T5168, RRID: AB_477579) was used. Secondary antibodies were Horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (1:5,000) (Cat# 7074S, RRID: AB_2099233) from Cell Signaling Technology (Danvers, MA, USA) and goat anti-mouse secondary antibodies (1:10,000) (Cat# G21040, RRID: AB_2536527) from Invitrogen (Carls bad, CA, USA) and used.
그 결과, 도 10에서와 같이 TAS1R3 siRNA 또는 lactisole을 처리하는 경우, mTOR의 인산화가 억제되고, PPARγ의 발현이 증가되었으며, 따라서 TAS1R3를 억제하는 경우 장벽을 강화시키는 밀착 연접 단백질 (tight junction protein) 및 항균 펩타이드(antimicrobial peptide)의 발현을 조절하여 실제로 염증성 장 질환을 치료할 수 있음을 알 수 있었다.As a result, as shown in FIG. 10, when TAS1R3 siRNA or lactisole was treated, mTOR phosphorylation was inhibited and PPARγ expression was increased. Therefore, when TAS1R3 was inhibited, it was found that inflammatory bowel disease can be actually treated by regulating the expression of tight junction proteins and antimicrobial peptides that strengthen the barrier.
실시예 6. 염증성 장 질환(IBD)을 가진 환자의 장 조직에서 TAS1R3 및 관련 분자들(mTOR/PPARγ)의 발현 분석Example 6. Expression analysis of TAS1R3 and related molecules (mTOR/PPARγ) in intestinal tissues of patients with inflammatory bowel disease (IBD)
실제로 염증성 장 질환 (IBD)을 가진 환자의 장 조직에서 TAS1R3 및 관련 분자들(mTOR/PPARγ)의 발현 양상을 확인하고자, 염증성 장 질환 환자 및 건강한 대조군의 장 생검(biopsy) mRNA 마이크로어레이 데이터 세트를 GEO 데이터베이스 (public database, available online: http://www.ncbi.nlm.nih.gov/geo)에서 다운로드 하여, 각 분자들의 발현 정도를 분석하였다. In order to actually confirm the expression patterns of TAS1R3 and related molecules (mTOR/PPARγ) in the intestinal tissue of patients with inflammatory bowel disease (IBD), biopsy mRNA microarray data sets of inflammatory bowel disease patients and healthy controls were downloaded from the GEO database (public database, available online: http://www.ncbi.nlm.nih.gov/geo ), and the expression levels of each molecule were analyzed.
(i) 활동성 IBD를 갖는 성인 환자의 장 생검 조직(not cultured cells) 일 것, (ii) 중재 또는 치료를 받지 않은 IBD 환자의 샘플일 것이라는 이 두 가지의 기준을 모두 포함하는 데이터세트를 스크리닝 한 결과, 총 4개의 mRNA 발현 데이터세트 [GSE160804, GSE126124, GSE95095, GSE75214 및 GSE53306]가 분석을 위해 선별되었다. After screening datasets that contained both criteria: (i) not cultured cells from adult patients with active IBD, and (ii) samples from IBD patients without intervention or treatment, a total of four mRNA expression datasets [GSE160804, GSE126124, GSE95095, GSE75214, and GSE53306] were selected for analysis.
원시(raw) 마이크로어레이 데이터는 GEO 데이터베이스에서 다운로드한 다음 배경 조정(Background correction), 분위수 정규화(Quantile normalization) 및 프로브 요약(Probe summarization)을 수행하는 강력한 다중 칩 분석(Robust multi-chip analysis, RMA) 알고리즘과 함께 Partek®버전 6.6(Partek®을 사용하여 사전 처리되었다. 유전자 차등 발현 분석(Differentially expressed genes)은 R/Bioconductor(available online: http://www.bioconductor.org/)로 수행되었으며, DEG를 선택할 때 FDR P < 0.001이 cut off 값으로 간주되었다.The raw microarray data was downloaded from the GEO database and then preprocessed using Partek® version 6.6 (Partek®) with a robust multi-chip analysis (RMA) algorithm to perform background correction, quantile normalization and probe summarization. Differentially expressed genes were analyzed using R/Bioconductor (available online: http://www.bioconductor.org /), and an FDR P < 0.001 was considered as the cut off value when selecting DEGs.
그 결과, 도 11에서와 같이, 염증성 장 질환(IBD) 환자에서도 본 발명의 Tas1r3 넉아웃 마우스에서 분석한 결과와 일치되게, 장 조직에서 미각 수용체 TAS1R3의 발현정도가 비정상적으로 높아져있는 것을 확인할 수 있었으며, 높아진 TAS1R3의 발현은 염증성 사이토카인 IL1B, IL6, TNF와 모두 일관적으로 유의한 양의 상관관계를 나타내는 것으로 확인되었다. As a result, as shown in FIG. 11, consistent with the results of the analysis in the Tas1r3 knockout mouse of the present invention, even in patients with inflammatory bowel disease (IBD), it was confirmed that the expression level of the taste receptor TAS1R3 was abnormally elevated in the intestinal tissue.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서, 이러한 구체적 기술은 단지 바람직한 실시양태일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As above, specific parts of the present invention have been described in detail, and for those skilled in the art, it will be clear that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereby. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

  1. TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 유효성분으로 포함하는 염증성 장 질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
  2. 제1항에 있어서, 상기 TAS1R3 발현 억제제는 TAS1R3 특이적인 안티센스올리고뉴클레오티드, siRNA, shRNA 또는 miRNA인 것을 특징으로 하는, 조성물.The composition according to claim 1, wherein the TAS1R3 expression inhibitor is a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA.
  3. 제1항에 있어서, 상기 TAS1R3 단백질 활성 억제제는 TAS1R3 특이적인 항체, 압타머 또는 안타고니스트(antagonist)인 것을 특징으로 하는, 조성물.The composition according to claim 1, wherein the TAS1R3 protein activity inhibitor is a TAS1R3-specific antibody, aptamer or antagonist.
  4. 제1항에 있어서, 상기 염증성 장 질환은 서구식 식이에 의해 유도되는 염증성 장 질환인 것을 특징으로 하는, 조성물.The composition according to claim 1, wherein the inflammatory bowel disease is inflammatory bowel disease induced by a western diet.
  5. 제1항에 있어서, 상기 조성물은 The method of claim 1, wherein the composition
    (i) 장 내 미생물군집(microbiome) 변화; (i) changes in the gut microbiome;
    (ii) 장 내 세포간 밀착 연접 강화; 및/또는(ii) strengthening the tight junctions between cells in the intestine; and/or
    (iii) 항균 펩타이드 분비 유전자 발현의 증가;를 유도하여 염증성 장 질환을 예방 또는 치료하는 것을 특징으로 하는, 조성물.(iii) an increase in antimicrobial peptide secretion gene expression; characterized in that for preventing or treating inflammatory bowel disease by inducing, the composition.
  6. 제5항에 있어서, 상기 장 내 미생물군집(microbiome) 변화는 장 내 혐기성 부티레이트 균의 확장 및/또는 장 내 유해균의 억제인 것을 특징으로 하는, 조성물.The composition according to claim 5, wherein the change in the intestinal microbiome is expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine.
  7. TAS1R3 발현 억제제 또는 TAS1R3 단백질 활성 억제제를 유효성분으로 포함하는 염증성 장 질환 예방 또는 개선용 식품용 조성물.A food composition for preventing or improving inflammatory bowel disease comprising a TAS1R3 expression inhibitor or a TAS1R3 protein activity inhibitor as an active ingredient.
  8. 제7항에 있어서, 상기 TAS1R3 발현 억제제는 TAS1R3 특이적인 안티센스올리고뉴클레오티드, siRNA, shRNA 또는 miRNA 인 것을 특징으로 하는, 조성물.The composition according to claim 7, wherein the TAS1R3 expression inhibitor is a TAS1R3-specific antisense oligonucleotide, siRNA, shRNA or miRNA.
  9. 제7항에 있어서, 상기 TAS1R3 단백질 활성 억제제는 TAS1R3 특이적인 항체, 압타머 또는 안타고니스트(antagonist)인 것을 특징으로 하는, 조성물.The composition according to claim 7, wherein the TAS1R3 protein activity inhibitor is a TAS1R3-specific antibody, aptamer or antagonist.
  10. 제7항에 있어서, 상기 염증성 장 질환은 서구식 식이에 의해 유도되는 염증성 장 질환인 것을 특징으로 하는, 조성물.The composition according to claim 7, wherein the inflammatory bowel disease is inflammatory bowel disease induced by a western diet.
  11. 제7항에 있어서, 상기 조성물은 The method of claim 7, wherein the composition
    (i) 장 내 미생물군집(microbiome) 변화; (i) changes in the gut microbiome;
    (ii) 장 내 세포간 밀착 연접 강화; 및/또는(ii) strengthening the tight junctions between cells in the intestine; and/or
    (iii) 항균 펩타이드 분비 유전자 발현의 증가;를 유도하여 염증성 장 질환을 예방 또는 개선하는 것을 특징으로 하는, 조성물.(iii) an increase in antibacterial peptide secretion gene expression; characterized in that for preventing or improving inflammatory bowel disease by inducing, the composition.
  12. 제11항에 있어서, 상기 장 내 미생물군집(microbiome) 변화는 장 내 혐기성 부티레이트 균의 확장 및/또는 장 내 유해균의 억제인 것을 특징으로 하는, 조성물.The composition according to claim 11, wherein the change in the intestinal microbiome is expansion of anaerobic butyrate bacteria in the intestine and/or suppression of harmful bacteria in the intestine.
  13. 다음 단계를 포함하는 염증성 장 질환 치료제 스크리닝 방법: A method for screening for drugs for inflammatory bowel disease comprising the following steps:
    (a) TAS1R3가 발현되거나 TAS1R3 단백질이 활성을 가지는 세포 또는 조직에 후보 물질을 처리하는 단계; (a) treating cells or tissues in which TAS1R3 is expressed or the TAS1R3 protein is active with a candidate substance;
    (b) TAS1R3 발현이 억제되거나 TAS1R3 단백질 활성이 억제되는 경우 상기 후보 물질을 염증성 장 질환 치료제로 선별하는 단계. (b) selecting the candidate substance as a therapeutic agent for inflammatory bowel disease when TAS1R3 expression is inhibited or TAS1R3 protein activity is inhibited.
  14. 제13항에 있어서, 상기 염증성 장 질환은 서구식 식이에 의해 유발되는 염증성 장 질환인 것을 특징으로 하는, 방법.14. The method of claim 13, wherein the inflammatory bowel disease is inflammatory bowel disease caused by a western diet.
PCT/KR2023/001053 2022-01-24 2023-01-20 Prevention or treatment of inflammatory bowel disease caused by western diets by inhibiting expression and activation of tas1r3 intestinal taste receptor WO2023140700A1 (en)

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