CN114058694A - Application of TRPV1 in screening or preparing medicines for preventing, relieving and/or treating liver diseases - Google Patents
Application of TRPV1 in screening or preparing medicines for preventing, relieving and/or treating liver diseases Download PDFInfo
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Classifications
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- C—CHEMISTRY; METALLURGY
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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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
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- 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
- A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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- 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
- G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
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- 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
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/08—Hepato-biliairy disorders other than hepatitis
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Abstract
The invention discloses an application of TRPV1 in screening or preparing a medicament for preventing, relieving and/or treating liver diseases, belonging to the field of gene function and application. According to the invention, C57/BL mice with TRPV1 gene overexpression and knockout are taken as experimental objects, and through researches on an acute liver injury model, a hepatic fibrosis model, a non-alcoholic fatty liver model and an alcoholic fatty liver model, the results show that compared with a control group of mice, the liver injury of TRPV1 gene overexpression mice is obviously inhibited, and the liver function is obviously improved, while compared with wild type mice, the pathological liver injury of TRPV1 gene knockout mice is obviously aggravated, and the liver function is obviously worsened. Aiming at the functions of the TRPV1, the specific agonist or the overexpression system thereof can promote the TRPV1 gene expression to intervene in liver diseases such as liver inflammation and liver fibrosis.
Description
Technical Field
The invention relates to the field of functions and applications of genes, in particular to application of TRPV1 in screening or preparing a medicament for preventing, relieving and/or treating liver diseases.
Background
The liver is an organ mainly having metabolic functions in the body, and has very complex physiological and biochemical functions, including the actions of removing oxidation, storing glycogen, synthesizing secretory proteins and the like. Common liver diseases comprise viral hepatitis, non-alcoholic fatty liver disease, alcoholic fatty liver, autoimmune liver disease and the like, liver injury caused by the diseases and liver fibrosis, even liver cirrhosis and liver cancer are important causes influencing human health, and the health of human bodies is seriously threatened.
With the change of living habits of people, the incidence rate of fatty liver diseases is on a trend of rising year by year in recent years, and the fatty liver diseases can develop into hepatic fibrosis, liver cirrhosis, liver cancer and the like along with the progress of disease course, and become global widely distributed diseases affecting public health in the last decade. Fatty liver can be classified into alcoholic fatty liver disease (ALD) and non-alcoholic fatty liver disease (NAFLD), wherein non-alcoholic fatty liver disease is a clinical syndrome with liver cell fat excessive deposition as a main pathological feature, and with the development of the disease course, the damage of liver cells of fatty liver gradually worsens, and can develop into non-alcoholic steatohepatitis (NASH), liver cirrhosis and hepatocellular carcinoma (HCC), which seriously threatens the health of human beings.
Hepatic fibrosis is a common pathological process of most chronic liver diseases and is an intermediate link for the evolution of various chronic liver diseases to cirrhosis. In recent years, the incidence of liver fibrosis is high, and if liver fibrosis is not treated in time, serious patients will cause cirrhosis and even liver cancer, and cirrhosis is one of the common diseases and the main causes of death in clinic, and seriously harms the health of Chinese people. It has been proved that if proper treatment is given at the early stage of the disease process, not only can the further development of the disease be prevented, but also the hepatic fibrosis can be reversed, and the method has positive significance for preventing the chronic hepatic disease from progressing to the cirrhosis.
However, no good-quality anti-hepatic fibrosis treatment drug is applied clinically, which means that finding an effective anti-hepatic fibrosis target is an important direction for treating hepatic fibrosis in the future, and therefore, research and search of a new treatment target are urgently needed in the field to develop a novel and high-safety drug for treating diseases such as hepatic injury, hepatic fibrosis and the like.
Transient receptor potential vanilloid 1 (TRPV 1) is one of the members of transient receptor potential channels, widely distributed in tissues and organs of mammals, is a non-selective cation channel, is mainly distributed in sensory nerve endings, and can release various neurotransmitters after being activated, thereby triggering a series of biological effects. In recent years, with the intensive research on TRPV1, TRPV1 has a vascular protection effect and an anti-vascular fibrosis effect, but no relevant research report on the effect of TRPV1 in liver inflammation, fibrosis and steatosis is found.
Disclosure of Invention
In order to solve the problem of few drugs for effectively treating liver diseases such as hepatic fibrosis in the prior art, the invention aims to research and determine the mutual relation between the expression of TRPV1 gene and the liver fibrosis diseases, provide the application of TRPV1 as a drug target or target gene in screening and preparing drugs for treating the liver diseases, and provide a new thought and method for clinically treating the liver diseases such as hepatic fibrosis.
In order to achieve the above objects, the present invention firstly provides use of an up-regulator of TRPV1 in the preparation of a medicament for preventing, alleviating and/or treating liver diseases.
The TRPV1 up-regulator has the effects of promoting the mRNA level of TRPV1, or improving the activity or level of TRPV1 protein, or enhancing the action of TRPV 1. Specifically, various chemical, physical, biological methods may be employed to achieve the foregoing objectives, including but not limited to:
(1) modulating a TRPV1 metabolic pathway to increase TRPV1 expression levels;
(2) an overexpression mode is adopted to improve the expression level of TRPV 1.
In one possible embodiment, modulating the TRPV1 metabolic pathway may be increasing TRPV1 activity or promoting transcription or expression of TRPV1 using a TRPV1 agonist, thereby up-regulating TRPV1 levels;
promoting transcription or expression of TRPV1 refers to: overexpresses TRPV1 or increases TRPV1 transcriptional activity. The regulation of TRPV1 transcription or expression may be performed by one skilled in the art using conventional methods.
In another possible embodiment, the level of TRPV1 expression may be increased by transfecting a form of TRPV1 overexpression system;
further, the TRPV1 overexpression system is a TRPV1 protein overexpression system constructed by taking a lentivirus system, an adenovirus system or a plasmid as a vector.
In another aspect, the present invention also provides an application of TRPV1 as a drug target in screening or preparing a drug for preventing, alleviating and/or treating liver diseases.
Preferably, the drug for treating liver diseases is a drug that enhances TRPV1 expression;
preferably, the liver diseases described in the present invention include acute liver injury, liver fibrosis, non-alcoholic fatty liver and alcoholic fatty liver.
Compared with the prior art, the invention has the following beneficial effects:
(1) the invention discovers a new function of the TRPV1 gene, namely that the TRPV1 gene has the functions of protecting acute liver injury, hepatic fibrosis, non-alcoholic fatty liver and alcoholic fatty liver.
(2) Based on the new function of the TRPV1, a drug screening model is constructed by taking the TRPV1 as a drug target, and the drug screening model is used for screening drugs for preventing, relieving and/or treating liver diseases such as liver inflammation and liver fibrosis; in addition, TRPV1 can be used as a target gene in gene therapy, and can be used for designing and preparing medicines or biological reagents for preventing, relieving and/or treating liver diseases such as liver inflammation, fibrosis and the like, so that the aim of treating related liver diseases is finally achieved.
Drawings
FIG. 1 shows a graph of the construction and identification results of TRPV1 knockout mice.
Figure 2 shows the effect of TRPV1 knock-out on acute liver injury, wherein:
a is WT and TRPV1-/-HE staining profile of mice;
b is WT and TRPV1-/-Serum ALT detection results of mice;
c is WT and TRPV1-/-The statistical results of the spleen weight, the spleen body ratio and the like of the mouse;
d is WT and TRPV1-/-And (3) detecting the gene expression of the mouse liver-related inflammatory factor.
FIG. 3 shows TRPV1 knock-out versus mouse CCl4The effects of liver fibrosis, wherein:
a is WT and TRPV1-/-Pathological staining graph and semi-quantitative analysis result of the mouse;
b is WT and TRPV1-/-Gene expression detection results of mouse liver-related fibrosis indexes;
c is WT and TRPV1-/-And (3) detecting the gene expression of the mouse liver related inflammatory factor index.
Figure 4 shows the effect of TRPV1 overexpression on liver fibrosis, wherein:
a is the expression detection result of LV-Con and LV-SMA-TRPV1 mice;
b is the result of semi-quantitative analysis of the expression of mouse TRPV1 and alpha-SMA protein by LV-Con and LV-SMA-TRPV 1;
C-D is the liver sirius red staining and statistical result of LV-Con and LV-SMA-TRPV1 mice, and the alpha-SMA immunohistochemical detection and statistical result;
e is the detection results of ALT and AST of LV-Con and LV-SMA-TRPV1 serum;
f is the gene expression detection result of LV-Con and LV-SMA-TRPV1 mouse liver related fibrosis indexes;
g is the gene expression detection result of the indexes of the inflammation factors related to the liver of the LV-Con mouse and the LV-SMA-TRPV1 mouse.
Figure 5 shows the effect of TRPV1 knockdown on MCD-induced non-alcoholic fatty liver, wherein:
a is WT and TRPV1-/-Results of liver sirius red staining and semi-quantitative analysis of mice;
b is WT and TRPV1-/-Oil red staining pattern and semi-quantitative analysis result of the mice;
c is WT and TRPV1-/-Serum ALT and AST detection results of the mice;
d is WT and TRPV1-/-Gene expression detection results of mouse liver-related fibrosis indexes;
e is WT and TRPV1-/-Gene expression detection results of mouse liver-related inflammatory factor indexes;
f is WT and TRPV1-/-And (4) carrying out western blot and gray level analysis statistical results on the mouse alpha-SMA.
Fig. 6 shows the effect of TRPV1 overexpression on MCD-induced non-alcoholic fatty liver disease, wherein:
a is the result of liver sirius red staining and semi-quantitative analysis of LV-Con and LV-SMA-TRPV1 mice;
b is oil red staining pattern and semi-quantitative analysis result of LV-Con and LV-SMA-TRPV1 mice;
c is the liver weight and the liver body ratio of LV-Con and LV-SMA-TRPV1 mice and the detection results of ALT and AST in serum;
d is the gene expression detection result of LV-Con and LV-SMA-TRPV1 mouse liver related fibrosis indexes;
e is the gene expression detection result of the indexes of relevant inflammatory factors of the mouse liver of LV-Con and LV-SMA-TRPV 1;
f is the statistical result of the Western blot and gray scale analysis of LV-Con and LV-SMA-TRPV1 mouse alpha-SMA.
Figure 7 shows the effect of TRPV1 knockdown on CDAA-induced nonalcoholic fatty liver, wherein:
a is WT and TRPV1-/-Pathological staining and semi-quantitative analysis results of sirius red liver of the mouse;
b is WT and TRPV1-/-The results of immunohistochemical staining and semi-quantitative analysis of mouse liver alpha-SMA;
c is WT and TRPV1-/-The results of the liver TC and serum TG detection of the mice;
d is WT and TRPV1-/-Gene expression detection results of mouse liver-related fibrosis indexes;
e is WT and TRPV1-/-Gene expression detection results of mouse liver-related inflammatory factor indexes;
f is WT and TRPV1-/-And (3) carrying out western blot detection and gray analysis statistical results on the mouse alpha-SMA.
Fig. 8 shows the results of measurement of TRPV1 expression in alcoholic fatty liver, in which:
a is a TRPV1 staining pattern and a semi-quantitative analysis result of mice in a control group (Pair-fed) group and an Alcohol group (Alcohol) group;
b is gene expression detection results of mouse TRPV1 and fibrosis indexes of the Pair-fed group and the Alcohol group;
c is the Western blot detection result of TRPV1 of mice in the Pair-fed group and the Alcohol group.
Detailed Description
The technical solution of the present invention is further described below with reference to the accompanying drawings and examples.
The inventors of the present application found in the study and analysis of clinical liver biopsy gene chips that: the expression of TRPV1 protein is down-regulated in liver fibrosis tissues. Based on the findings, the inventors of the present application continued to study the expression, distribution, functions and the like of TRPV1 in liver diseases such as hepatic fibrosis, and found that, by taking a TRPV1 gene overexpressed and knocked-out C57/BL mouse as an experimental subject, and by studying an acute liver injury model, a hepatic fibrosis model, a non-alcoholic fatty liver model and an alcoholic fatty liver model, liver injury of a TRPV1 gene overexpressed mouse is significantly suppressed and liver function is significantly improved compared with a non-overexpressed mouse, and liver pathological injury of a TRPV1 gene knocked-out mouse is significantly aggravated, inflammation, hepatic fibrosis and the like are significantly aggravated and liver function injury is significantly aggravated compared with a normal mouse.
Therefore, the TRPV1 gene provided by the invention can be used as a drug target to construct a drug screening model (including a cell model or an animal model) for screening drugs for preventing, relieving and/or treating liver diseases such as acute liver injury, hepatic fibrosis, non-alcoholic fatty liver, alcoholic fatty liver and the like. The molecule of TRPV1 can be specifically excited, so that a novel therapeutic molecule is provided for treating liver diseases such as acute liver injury, hepatic fibrosis, non-alcoholic fatty liver, alcoholic fatty liver and the like.
The TRPV1 gene can also be used as a target gene in gene therapy, and the medicine and/or biological reagent for preventing, relieving and/or treating liver diseases such as acute liver injury, hepatic fibrosis, non-alcoholic fatty liver, alcoholic fatty liver and the like is designed and prepared, so that the purpose of preventing, relieving and/or treating the liver diseases such as hepatic fibrosis and the like is achieved through a gene engineering technology.
Therefore, the invention provides the application of the TRPV1 up-regulator in preparing the medicine for protecting the liver function, or preventing, relieving and/or treating liver diseases such as acute liver injury, hepatic fibrosis, non-alcoholic fatty liver, alcoholic fatty liver and the like. The TRPV1 up-regulator is preferably one of an overexpression plasmid of TRPV1 gene, virus-mediated TRPV1 overexpression transfection or other agonists or biological agents capable of promoting TRPV1 expression.
The following are detailed experimental procedures and data of the present invention. The chemical reagents used in the following examples are conventional and are commercially available. The experimental methods not specifically described are all the conventional ones known in the art.
The animal and various study index measurements used in the following examples were as follows:
(I) laboratory animals
Experimental animals: selecting mice of 8-week-old, 18g-20g in weight and male C57/BL strain with background, including wild type mice (WT) and TRPV1 gene knockout mice (TRPV 1) constructed by the method-/-). FIG. 1 is a graph showing the results of construction and identification of TRPV1 knockout mice, WBThe results show that TRPV1 knockout mice were successfully constructed in the present application.
Animal feeding and model construction were conducted at the animal center of the university of medicine, pluta hospital, and the animal center of the university of Shanghai, Life sciences college.
(II) detection method
1. Taking and leaving specimen
After the model building is finished, 2% sodium pentobarbital (2ml/kg mouse weight) is used for intraperitoneal injection and anesthesia, the supine fixation is carried out, the abdominal cavity is opened, a needle is inserted into the inferior vena cava to take blood, the blood is kept stand for 1h, the centrifugation is carried out for 10min at 4 ℃ and 3000rpm, the serum is separated, and the blood is stored at-80 ℃ and used for detecting indexes such as liver function, blood fat and the like.
Picking up liver, preparing frozen tissue and fixing with 10% neutral formalin for histopathological detection, and freezing and storing the rest liver tissue at-80 ℃ for detection of indexes such as Real-time PCR, immunoblotting and the like.
2. Detecting the index
General conditions of animals: including body weight, liver weight, spleen weight, etc.; mouse liver function (ALT, AST) and blood lipid (TC, TG) assays; liver tissue HE, oil red staining; inflammatory factors and fibrosis indexes Real-time PCR; alpha-SMA immunohistochemistry and immunoblotting, etc.
3. Pathological staining of liver tissue
(1) HE staining
Mouse liver tissue is fixed with 10% formalin, and is sliced after being embedded by dehydrated paraffin, the slices are placed in a full-automatic dyeing machine for HE dyeing, pathological morphological change of the liver tissue is observed by a fluorescence microscope, and Image software is used for statistical analysis.
(2) Oil red dyeing
After being frozen and embedded, the liver tissue of the mouse is sliced and kept stand for 15min at room temperature, and the weight ratio of oil red to distilled water is 3: diluting at a ratio of 2, cleaning with 60% isopropanol, dyeing with oil red for 15min, air drying, cleaning with distilled water, counterstaining with hematoxylin, sealing with glycerol gelatin, and performing microscopic examination.
(3) Immunohistochemistry
Mouse liver tissue is fixed by 10% formalin, is sliced after being embedded by dehydrated paraffin, is dewaxed to water, is repaired by microwave at the temperature higher than 92 ℃, is cooled, is washed by PBS for 5min multiplied by 3 times, is sealed for 30min at the temperature of 5% BSA37 ℃, is dripped with primary antibody, is washed by PBS for 3min multiplied by 3 times at the temperature of 4 ℃, and is added with secondary antibody for room temperature of 1 h. SABC was added dropwise at 37 ℃ for 20 min. Washing with PBS for 5min × 3 times, DAB developing, washing with distilled water, counterstaining with hematoxylin, dehydrating with anhydrous ethanol, sealing after xylene is transparent, and performing microscopic examination.
4. Gene expression of inflammation-related factor detected by Real-time fluorescence quantitative PCR (Real-time PCR) method
Weighing about 50mg of mouse liver tissue, extracting total RNA by a Trizol method, and determining the concentration and the purity of the total RNA by a nucleic acid quantitative analyzer, wherein the value of A260 nm/A280 nm is 1.8-2.1. Synthesis of cDNA by reverse transcription was performed with reference to the reverse transcription kit instructions. The target genes are respectively amplified by PCR by using cDNA as a template, and the primer sequences are synthesized by Shanghai biological engineering Co. Reaction conditions are as follows: data were derived for statistical analysis at 95 ℃ for 30sec, 95 ℃ for 5sec, and 60 ℃ for 30sec for 45 cycles.
5. Western blot detection of protein expression
100mg of liver tissue was taken, pre-cooled RIPA (containing protease inhibitor) was added to the homogenate, centrifuged at 12000rpm for 15 minutes at 4 ℃ and a radius of centrifugation of 6cm, and the supernatant was retained for protein quantification. Taking 20 mu g of sample, carrying out 12% SDS-PAGE electrophoresis, transferring 1 hour to a PVDF membrane at 100V, and sealing for 1 hour; the primary antibody is incubated at 4 ℃ overnight and the secondary antibody is incubated for 45 minutes at room temperature, ECL is used for developing, and Western blot protein expression gray value is analyzed by using Image J.
6. Statistical method
Statistical analysis was performed using SPSS 23.0 software. Data are expressed as mean ± standard deviation, comparisons among groups were tested with a single sample t, with differences of P < 0.05 being statistically significant and P < 0.01 suggesting significant statistical differences.
Example 1 TRPV1 knock-out aggravates acute liver injury
(I) procedure of experiment
Constructing a TRPV1 knockout mouse acute liver injury model: 4 male C57/BL wild-type mice at 8 weeks of age served as WT group (control group), and 4 male TRPV1 at the same week of age-/-Mouse as TRPV1-/-Group (TRPV1 gene knock-out group).
Two groups of mice were given LPS (10mg/kg body weight) for intraperitoneal injection, and the material was sacrificed 18h later. Weighing the weight of the mouse and the weight of the spleen, and calculating the spleen-body ratio. And (3) reserving serum and liver for relevant detection: liver function test of mouse and expression of inflammatory factor.
(II) results of the experiment
The results are shown in FIG. 2 for TRPV1 compared to the WT group-/-The spleen weight of the group mice was significantly reduced, the spleen-to-body ratio was significantly reduced (C of fig. 2), and the serum liver function (ALT) was also significantly lower than that of the WT group (B of fig. 2); in addition, HE staining results showed: the hepatocytes of the WT group mice were aligned, and inflammatory cells in the tract area were less infiltrated; and TRPV1-/-The hepatocytes of the group mice were loosely arranged, the central venous structure was deformed, and there was massive infiltration of inflammatory cells in the zone of the sink (fig. 2 a); the PCR detection result shows that: TRPV1 compared to WT group-/-The expression of group inflammatory factors (Tnf-alpha, Mcp1, Il-1 beta, Cxcr1, Ccl19, Cxcl9, Cxcl11, Cxcl19, Ccl3 and the like) is obviously increased, and TRPV1-/-The inflammatory response was markedly increased in the group mice.
The above results indicate that the knock-out of TRPV1 aggravates acute liver injury, and conversely demonstrate that TRPV1 has a protective effect in acute liver injury.
Example 2 TRPV1 knock-out aggravates hepatic fibrosis
(I) procedure of experiment
Constructing a TRPV1 knockout mouse liver fibrosis model: 4 male C57/BL mice 8 weeks old were used as WT group, and 4 male TRPV1 mice of the same week old-/-Mouse as TRPV1-/-And (4) grouping. Intraperitoneal injection of CCl4(10％CCl40.04 ml/tube), 3 times per week for 6 consecutive weeks. The material is sacrificed and taken 72 hours after the last injection, and liver and serum are taken for relevant detection: pathological detection of sirius red staining and alpha-SMA expression; the expression of inflammatory factors and fibrosis indices were detected by PCR.
(II) results of the experiment
Results are shown in fig. 3, sirius red staining shows: the liver tissue of WT mice has narrow fiber intervals and loose and discontinuous arrangement, and TRPV1-/-The liver lobules are divided by the hyperplastic collagen fibers to form thicker intervals, and the liver lobules have abnormal structures to form false lobules; semi-quantitative analysis and immunohistochemistry showed: and WT groupIn contrast, TRPV1-/-The positive staining area of the group sirius red is obviously increased, the expression of the alpha-SMA is obviously increased, and the difference has statistical significance (A of figure 3); in addition, the PCR assay results showed: TRPV1 compared to WT group-/-The fibrosis indices (alpha-Sma, Col1 (alpha) 1, Col4 (alpha) 1, Desmin, Timp1) and the expression of inflammatory factors (Tnf-alpha, Cd68, Il-1 beta, Il-6, Mcp1, Cxcr1) of the groups were significantly increased (B, C of FIG. 3), indicating that TRPV1-/-The inflammatory response and the degree of fibrosis in the group of mice increased significantly.
The results show that the TRPV1 knockout can aggravate the hepatic fibrosis of mice, and the TRPV1 is proved to have a protective effect on the hepatic fibrosis.
Example 3 alleviation of liver fibrosis by overexpression of TRPV1
(I) procedure of experiment
Constructing a mouse liver fibrosis model with over-expressed TRPV 1: 12 male C57/BL mice at 8 weeks of age were randomized into control (LV-Con) and overexpression (LV-SMA-TRPV1) groups of 6 mice each. Two groups of mice were injected with 10% CCl4Olive oil, 0.04 ml/stick, 3 times per week for 6 weeks; mice injected with CCl4After 2 weeks of molding, the group of LV-SMA-TRPV1 is injected with TRPV1 lentivirus (LV-SMA-TRPV1) in tail vein; LV-Con group mice were injected with an equal volume of control virus (LV-Con). Last injection of CCl4The materials were sacrificed and taken 72 hours after olive oil and relevant assays were performed: detecting liver function and pathological changes of the mouse; immunohistochemical detection of alpha-SMA expression; PCR detects the expression of fibrosis index and inflammatory factor.
(II) results of the experiment
The results are shown in FIG. 4, and compared with LV-Con group, the TRPV1 expression of LV-SMA-TRPV1 group mice is obviously improved, which indicates that LV-SMA-TRPV1 virus is successfully expressed (A of FIG. 4); pathological staining results show that sirius red staining of the LV-SMA-TRPV1 group is remarkably reduced, and alpha-SMA expression is remarkably reduced (C, D of FIG. 4); compared with the LV-Con group, the serum ALT and AST of the mice in the LV-SMA-TRPV1 group are obviously reduced, and the liver function of the mice over-expressing TRPV1 is obviously improved (E of figure 4); in addition, the PCR assay results showed: compared with the LV-Con group, the expression of fibrosis indexes (alpha-SMA, Col1 (alpha) 1, Col4 (alpha) 1 and Desmin) and inflammatory factors (Tnf-alpha, Cd68, Il-1 beta, Il-6, Mcp1, Cxcr1 and Cxcl15) of the LV-SMA-TRPV1 group is also obviously reduced (F, G of figure 4), and the inflammatory response and the fibrosis degree of the mice of the LV-SMA-TRPV1 group are obviously relieved.
The results show that the over-expression of TRPV1 can obviously relieve hepatic fibrosis, and the over-expression of TRPV1 has a protective effect on hepatic fibrosis.
Example 4 TRPV1 knockout exacerbates MCD-induced non-alcoholic fatty liver disease
(I) procedure of experiment
Construction of TRPV1 knockout mouse non-alcoholic fatty liver model (MCD induction): 4 male C57/BL mice 8 weeks old were used as WT group, and 4 male TRPV1 mice of the same week old-/-Mouse as TRPV1-/-And (4) grouping. Mice were fed MCD feed (purchased from nanton telofil) and sacrificed for 6 consecutive weeks before the relevant testing: detecting biochemical indexes of the mouse serum; pathological detection of sirius red staining and oil red staining; detecting the expression of inflammatory factors and fibrosis indexes by PCR; immunoblotting was used to detect protein expression of α -SMA.
(II) results of the experiment
The results are shown in FIG. 5 for TRPV1 compared to the WT group-/-ALT and AST values of the mice in the group are obviously increased (C in figure 5); sirius red staining and oil red staining showed: TRPV1 compared to WT group-/-The dyeing of the sirius red is obviously increased, lipid droplets are obviously increased and increased, and the lipid deposition is obvious (A, B in figure 5); in addition, the PCR results showed: TRPV1-/-The expression of fibrosis indexes (alpha-Sma, Col1 (alpha) 1, Col4 (alpha) 1 and Timp1) and inflammatory factors (Tnf-alpha, Cd68, Il-1 beta, Mcp1, Ccl3, Cxcr1 and Cxcl2) of the mice in the group are obviously increased compared with that in the WT group (D, E in figure 5), and the expression of TRPV1 and the expression of inflammatory factors (Tnf-alpha, Cd68, Mcp1, Ccl3 and Cxcl2) are obviously increased compared with that in the WT group (D, E in figure 5), and the expression of the inflammatory factors is obviously increased-/-The inflammatory response and the degree of fibrosis in the group of mice increased significantly; at the same time, the WB results show that TRPV1-/-The fibrosis index of group a-SMA expression was significantly increased, the difference was statistically significant (fig. 5F).
The results show that the knockout of TRPV1 aggravates hepatic steatosis and fibrosis of mice in an MCD non-alcoholic fatty liver model, and the TRPV1 is proved to have a protective effect on the hepatic steatosis and the fibrosis.
Example 5 alleviation of MCD induced non-alcoholic fatty liver disease by overexpression of TRPV1
(I) procedure of experiment
Construction of TRPV 1-overexpressed mouse non-alcoholic fatty liver model (MCD induction): 10 male C57/BL mice 8 weeks old were randomized into 2 groups, designated as control group (LV-Con) and TRPV1 virus overexpression group (LV-SMA-TRPV1), each of which was 5 mice. Two groups were fed with MCD feed for 6 weeks; after the mice are fed with MCD feed for 1 week, LV-SMA-TRPV1 group is injected with LV-SMA-TRPV1 virus through caudal vein (1.5 x 10^ per mouse7TU); the LV-Con group was injected with an equal volume of control virus. The material was sacrificed after 6 weeks for relevant testing: weighing the weight and the liver weight of the mouse and calculating the liver-body ratio; detecting biochemical indexes of the mouse serum; pathological detection of sirius red staining and oil red staining; detecting inflammatory factors and fibrosis indexes by PCR; immunoblotting was used to detect protein expression of α -SMA.
(II) results of the experiment
The results are shown in fig. 6, compared with the LV-Con group, the liver weight and the liver volume ratio of the LV-SMA-TRPV1 group mice are significantly reduced, the serum ALT and AST are significantly reduced, and the liver function of the TRPV1 over-expressed mice is significantly improved (C in fig. 6); sirius red staining shows: the control group mice had obvious sirius red staining, while the over-expression group sirius red staining was significantly reduced; oil red staining showed: the control group had more lipid droplets, varied in size and significant in fat deposition, while the TRPV 1-overexpressed group had significantly reduced lipid droplets (A, B in fig. 6); the PCR results show that: compared with the LV-Con group, the expression of fibrosis indexes (alpha-SMA, Col1 (alpha) 1, Desmin, Timp1) and inflammatory factors (Tnf-alpha, Cd68, Il-1 beta, Il-6, Mcp1, Cxcr1 and Cxcl2) of the LV-SMA-TRPV1 group is remarkably reduced, which indicates that the inflammatory response and the fibrosis degree of mice of the LV-SMA-TRPV1 group are remarkably relieved (D, E of figure 6); meanwhile, WB results show: after overexpression of TRPV1, α -SMA expression was significantly down-regulated (F in fig. 6), further demonstrating that overexpression of TRPV1 can reduce liver steatosis, inflammation, and fibrosis in mice in the MCD non-alcoholic fatty liver model.
The results show that the overexpression of the TRPV1 can obviously reduce liver steatosis, inflammation and fibrosis, and the overexpression of the TRPV1 has a protective effect on the liver steatosis and the fibrosis.
Example 6 TRPV1 knockout exacerbates CDAA-induced non-alcoholic fatty liver disease
(I) procedure of experiment
Construction of TRPV1 knockout mouse non-alcoholic fatty liver model (CDAA induction): 6 male C57/BL mice at 8 weeks of age were used as WT group, and 6 male TRPV1 at the same week of age-/-Mouse as TRPV1-/-And (4) grouping. Mice were fed with CDAA (purchased from nanton telofil) feed and sacrificed for 18 consecutive weeks before the relevant tests: detecting the serological indexes of the mice; pathologically detecting the positive expression of stellera chamaejasme red and alpha-SMA immunohistochemistry; detecting inflammatory factors and fibrosis indexes by PCR; immunoblotting was used to detect protein expression of α -SMA.
(II) results of the experiment
The results are shown in FIG. 7 for TRPV1 compared to the WT group-/-Group TG, TC levels were significantly elevated (C of fig. 7); TRPV1-/-The liver of the group is obviously stained by sirius red, and fat vacuoles are obviously increased; a-SMA staining was significantly increased, with the difference being statistically significant (A, B of fig. 7). The PCR test result shows that: TRPV1-/-The fibrosis indexes (alpha-Sma, Col1 (alpha) 1, Col4 (alpha) 1, Desmin, Timp1) and the expression of inflammatory factors (Tnf-alpha, Il-1 beta, Il-6, Cxcr1 and the like) of the mice in the group are obviously increased, and the TRPV1-/-The inflammatory response and the degree of fibrosis in the group mice increased significantly (D, E of fig. 7); at the same time, the WB results show that TRPV1-/-The group had significantly increased α -Sma expression, with the differences being statistically significant (fig. 7F).
The results show that the knockout of TRPV1 aggravates the hepatic fibrosis and fatty degeneration of mice in a CDAA-induced non-alcoholic fatty liver model, and the reverse side proves that TRPV1 has a protective effect on the hepatic fibrosis and fatty degeneration.
Example 7 expression of TRPV1 in alcoholic fatty liver
(I) procedure of experiment
Constructing an alcoholic fatty liver model: male C57/BL mice, 8 weeks old, 12 were randomized into control (Pair-fed) and Alcohol (Alcohol) groups of 6 mice each. The feed is purchased from Nantong Telofu feed science and technology Limited, and liquid feed is prepared according to the instruction: Lieber-Decari control liquid feed (TP4030C) and Lieber-Decari alcohol liquid feed (TP 4030B). Liquid control feed (36% high-fat feed) is fed in the whole molding process of the Pair-fed group, and the method of feeding chronic ethanol for 4 weeks and adding acute ethanol for intragastric administration is adopted in the Alcohol group: control liquid feed was first administered to mice in the Alcohol group for three days, and then the control feed was administered with alcoholic feed at a ratio of 2: 1. 1: 1. 1: 2 proportion for 3 days, and then entering a complete alcohol feed feeding period (4 weeks in total). Intragastric administration on day 32: gavage with 31.5% ethanol in the Alcohol group at a dose of 5 g/kg body weight; the Pair-fed group was gavaged with 45% dextrin at a dose of 9 g/kg body weight, and mice were sacrificed after 9h for relevant testing: immunohistochemical detection of positive expression of TRPV 1; PCR detecting the expression of TRPV1, alpha-Sma and Col1 (alpha) 1 and counting the correlation; WB detects protein expression of TRPV 1.
(II) results of the experiment
As a result, as shown in fig. 8, the expression of TRPV1 protein was detected by immunohistochemistry, and the results showed that: compared with the Pair-fed group, the expression of TRPV1 in the Alcohol group is obviously reduced, and the difference is statistically significant (A in figure 7); the PCR results further show that: the expression of the TRPV1 in the Alcohol group was significantly reduced, the expression of fibrosis indexes α -Sma and Col1(α)1 was significantly increased, and was significantly negatively correlated with the expression of TRPV1 (fig. 8B). The WB results also show: expression of the Alcohol group TRPV1 protein was significantly reduced (C of fig. 8).
The results show that the expression of TRPV1 is down-regulated in alcoholic fatty liver, and the TRPV1 is proved to have a protective effect on alcoholic fatty liver.
The research results show that the over-expression of the TRPV1 gene can improve the inflammation and fibrosis degree of acute liver injury, hepatic fibrosis, non-alcoholic fatty liver and promote the recovery of liver function; the knockout of TRPV1 gene can aggravate acute liver injury, hepatic fibrosis, inflammation and fibrosis degree of non-alcoholic fatty liver and alcoholic fatty liver, and aggravate liver injury. The TRPV1 is proved to have protective effect in acute liver injury, hepatic fibrosis, non-alcoholic fatty liver and alcoholic fatty liver.
In view of this, the present invention provides: TRPV1 is used as a drug target to prepare TRPV1 agonists for preventing, relieving and/or treating acute liver injury, hepatic fibrosis, non-alcoholic fatty liver and alcoholic fatty liver, and comprises an overexpression plasmid of TRPV1 gene, virus-mediated TRPV1 overexpression transfection or other drugs or biological preparations capable of promoting TRPV1 expression.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (7)
- Use of a TRPV1 up-regulator for the preparation of a medicament for the prevention, alleviation and/or treatment of liver disease.
- 2. The use as claimed in claim 1, wherein the TRPV1 up-regulator has the effect of promoting the mRNA level of TRPV1, or increasing the activity or level of TRPV1 protein, or enhancing the effect of TRPV 1.
- 3. The use of claim 1, wherein the up-regulator is selected from the group consisting of a TRPV1 agonist and a TRPV1 overexpression system.
- 4. The use as claimed in claim 3, wherein the TRPV1 overexpression system is a TRPV1 protein overexpression system constructed by using a lentiviral system, an adenoviral system or a plasmid as a vector.
- Use of TRPV1 as a drug target in the screening or manufacture of a medicament for the prevention, alleviation and/or treatment of liver disease.
- 6. The use according to claim 5, wherein the medicament for the treatment of a liver disease is a medicament that enhances the expression of TRPV 1.
- 7. The use of any one of claims 1 to 6, wherein the liver disease comprises acute liver injury, liver fibrosis, non-alcoholic fatty liver disease and alcoholic fatty liver disease.
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