CN117385019A - ALKBH5 as marker and therapeutic target of metabolic diseases - Google Patents
ALKBH5 as marker and therapeutic target of metabolic diseases Download PDFInfo
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- CN117385019A CN117385019A CN202311291056.2A CN202311291056A CN117385019A CN 117385019 A CN117385019 A CN 117385019A CN 202311291056 A CN202311291056 A CN 202311291056A CN 117385019 A CN117385019 A CN 117385019A
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Abstract
The present invention provides a method for detecting, preventing or treating metabolic diseases (including type 2 diabetes, hyperlipidemia, metabolic fatty liver, liver cancer, etc.), which comprises detecting or inhibiting liver ALKBH5. Compared with the existing medicines for treating type 2 diabetes and hyperlipidemia, such as metformin and statin, the method for preventing or treating metabolic diseases (inhibiting liver ALKBH 5) has the advantages of reducing blood sugar and blood lipid, relieving metabolic fatty liver and the like, and can be used for preventing and treating metabolic diseases.
Description
Technical Field
The present invention relates to a method for preventing or treating metabolic diseases (including type 2 diabetes, hyperlipidemia, metabolic fatty liver, liver cancer, etc.), which comprises inhibiting liver ALKBH5. The invention also relates to a medicament for preventing and treating metabolic diseases.
Background
Metabolic diseases include obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver, etc., and metabolic fatty liver promotes the occurrence of liver cancer. Metabolic disease is one of the major diseases threatening human health. The incidence rate of metabolic diseases of adult groups in China is over 50 percent, the metabolic diseases are continuously increased, and the prevention and the treatment of the metabolic diseases become a great strategic requirement in China.
The most currently used hypoglycemic agent is metformin, but metformin cannot reduce blood lipid and relieve non-alcoholic fatty liver; the GLP-1 agonist liraglutide and other medicaments can promote insulin secretion and inhibit appetite, further control blood sugar and weight, and are used for blood sugar and weight management of type 2 diabetes and obesity patients; lipid lowering drugs are mostly statins and PCSK inhibitors, but statins and PCSK inhibitors cannot lower blood glucose and alleviate non-alcoholic fatty liver disease; at present, no effective medicine for treating the metabolic fatty liver exists, and no medicine for simultaneously reducing blood sugar and blood fat and treating the metabolic fatty liver and liver cancer exists. Therefore, new strategies need to be established to develop new drugs to avoid or alleviate the occurrence and development of metabolic diseases and to treat metabolic diseases.
Finding drug targets that can be applied to hyperglycemia, hyperlipidemia, metabolic fatty liver and liver cancer simultaneously is a key to solve this problem, i.e. finding key driving molecules for metabolic disease occurrence.
Human ALKBH5 is a 2-oxoglutarate (2 OG) and ferrous-dependent Nucleic Acid Oxygenase (NAOX), an m6A RNA modified demethylase, having 394 amino acids in its full length, containing the active site motif HXDXnH (including residues His204, asp206 and His 266) for Fe (II) binding, RXXnR for 2OG binding and substrate recognition, and an additional loop (βIV-V) which is considered to have single stranded RNA selectivity (Xu, chao et al J Biol chem.2014;289 (25): 17299-17311). The conserved DSBH core sheet of albh 5 consists of eight antiparallel β -strands β0i—viii (β16-13) which form two β2-sheets: master beta 3-sheet (chains beta 76, 8, 11 and 13) and slave beta-sheet (chains beta 7, 9, 10 and 12). The other three beta-chains, beta 1, beta 2 and beta 3, extend the main beta-sheet group. DSBH is flanked by three helices β41, β52 and β63.DSBH acts as a scaffold for three Fe (II) linking residues His204, asp206 and His266, which constitute the highly conserved hdxnh motif coordinated metal ion; the 2OG binding site is located in a cavity surrounded by β -sheets of two DSBHs, the more open end of which allows interaction between the substrate and the active site; m is m 6 The A base may be in the pocket containing Arg130 and Tyr139, his204 (His 205Ala in mice) is necessary for the demethylase activity of ALKBH5 (Zheng, guanqun et al mol cell 2013;49 (1): 18-29;Aik,WeiShen et al.Nucleic Acids Res.2014;42 (7): 4741-4754.). ALKBH5 is widely present in a number of tissues and expressed relatively high in liver, testis, lung, spleen and kidney. ALKBH5 knockout mice are viable and have problems with sperm development (Zheng, guanqun et al mol cell 2013;49 (1): 18-29). The mechanism of action of ALKBH5 in hepatic glycolipid metabolic homeostasis and metabolic disorders is unclear, nor is it clear whether ALKBH5 is a key driver for metabolic disorders, nor is targeting of hepatic ALKBH5 possible to prevent or treat metabolic disorders even more unclear.
Disclosure of Invention
Through intensive research, the inventor searches for a key driver for metabolic diseases starting from metabolic state changes caused by different eating habits, and finds out that RNA binding protein ALKBH5 is the key driver for metabolic diseases. A novel method for preventing or treating metabolic diseases is established by utilizing liver specific gene knockout or GalNac coupling modified siRNA to inhibit liver ALKBH5. The inventors have established a method of preventing or treating metabolic diseases, inhibition of liver ALKBH5.
Specifically, the inventor finds that ALKBH5 is abnormally high expressed in the liver of mice and human with metabolic diseases, and the specific knockout of the liver of the mice can resist the onset of the metabolic diseases, including reducing blood sugar and blood fat and relieving metabolic fatty liver and liver cancer. These data suggest that liver ALKBH5 is a key driver for metabolic diseases, and inhibition of liver ALKBH5 can avoid or alleviate metabolic diseases. We designed GalNac (N-acetylgalactosamine) coupled modified siRNA, and target db/db mouse liver ALKBH5, and found that db/db mouse type 2 diabetes, hyperlipidemia and metabolic fatty liver are greatly relieved.
Thus, according to one aspect of the present invention there is provided the use of a reagent (alone) for detecting the expression level of ALKBH5 in the manufacture of a diagnostic agent or diagnostic kit for metabolic disorders.
In one embodiment, the reagent is a specific probe, gene chip, PCR primer, or antibody or Western Blotting reagent for detecting the alk bh5 protein.
In one embodiment, the metabolic disease is selected from obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver, liver cancer (hepatocellular carcinoma), or a combination thereof, preferably the combination is a combination of hyperglycemia (type 2 diabetes), hyperlipidemia, and metabolic fatty liver, and/or the agent detects an alk bh5 expression level in the liver of a subject.
According to another aspect of the present invention there is provided the use of an agent that reduces or inhibits the expression of ALKBH5 (either as the sole active ingredient or in combination with other active agents for the treatment of metabolic disorders) in the manufacture of a medicament for the prevention or treatment of metabolic disorders.
In one embodiment, the agent that reduces or inhibits expression of albh 5 is selected from the group consisting of: gapmer, antisense RNA, siRNA, esiRNA, shRNA, miRNA, RNA aptamer, TALEN, CRISPR, zinc finger nuclease, small molecule compound, alk bh 5-specific antibody, and (liver) alk bh5 knockout or knockdown reagent.
In one embodiment, the metabolic disease is selected from obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver, liver cancer (hepatocellular carcinoma) or a combination thereof, preferably the combination is a combination of hyperglycemia (type 2 diabetes), hyperlipidemia and metabolic fatty liver.
In one embodiment, the agent reduces or inhibits the expression level of alk bh5 in the liver of the subject.
In one embodiment, the agent is an N-acetylgalactosamine (GalNAc) coupled modified siRNA, preferably GalNAc-siAlkbh5, the sense and antisense strands (5 '. Fwdarw.3') of which are c-c-accAgAgAgAugcucuavauL (SEQ ID NO: 1) and a-U-cugAagCauagCuGggugg-U-a (SEQ ID NO: 2), respectively, wherein the capital letters represent 2-deoxy-2-fluoro (2-F) modifications, the lowercase letters represent 2-O-methyl (2-OMe) modifications, -PS (phosphothioase, phosphorothioate) linkages, L represents GalNAc linkages.
In one embodiment, the subject is a mammal, preferably a human.
According to another aspect of the present invention, there is provided a method of screening for a drug for preventing or treating a metabolic disease, the method comprising the steps of:
1) Determining the expression level of ALKBH5 in the cell over-expressing ALKBH 5;
2) Contacting the candidate compound with the cells of step 1);
3) Determining the expression level of alk bh5 in the cell after step 2); and
4) Comparing the expression levels of ALKBH5 determined in step 1) and step 3), wherein a decrease in the expression level of ALKBH5 indicates that the candidate compound has the potential to prevent or treat metabolic disorders,
preferably the cells are liver cells, more preferably liver cells from a subject suffering from a metabolic disease, still more preferably the metabolic disease is selected from obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver and liver cancer (hepatocellular carcinoma), preferably the combination is a combination of hyperglycemia (type 2 diabetes), hyperlipidemia and metabolic fatty liver, more preferably the subject is a mammal, most preferably a human.
According to another aspect of the present invention there is provided a method of evaluating the effect of an agent in the treatment and/or prophylaxis of a metabolic disease, wherein the method comprises testing whether the agent is capable of reducing expression of ALKBH5 in a liver cell sample from a subject with a metabolic disease and if so, indicating that the agent is suitable for the treatment and/or prophylaxis of the metabolic disease. In a preferred embodiment, the metabolic disease is selected from obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver and liver cancer (hepatocellular carcinoma), preferably the combination is a combination of hyperglycemia (type 2 diabetes), hyperlipidemia and metabolic fatty liver, more preferably the subject is a mammal, most preferably a human.
The invention has the following advantages: 1) The targeted single molecule ALKBH5 can prevent or treat metabolic diseases, namely, simultaneously reduce blood sugar and blood fat and relieve metabolic fatty liver; 2) The delivery route (GalNac-coupled modified siRNA) has been used clinically, with safety, durability and reliability; 3) Inhibiting liver ALKBH5 has safety.
Compared with the existing hypoglycemic and lipid-lowering drugs, the novel method for preventing or treating the metabolic diseases has the advantages of simultaneously lowering blood sugar and lipid, relieving the metabolic fatty liver, ensuring that the delivery route is safe and durable and effective, ensuring that the target spot has safety and the like, and can be used for preventing or treating the metabolic diseases.
Drawings
Figure 1. Abnormally high expression of alk bh5 in the liver of HFD-induced obese mice;
FIG. 2 shows abnormally high expression of ALKBH5 in db/db mouse liver;
FIG. 3 shows abnormally high expression of ALKBH5 in the liver of human diabetic patients;
FIG. 4.ALKBH5-HKO mice were successfully constructed;
FIG. 5 ALKBH5-HKO mice were euglycemic and resistant to HFD-induced hyperglycemia compared to control mice;
FIG. 6 shows that ALKBH5-HKO mice significantly increased glucose tolerance levels compared to control mice;
FIG. 7 shows a significant reduction in lactate-induced glucose production in ALKBH5-HKO mice compared to control mice;
FIG. 8 shows a significant decrease in glucagon sensitivity in ALKBH5-HKO mice compared to control mice;
FIG. 9.ALKBH5-HKO reduces glucagon signaling pathway;
FIG. 10 further illustrates that ALKBH5-HKO reduces the glucagon signaling pathway, which is responsible for the reduced blood glucose in ALKBH5-HKO mice and is resistant to HFD-induced hyperglycemia;
FIG. 11 shows that serum TG levels were significantly reduced in both normal and HFD-bred mice compared to control mice;
FIG. 12 shows that there was no significant change in serum total cholesterol levels in ALKBH5-HKO mice under normal feeding conditions and that serum total cholesterol levels in ALKBH5-HKO mice were significantly reduced under HFD feeding conditions, as compared to control mice;
FIG. 13 shows that serum low density lipoprotein cholesterol levels of ALKBH5-HKO mice were not significantly altered under normal feeding conditions and were significantly reduced under HFD feeding conditions compared to control mice;
FIG. 14 shows that ALKBH5-HKO mice serum ALT is not significantly different from control mice under normal feeding conditions, but ALKBH5-HKO mice serum ALT levels are significantly reduced from control mice under HFD feeding conditions;
FIG. 15 ALKBH5-HKO mice were able to combat HFD induced fatty liver;
FIG. 16 ALKBH5-HKO mice were resistant to HFD-induced liver lipid accumulation;
FIG. 17 DEN-induced liver cancer model, ALKBH5 flox/flox Compared with mice, the liver weight of ALKBH5-HKO mice is obviously reduced;
DEN-induced ALKBH5 flox/flox The mice have larger liver cancer, the DEN induced ALKBH5-HKO liver cancer is smaller and less,the liver is more normal;
FIG. 19 ALKBH5 induced with DEN flox/flox Compared with the liver cancer of mice, the number of DEN-induced ALKBH5-HKO mice liver tumors is obviously reduced;
FIG. 20 DEN-induced ALKBH5 flox/flox Compared with the liver cancer of the mice, the size of the liver tumor of the DEN-induced ALKBH5-HKO mice is obviously reduced;
FIG. 21 ALKBH5 induced with DEN flox/flox Compared with the mouse liver cancer, DEN-induced ALKBH5-HKO mouse liver cancer Ki-67 positive signals are obviously reduced, and the cell proliferation capacity is obviously weakened;
FIG. 22 ALKBH5 induced with DEN flox/flox Compared with the liver cancer of mice, the content of the liver TG of the liver cancer of the DEN-induced ALKBH5-HKO mice is obviously reduced;
FIG. 23 shows that ALKBH5-HKO mice reduced mTORC1 activity by reducing EGFR and its phosphorylated forms, promote autophagy of the liver, reduce lipid droplet accumulation, and reduce lipid synthesis;
FIG. 24.ALKBH5-HKO reduces EGFR/PI 3K/mTorrC 1 signaling pathway;
FIG. 25 GalNAc-siAlkbh5 can significantly knock down db/db BKS mouse liver ALKBH5;
FIG. 26 shows a significant reduction in fasting blood glucose in GalNAc-siAlkbh5db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 27 shows a significant improvement in glucose tolerance in GalNAc-siAlkbh5db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 28 shows a significant reduction in lactate-induced glucose production in GalNAc-siAlkbh5db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 29 shows that GalNAc-siAlkbh5db/db BKS mice have significantly reduced glucagon sensitivity compared to control GalNAc-siCon db/db BKS mice;
FIG. 30.GCGR signal pathway reduction is a significant cause of GalNAc-siAlkbh5 hypoglycemic;
FIG. 31.GCGR/cAMP signal pathway decrease is a significant cause of GalNAc-siAlkbh5 hypoglycemic;
FIG. 32 shows a significant reduction in serum triglyceride levels in GalNAc-siAlkbh5db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 33 shows that GalNAc-siCon db/db BKS mice have significantly reduced serum total cholesterol levels compared to control GalNAc-siCon db/db BKS mice;
FIG. 34 shows that serum low density lipoprotein cholesterol levels were significantly reduced in GalNAc-siAlkbh5db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 35 serum ALT levels were significantly reduced in GalNAc-siAlkbh5 db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 36 shows a significant decrease in liver TG in GalNAc-siAlkbh5 db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 37 shows a significant reduction in liver lipid droplets in GalNAc-siAlkbh5 db/db BKS mice compared to control GalNAc-siCon db/db BKS mice;
FIG. 38 shows that EGFR/AKT/mTorrC 1 signaling pathway is decreased, lipid synthesis is decreased, and autophagy is increased, which are important causes of the decrease of fatty liver and blood lipid by GalNAc-siAlkbh 5;
FIG. 39 shows that EGFR/PI 3K/mORC 1 signaling pathway is reduced by GalNAc-siAlkbh5, which is a major cause of fatty liver and blood lipid lowering.
Detailed Description
Unless otherwise indicated, terms used herein have the ordinary technical meaning as understood by those skilled in the art. For definitions and terms in the art, reference is made to Sambrook et al, molecular Cloning: ALaboratory Manual, version 2, cold Spring Harbor Press, plainsview, new York (1989); and Ausubel et al CurrentProtocols in Molecular Biology (supply 47), john Wiley & Sons, new York (1999).
The term "ALKBH5 (Alkylation repair homolog protein) refers to a 2-oxoglutarate (2 OG) and ferrous-dependent Nucleic Acid Oxygenase (NAOX), an m6A RNA modified demethylase.
With respect to expression of ALKBH5, it is meant expression at three levels thereof: one is expression at the DNA level; secondly, expression at the RNA level; and thirdly, expression at the protein level.
The term "overexpression" refers to the fact that a gene may not be properly "turned off" or transcribed at a high rate when the strict control of gene expression (transcription) is disturbed. High-speed transcription results in the production of large amounts of mRNA. By over-expression of ALKBH5 in the present invention is meant that its DNA, RNA or protein expression level is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 200% or 300% higher than that of the control (normal or healthy tissue/cell), or even 4, 5, 6, 7, 8, 9, 10 times or more the expression level of ALKBH5 in the control.
Techniques and reagents for detecting gene expression levels are well known to those skilled in the art. In the present invention, it is preferable that the reagent is selected from a specific probe (preferably a nucleic acid probe, with a detection label, usually complementary to the gene of interest) of the ALKBH5 gene, a gene chip or a PCR primer for PCR-specific amplification reaction.
Techniques and reagents for detecting protein expression levels are well known to those skilled in the art. In the present invention, it is preferable that the reagent is selected from an antibody or a Western Blotting reagent for detecting ALKBH5 protein.
The term "reducing or inhibiting expression of ALKBH 5" refers to reducing the expression level of DNA, RNA or protein of ALKBH5 to 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, or 10% or less, for example 5% or less, 2% or less, 1% or less, or even 0% of the original expression level. In one embodiment, expression of ALKBH5 may be reduced or inhibited by gene knockout or knockdown.
The term "knockout" refers to a genetic engineering technique in which an endogenous normal homologous gene is replaced by homologous recombination using an exogenous mutated gene, thereby inactivating the endogenous gene to express the mutant's trait.
The term "knockdown" refers to the effect of preventing gene expression by degrading mRNA of a target gene having a homologous sequence. The double-stranded small RNA is utilized to efficiently and specifically degrade homologous mRNA in cells, so that the expression of target genes in the body is blocked, and the cells have a target gene deletion phenotype. It is different from gene knockout to make the target gene permanently expressed and silenced, but can be used for preventing gene expression by degrading mRNA of target gene with homologous sequence.
Techniques for gene knockout or knockdown are well known in the art and include, but are not limited to, retroviral gene transfer, resulting in mutations such as point mutations, insertions, deletions, frameshifts, or missense mutations. Another way in which the gene can be knocked out is by using zinc finger nucleases. Zinc Finger Nucleases (ZFNs) are artificial restriction enzymes produced by fusing a zinc finger DNA binding domain to a DNA cleavage domain. The zinc finger domain can be engineered to target DNA sequences of interest, which can target zinc finger nucleases to unique sequences in complex genomes. Other genomic customization techniques that can be used to knock out genes are TAL effector nucleases (TALENs). Another technique is the genome editing technique CRISPR/Cas system, which can be used to achieve RNA-guided genome engineering.
Techniques to achieve "reduced or inhibited expression of ALKBH 5" may also include the use of gapmers, antisense RNA, siRNA, esiRNA, shRNA, miRNA, RNA aptamers, or ALKBH 5-specific antibodies.
"antisense RNA" refers to RNA molecules that are complementary to mRNA, and also includes RNA molecules that are complementary to other RNAs. Since ribosomes are unable to translate double-stranded RNA, antisense RNA binds specifically to mRNA complementarily, i.e., inhibits translation of the mRNA. The antisense construct can be delivered, for example, as an expression plasmid that, when expressed in a cell, produces RNA that is complementary to at least one unique portion of cell (preferably liver cell) alk bh 5.
Another particular form of antisense RNA strategy is gapmer. Gapmer is a chimeric antisense oligonucleotide comprising a central segment (central block) of deoxynucleotide monomers of sufficient length to induce cleavage by RNase H. The design and synthesis of gapmers is well known to those skilled in the art and can be accomplished by commercial companies (e.g., exiqon, isis pharmaceuticals).
"Small interfering RNAs (siRNAs), sometimes referred to as short interfering RNAs or silencing RNAs, are a class of double-stranded RNA molecules that are about 20-25 base pairs in length and function via an RNA interference (RNAi) pathway. It interferes with post-transcriptional degraded mRNA of a specific gene expressing a nucleotide sequence complementary to it, thereby preventing translation. The siRNA of the present invention may target any segment of about 19 to 25 contiguous nucleotides in the target sequence of the albh 5 gene, examples of which are provided herein. Techniques for selecting target sequences for siRNA are well known in the art. GalNAc-coupled modified siRNA has been widely used in clinic and proved to be a safe and effective method for inhibiting liver targets. Preferably, the present invention uses GalNac (N-acetylgalactosamine) coupling modified siRNA.
"short hairpin RNA" (short hairpin RNA, abbreviated shRNA) is an RNA sequence comprising two short inverted repeats that can silence gene expression via RNA interference (RNAi).
The full name of "esiRNA" is endonucleonucleotide-prepared siRNAs, which is an siRNA mixture generated by cutting long double-stranded RNA (dsRNA) by RNase III (a ribonuclease) of escherichia coli, has a length of 18-25bp, and can be used for efficiently knocking out the expression level of a target gene.
The present invention is based on the following unexpected findings: namely ALKBH5 can be used as a marker and a treatment target of metabolic diseases. Therefore, the invention provides application of a reagent for detecting ALKBH5 expression level in preparation of a diagnostic agent or a diagnostic kit for metabolic diseases. The invention also provides application of the reagent for reducing or inhibiting ALKBH5 expression in preparation of medicaments for treating metabolic diseases. In addition, the present invention provides a method of screening for a drug for the treatment of metabolic disorders, the method comprising the step of determining whether a candidate compound is capable of reducing or inhibiting expression of ALKBH5 in liver cells.
The invention will now be described in further detail with reference to the drawings and examples, which are not intended to limit the scope of the invention. The chemicals used in the following reactions are all commercially available products unless otherwise indicated.
Unpaired student's t-test was used as a statistical analysis in the present invention. Statistical calculations were performed using Microsoft Excel. When P <0.05, the P value is significant.
Example 1: ALKBH5 induces expression in the liver of obese mice in HFD.
After normal feed (rat maintenance feed MD17121, jiangsu middleson) was fed for 8 weeks, HFD high fat feed (PD 6001, one-mouse two biotechnology limited, constant state) was replaced for 12 male mice (C57 BL/6J, beijing villous laboratory animal technologies limited, 42.33± 5.355 g) fed for 8 weeks and 16 male mice (C57 BL/6J, beijing villous laboratory animal technologies limited, 32.125 ±2.775 g) fed for 16 weeks with normal feed, starved for 20 hours, sacrificed, liver collected, and Western Blot examined for alk bh5 expression.
[ materials and methods ]:
HFD raised 8 week male mice and normal feed raised the same week old mice.
The Western Blot details are as follows:
1) Early preparation
Preparing the protein adhesive: preparing separating gel according to the required concentration, adding 1ml of 20% ethanol liquid seal, standing at room temperature until solidification, pouring out ethanol, wiping, pouring concentrated gel, and inserting a comb according to the experiment requirement.
Preparing an electrophoresis liquid: 200ml 5 XTris/Gly, 800ml dH 2 O,10ml of 10% SDS was stored at room temperature for further use.
Preparing a transfer film liquid: 200ml 5 XTris/Gly, 200ml methanol, 600ml dH 2 O, precooling at 4 ℃ for standby.
Preparing a sealing liquid: 5g of skimmed milk powder is added into 95ml of PBST, and the mixture is uniformly mixed and stored at 4 ℃ for standby.
Protein sample preparation: taking liver tissue, adding 10 times volume of RIPA Buffer, grinding, performing ice lysis for 30min, performing votex 1 time every 10min, centrifuging at 4deg.C for 10min at 12000rmp/min, transferring supernatant to new EP tube, adding Loading Buffer, and decocting at 100deg.C for 5min.
2) Electrophoresis
And respectively adding a marker and a sample into the sample loading hole slowly and uniformly, carrying out 100V constant-pressure electrophoresis, and determining the electrophoresis time according to the molecular weight of the target protein.
3) Transfer film
The membrane transferring clamp is clamped according to the sequence of black surface, foam cushion, filter paper, albumin glue, NC membrane, filter paper, foam cushion and white surface, air bubbles are emptied, membrane transferring is carried out at constant pressure of 100V, and membrane transferring time is determined according to the molecular weight of target protein.
4) Closure
NC membrane was immersed in the blocking solution, and blocked for 1 hour at room temperature with a horizontal shaker at 10 rpm/min.
5) First antibody
The blocking solution was decanted, the blocking solution was washed with PBST, the primary antibody (anti-ALKBH 5, proteintech, 1:5000) was diluted with antibody dilution (PBST solution containing 3% BSA), primary antibody was added, incubated overnight at 10rpm/min 4℃on a horizontal shaker, and the PBST membranes were recovered and washed 3 times for 10min each.
6) Secondary antibody
The secondary antibody (horseradish enzyme labeled goat anti-mouse IgG (H+L), zhonghua gold bridge, 1:5000 dilution) was diluted with blocking solution, horizontal shaker 10rpm/min room temperature secondary antibody 1H, PBST washed 3 times for 10min each.
7) Color development
And uniformly dripping a developing solution on the NC film, and developing by using a chemiluminescent imager.
[ results are shown in FIG. 1]
Results (results): compared to normal feed-fed mice, alk bh5 is abnormally high expressed in the liver of HFD-induced obese mice.
Example 2: expression of ALKBH5 in the liver of db/db mice.
10 male mice at 8 weeks of age db/db (diabetes) and 10 male mice at 8 weeks of age control wild type. Purchased from karwensi laboratory animals, inc., starvation for 20 hours, sacrifice, liver collection, western Blot detection of alk bh5 expression.
[ materials and methods ]:
db/db mice and control wild-type mouse livers.
The Western Blot procedure is detailed in example 1.
See fig. 2 for results:
results (results): compared with wild type mice, ALKBH5 is abnormally high expressed in the liver of db/db mice.
Example 3: expression of ALKBH5 in human diabetic liver.
7 groups of liver tissues of human liver cancer patients, diabetes patients and control liver cancer patients are respectively from university of Harbin medical science, and Western Blot is used for detecting ALKBH5 expression.
[ materials and methods ]:
the Western Blot procedure is detailed in example 1.
See fig. 3 for results:
results (results): ALKBH5 is abnormally high expressed in the liver of human diabetics.
Example 4: construction of ALKBH5-HKO mice.
[ materials and methods ]:
ALKBH5 flox/flox mice (commercially available from Jiangsu Jiugang Biotech Co., ltd.) were hybridized with Alb-Cre mice (commercially available from Jiangsu Jiugang Biotech Co., ltd.). As shown in FIG. 4, A, loxP sequences (ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT) (SEQ ID NO: 3) are added to both ends of a wild mouse Alkbh5 sequence (NC_ 000077.7) by CRISPR/CAS9 technology, and the expression of ALKBH5 is not affected under normal conditions, namely ALKBH5 flox/flox And (3) a mouse. When ALKBH5 flox/flox After hybridization of mice with Alb-Cre mice (mice that express Cre enzyme specifically in the liver), cre enzyme can cleave off the LoxP site, resulting in specific knockout of alk bh5 in the liver, i.e., alk bh5-HKO mice.
The specific hybridization strategy is: ALKBH5 flox/flox Mouse and Alb-Cre +/- Mice are hybridized to obtain ALKBH5 flox/- Alb-Cre +/- Mice, which are associated with ALKBH5 flox/flox Backcrossing the mice to obtain ALKBH5 flox/flox Alb-Cre +/- The mice are ALKBH5-HKO mice. ALKBH5-HKO mice and ALKBH5 flox/flox The mice are hybridized to obtain the ALKBH5-HKO mice and ALKBH5 with equal amounts flox/flox Mice (control mice).
The gene identification method is as follows:
ALKBH5-HKO mice (genotype: ALKBH 5) flox/flox Alb-Cre +/- ): ALKBH5 primer PCR reaction yielded a single 411bp band, and Alb-cre primer PCR yielded a single 250bp band.
ALKBH5 flox/flox Mouse (genotype: ALKBH 5) flox/flox Alb-Cre -/- ): ALKBH5 primer PCR reactions gave a single 411bp band, and Alb-cre primer PCR did not give a single 250bp band.
ALKBH5 flox/- Alb-Cre +/- Mice: ALKBH5 primer PCR reaction gave 307bp and 411bp bands, and Alb-cre primer PCR gave a single 250bp band.
The ALKBH5 mouse gene identification primers are as follows:
ALKBH5-F:ACCTGGAGTCCCATAAGATTCATCC(SEQ ID NO:4)
ALKBH5-R:TTCAGGTCAGTTGGGATTACCAGG(SEQ ID NO:5)
the Alb-cre mouse gene identification primers are as follows:
Cre-F:GCATAACCAGTGAAACAGCATTGCTG(SEQ ID NO:6)
Cre-R:GGACATGTTCAGGGATCGCCAGGCG(SEQ ID NO:7)
the results of the gene identification are shown in FIG. 4, B: ALKBH5 -/- The mice are 307bp single bands, no cre bands and ALKBH5 flox/- Mice were 307bp and 411bp bands and single cre band, and ALKBH5-HKO mice were 411bp single band and single cre band.
ALKBH5-HKO mice were verified by Western Blot, which is described in example 1 (hereinafter the same) and the results are shown in FIGS. 4 and C.
See fig. 4 for results:
results (results): ALKBH5-HKO mice were successfully constructed.
Example 5: effects of ALKBH5-HKO on blood glucose in mice.
Normal feed (rat maintenance feed MD17121, jiangsu meidi sen) raised the alk bh5-HKO male rats obtained in example 4 and control alk bh5 flox/flox Male mice were starved for 6 hours (8:00-14:00 a) 7 to 8 weeks old each, and blood glucose was measured.
Normal feed (rat maintenance feed MD17121, jiangsu meidi sen) feeding the alk bh5-HKO mice obtained in example 4 and control alk bh5 flox/flox Mice were kept for a further 12 weeks with HFD feed (PD 6001, changzhou mouse-two Biotech Co., ltd.) 6 weeks later, starved for 6 hoursBlood glucose was measured at time (8:00-14:00 a).
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Glucometer (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury, qiangsheng).
See fig. 5 for results:
results (results): compared to control mice, alk bh5-HKO mice were euglycemic and resistant to HFD-induced hyperglycemia.
Example 6: effects of ALKBH5-HKO on glucose tolerance in mice.
7 male mice of ALKBH5-HKO and control ALKBH5 were bred according to the breeding method in example 5 flox/flox Male mice were 8 to 8 weeks old and were fed with HFD feed ALKBH5-HKO male and control ALKBH5 flox/flox The male mice were starved for 6 hours (8:00-14:00 a) for 12 weeks each, and glucose tolerance experiments were performed.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Glucometer (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury, qiangsheng).
Glucose (G116302, aladin), sodium chloride injection (Harbin triple pharmaceutical Co., ltd.) and sterile syringes (1 mL, jiangsu medical devices Co., ltd.).
The detailed method for glucose tolerance test is as follows:
1) 0 point blood glucose test
After 6 hours of starvation, the mouse was gently sheared at the tip of the tail, a drop of filled blood was gently squeezed out, and blood glucose and body weight were recorded by blood glucose meter.
2) Tolerance test
A glucose solution of 0.1g/ml was prepared with physiological saline, and the solution was injected at a dose of 1g/kg (e.g., 200. Mu.L for 20g mice) according to the weight of the mice, and blood glucose was measured 15min, 30min, 60min, and 120min after the injection, respectively, and recorded.
See fig. 6 for results:
results (results): under normal and high fat diet, alk bh5-HKO mice significantly increased glucose tolerance levels compared to control mice.
Example 7: effect of ALKBH5-HKO on lactic acid tolerance in mice.
7 male mice of ALKBH5-HKO and control ALKBH5 were bred according to the breeding method in example 5 flox/flox Male mice were 8 to 8 weeks old and were fed with HFD feed ALKBH5-HKO male and control ALKBH5 flox/flox Male mice were starved for 6 hours (8:00-14:00 a) for 12 weeks each, and lactic acid tolerance experiments were performed.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Glucometer (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury, qiangsheng).
Sodium lactate (71718, sigma), sodium chloride injection (haerbin trigeminy pharmaceutical products Co., ltd.) and sterile syringes (1 mL, jiangsu medical devices Co., ltd.).
The detailed method for lactic acid tolerance test is as follows:
1) 0 point blood glucose test
After 6 hours of starvation, the mouse was gently sheared at the tip of the tail, a drop of filled blood was gently squeezed out, and blood glucose and body weight were recorded by blood glucose meter.
3) Tolerance test
A sodium lactate solution (0.1 g/ml) was prepared with physiological saline, and the solution was injected at a dose of 1g/kg (e.g., 200. Mu.L for 20g mice) according to the weight of the mice, and blood glucose was measured 15min, 30min, 60min, and 120min after the injection, respectively, and recorded.
See fig. 7 for results:
results (results): in the case of normal diet and high fat diet, alk bh5-HKO mice had significantly reduced glucose production induced by lactic acid compared to control mice.
Example 8: effects of ALKBH5-HKO on glucagon tolerance in mice.
7 male mice of ALKBH5-HKO and control ALKBH5 were bred according to the breeding method in example 5 flox/flox Male mice were 8 to 8 weeks old and were fed with HFD feed ALKBH5-HKO male and control ALKBH5 flox/flox Male mice were starved for 6 hours (8:00-14:00 a) for 12 weeks each, and glucagon tolerance experiments were performed.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Glucometer (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury, qiangsheng).
Glucagon (GP 21258, GLPBIO), sodium chloride injection (halbine trigeminy pharmaceutical products inc.) and sterile syringes (1 mL, jiangsu medical devices inc.).
The detailed method for glucagon tolerance experiments is as follows:
1) 0 point blood glucose test
After 6 hours of starvation, the mouse was gently sheared at the tip of the tail, a drop of filled blood was gently squeezed out, and blood glucose and body weight were recorded by blood glucose meter.
4) Tolerance test
A glucagon solution of 1. Mu.g/ml was prepared with physiological saline and injected at a dose of 10. Mu.g/kg according to the body weight of the mice (e.g., 200. Mu.L for 20g mice), and blood glucose was measured 15min, 30min, 60min, 120min after the injection, respectively, and recorded.
See fig. 8 for results:
results (results): under normal and high fat diet, alk bh5-HKO mice had significantly reduced glucagon sensitivity compared to control mice.
Example 9: effect of ALKBH5-HKO on the mouse GCGR/PKA/CREB signaling pathway.
ALKBH5-HKO male mice and control ALKBH5 were bred according to the breeding method in example 5 flox/flox Male mice were sacrificed 7 to 8 weeks old each, livers were collected, western Blot.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Western Blot detects expression of glucagon receptor (Glucagon Receptor, GCGR) (GCGR, proteintech, 26784-1-AP), PKA Substrate phosphorylation level (Phospho- (Ser/Thr) PKA Substrate, CST, 9621), cAMP response element binding protein (CREB, proteintech, 12208-1-AP) and phosphorylated form p-CREB (Phospho-CREB (Ser 133), CST, 9198) as described in example 1.
See fig. 9 for results:
results (results): in normal and HFD-bred ALKBH5-HKO mice, liver GCGR, p-PKA Sub and p-CREB levels were significantly reduced. It was demonstrated that ALKBH5-HKO reduced the glucagon signaling pathway.
Example 10: effects of ALKBH5-HKO on mouse cAMP.
ALKBH5-HKO male mice and control ALKBH5 were bred according to the breeding method in example 5 flox/flox Male mice were sacrificed 7 to 8 weeks old each, livers were collected and assayed for cAMP content.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
The cAMP ELISA kit (581001, cayman) was tested as follows: the kit is restored to room temperature
1) Preparation of buffer
ELISA Buffer: 1 bottle of 10mL ELISA Buffer (10X) was added to 90mL ddH 2 And (3) mixing uniformly, and storing at 4 ℃ for later use.
Wash Buffer: 2.5mL Wash Buffer was added to 997mL ddH 2 Adding 0.5ml polysorbate into O, mixing, and storing at 4deg.C.
cAMP AchE tracker: cAMP AchE Tracer was taken, 30mL ELISA Buffer was added, and 300. Mu.L Tracer was added and stored at 4℃for further use.
cAMP ELISA Antiserum: cAMP ELISA Antiserum, 30mL ELISA Buffer was added, 300. Mu.L of tracer was added, and stored at 4℃for further use.
Ellman's Reagent: 1 tube Ellman's Reagent was dissolved in 50mL ddH 2 O, the existing preparation and the light shielding.
2) Sample preparation
Taking 20mg liver sample, adding 200 μl of 0.1M HCl, grinding, cracking on ice for 10min, adding 400 μl of ELISA Buffer, voltex, centrifuging at 4deg.C for 10min, transferring supernatant to new EP tube, and placing in ice box for use.
3) Detection of
The required number of ELISA plates were taken and Blk, TA, NSB, B and Standard, sample wells were set. Sequentially adding according to the following volume:
Blk: blank space
TA: blank space
NSB:100μL ELISA Buffer,50μL Tracer
B0:50μL ELISA Buffer,50μL Tracer,50μL Antiserum
Standard:50μL Standard,50μL Tracer,50μL Antiserum
Sample:50μL Sample,50μL Tracer,50μL Antiserum
Cover the sealing plate film and incubate in 4 ℃ wet box for 18h.
The well plate samples were spun dry and patted dry on a clean paper towel.
200 μl of Wash Buffer was added to each well, gently shaken, the liquid was spun dry and patted dry on paper towels, and repeated 5 times.
200. Mu.L of Ellman' Reagent was added to each well, while 5. Mu.L of Tracer was added to the TA wells.
Covering a sealing plate film, avoiding light, developing by using a horizontal shaking table at 10rpm/min, reading a light absorption value of 405/420nm at any time, and cleaning again if the light absorption value of B0 is more than 1.5 and is within the range of 0.3-1.
Drawing a standard curve by using a standard substance, taking in a sample value, and measuring the protein concentration of the sample by using a BCA method, wherein the ratio is the cAMP content.
See fig. 10 for results:
results (results): liver cAMP levels were significantly reduced in normal and HFD-bred alk bh5-HKO mice. Further illustrating that ALKBH5-HKO reduces the glucagon signaling pathway, which is responsible for the reduced blood glucose in ALKBH5-HKO mice and is resistant to HFD induced hyperglycemia.
Example 11: effects of ALKBH5-HKO on serum triglyceride levels in mice.
ALKBH5-HKO mice and control ALKBH5 were bred according to the breeding method in example 5 flox/flox Mice were sacrificed, blood was collected, centrifuged at 4000rpm/min at 4℃for 10min, and the supernatant, i.e., serum, was transferred and frozen at-80℃for further use.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Triglyceride (TG) test boxes (F001-1-1, built in Nanjing) were tested according to the methods listed in the kit instructions.
Serum is taken out from a refrigerator at the temperature of minus 80 ℃, melted on ice, and is evenly mixed with votex, immediately separated and placed in an ice box for standby.
TG determination: 2.5. Mu.L of dH was added to each 96-well plate 2 O, standard substance and serum to be detected are added with 250 mu L of detection liquid, mixed by gentle shaking, reacted for 10min at 37 ℃ and read the light absorption value of 510 nm.
The calculation method comprises the following steps: standard, determination of serum absorbance-dH 2O absorbance, recorded as delta Standard substance And delta Serum ,△ Serum /△ Standard substance * The standard concentration is the serum TG level.
See fig. 11 for results:
results (results): serum TG levels were significantly reduced in both normal and HFD-bred alk bh5-HKO mice compared to control mice.
Example 12: effect of ALKBH5-HKO on the serum total cholesterol content of mice.
ALKBH5-HKO mice and control ALKBH5 were bred according to the breeding method in example 5 flox/flox And (3) a mouse. Serum was prepared as in example 11.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Total cholesterol (T-CHO) test boxes (A111-1-1, built in Nanjing) were tested in the exact manner set forth in the kit instructions.
T-CHO assay: 2.5. Mu.L of dH was added to each 96-well plate 2 O, standard substance and serum to be detected are added with 250 mu L of detection liquid, mixed by gentle shaking, reacted for 10min at 37 ℃ and read the light absorption value of 500 nm.
The calculation method comprises the following steps: standard, determination of serum absorbance-dH 2O absorbance, recorded as delta Standard substance And delta Serum ,△ Serum /△ Standard substance * The standard concentration is the serum T-CHO level.
See fig. 12 for results:
results (results): the serum total cholesterol level of the ALKBH5-HKO mice did not significantly change under normal feeding conditions compared to control mice, and was significantly reduced under HFD feeding conditions.
Example 13: effects of ALKBH5-HKO on mouse serum Low Density lipoprotein cholesterol levels.
ALKBH5-HKO mice and control ALKBH5 were bred according to the breeding method in example 5 flox/flox And (3) a mouse. Serum was prepared as in example 11.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
A kit for measuring low density lipoprotein cholesterol (LDL-C) (A113-1-1, built in Nanjing) is used for detection according to the method listed in the specification of the kit.
LDL-C assay: 2.5. Mu.L of dH was added to each 96-well plate 2 O, a standard substance and serum to be detected are added into 180 mu L of a first detection solution of the kit, mixed by light shaking, reacted for 5min at 37 ℃, read the light absorption value of 546nm, recorded as A1, added into 60 mu L of a second detection solution of the kit, mixed by light shaking, reacted for 5min at 37 ℃, read the light absorption value of 546nm, recorded as A2.
The calculation method comprises the following steps: Δa=a2-A1, standard, measured serum absorbance-dH 2O absorbance, recorded as Δa Standard substance And DeltaA Serum ,△A Serum /△A Standard substance * The standard concentration is the serum LDL-C level.
See fig. 13 for results:
results (results): the serum low-density lipoprotein cholesterol level of the ALKBH5-HKO mice is not significantly changed under normal feeding conditions compared with the control mice, and is significantly reduced under HFD feeding conditions.
Example 14: effects of ALKBH5-HKO on mouse serum ALT.
ALKBH5-HKO mice and control ALKBH5 were bred according to the breeding method in example 5 flox/flox And (3) a mouse. Serum was prepared as in example 11.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
GPT (ALT) kit (C009-2-1, built in Nanjing).
Serum is taken out from a refrigerator at the temperature of minus 80 ℃, melted on ice, and is evenly mixed with votex, immediately separated and placed in an ice box for standby. The kit is restored to room temperature, and the matrix solution is preheated at 37 ℃.
Control wells: 10. Mu.L of matrix solution was added to the 96-well plate.
Experimental hole: after 10. Mu.L of matrix solution was added to the 96-well plate, 2.5. Mu.L of serum was added thereto, and the mixture was pipetted and homogenized.
Incubate at 37℃for 30min.
10 mu L of phenylhydrazine solution is added into the control hole and the experimental hole respectively, the mixture is gently shaken and mixed, 2.5 mu L of serum corresponding to the experimental hole is added into the control hole, and the mixture is sucked and beaten and mixed.
Incubate at 37℃for 20min.
100 mu L of 0.4M NaOH is added into the control hole and the experimental hole respectively, the mixture is gently shaken and mixed uniformly, the mixture is kept stand for 15min at room temperature, and the light absorption value of 510nm is read.
The calculation method comprises the following steps: and (V) Measurement value Experimental Kong Xiguang value-control Kong Xiguang value, will delta Measurement value The introduced standard yeast is serum ALT (serum alanine aminotransferase) level.
See fig. 14 for results:
results (results): there was no significant difference in serum ALT from the alk bh5-HKO mice compared to control mice under normal feeding conditions, but the alk bh5-HKO mice serum ALT levels were significantly reduced compared to control mice under HFD feeding conditions.
Example 15: effects of ALKBH5-HKO on mouse liver TG.
ALKBH5-HKO mice and control ALKBH5 were bred according to the breeding method in example 5 flox/flox Mice were sacrificed, livers were collected, and changes in liver TG were detected.
[ materials and methods ]:
normal feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox Mice, HFD-feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
The liver TG (triglyceride) detection detailed method is as follows:
1) TG separation
60mg of liver tissue was taken, 1.5mL of 1% acetic acid was added thereto, and the mixture was homogenized.
Transfer 200. Mu.L of homogenate to a fresh EP tube and add 800. Mu.L of chloroform/methanol (2:1) mixture.
Votex 4-6 times, 30s each time, standing for 5min at room temperature, centrifuging 10000g at room temperature for 10min.
Transfer the lower organic solution of the EP tube to a new EP tube, place in a fume hood and dry overnight.
2) Lipolysis
200. Mu.L of 3M KOH was added to the dried EP tube and resuspended and incubated at 70℃for 1 hour.
Cooled to room temperature and 600. Mu.L of 1M MgCl is added 2 Volex, incubate on ice for 10min, centrifuge for 10min at 12000rpm/min, transfer supernatant to new EP tube for use.
3) TG detection
Add 40mL ddH to glycerol reagent (F6428, sigma) 2 And O, gently reversing, mixing uniformly, shading, subpackaging and storing at-20 ℃ for later use.
mu.L of ddH was added to each 96-well plate 2 O、4μL ddH 2 O+4mu L standard substance, 8mu L standard substance and 8mu L sample to be detected are added with 80 mu L glycerol reagent, mixed by gentle shaking, incubated for 15min at room temperature, and the absorbance at 540nm is read.
And (3) preparing standard curve by taking the 540nm absorbance value of the standard product as an X axis and the glycerol concentration as a Y axis, and bringing the 540nm absorbance value of the sample to be tested into the standard curve to obtain the glycerol concentration.
4) Calculation of
The calculation formula is as follows: (0.07687) glycerol concentration)/liver weight to obtain liver TG level (mg/g).
See fig. 15 for results:
results (results): there was no significant difference in the liver TG of the alk bh5-HKO mice compared to control mice under normal feeding conditions, but the liver TG levels of the alk bh5-HKO mice were significantly reduced compared to control mice under HFD feeding conditions. This suggests that ALKBH5-HKO mice are resistant to HFD induced fatty liver.
Example 16: effect of ALKBH5-HKO on mouse liver lipid accumulation.
The ALKBH5-HKO mice obtained in example 4 and the control ALKBH5 were fed with normal feed flox/flox Mice were kept on HFD feed for 12 weeks after 6 weeks, starved for 20 hours, sacrificed, livers were collected, 4% paraformaldehyde solution was fixed for 4 hours, and H was performed&E, dyeing and photographing.
[ materials and methods ]:
HFD feed-raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
Study grade inverted fluorescence microscope imager (IX 71+DP74, olympus) was photographed.
See fig. 16 for results:
results (results): under HFD feeding conditions, the liver lipid droplet size was significantly reduced in ALKBH5-HKO mice compared to control mice. This suggests that ALKBH5-HKO mice are resistant to HFD-induced liver lipid accumulation.
Example 17: effects of ALKBH5-HKO on Diethylnitrosamine (DEN) induced liver cancer.
ALKBH5-HKO mice obtained in example 4 and control ALKBH5 flox/flox Mice were injected with diethylnitrosamine (Sigma, 55-1-8-5, 50 mg/kg) at 14 days, fed with normal feed for 8 weeks, fed with HFD feed for 45 weeks, induced mice to develop liver cancer, sacrificed and the livers collected.
[ materials and methods ]:
diethylnitrosamine (Sigma, 55-1-8-5)
ALKBH5-HKO mice and control ALKBH5 flox/flox A mouse
Electronic analytical balance (ESJ-B, dragon electronic)
See fig. 17 for results:
[ result]: DEN-induced liver cancer model, ALKBH5 flox/flox Compared with mice, the liver weight of ALKBH5-HKO mice is obviously reduced.
Example 18: effects of ALKBH5-HKO on the morphology of Diethylnitrosamine (DEN) induced liver cancer.
DEN-induced ALKBH5-HKO and control ALKBH5 were obtained as in example 17 flox/flox Liver cancer mice were sacrificed, livers were collected, and photographed.
[ materials and methods ]:
DEN-induced ALKBH5-HKO and control ALKBH5 flox/flox Liver cancer mice
See fig. 18 for results:
[ result]: DEN-induced ALKBH5 flox/flox The mice have larger liver cancer, the DEN induced ALKBH5-HKO liver cancer is smaller and less, and the liver is more normal.
Example 19: effect of ALKBH5-HKO on the number of Diethylnitrosamine (DEN) -induced hepatoma tumors.
DEN-induced ALKBH5-HKO and control A were obtained as in example 17LKBH5 flox/flox Liver cancer mice were sacrificed, livers were collected, and photographed.
[ materials and methods ]:
DEN-induced ALKBH5-HKO and control ALKBH5 flox/flox Liver cancer mice
See fig. 19 for results:
[ result]: ALKBH5 induced with DEN flox/flox Compared with the liver cancer of mice, the number of DEN-induced ALKBH5-HKO mice liver tumors is obviously reduced.
Example 20: effect of ALKBH5-HKO on Diethylnitrosamine (DEN) induced liver cancer tumor size.
DEN-induced ALKBH5-HKO and control ALKBH5 were obtained as in example 17 flox/flox Liver cancer mice were sacrificed, livers were collected, and photographed.
[ materials and methods ]:
DEN-induced ALKBH5-HKO and control ALKBH5 flox/flox Liver cancer mice
See fig. 20 for results:
[ result]: ALKBH5 induced with DEN flox/flox Compared with the liver cancer of the mice, the size of the liver tumor of the DEN-induced ALKBH5-HKO mice is obviously reduced.
Example 21: effect of ALKBH5-HKO on Diethylnitrosamine (DEN) to induce proliferation of liver cancer hepatic parenchymal cells in mice.
DEN-induced ALKBH5-HKO and control ALKBH5 were obtained as in example 17 flox/flox Liver cancer mice were sacrificed, livers were collected, sectioned, immunofluorescent stained, and photographed.
[ materials and methods ]:
DEN-induced ALKBH5-HKO and control ALKBH5 flox/flox Liver cancer mice
Study grade inverted fluorescence microscope imager (IX 71+DP74, olympus) was photographed.
The Ki-67 immunofluorescence staining method is as follows:
1) Fixing
The cut slide was taken out of the refrigerator at-80 ℃, dried in the air, fixed with 4% paraformaldehyde solution for 30 minutes, washed with PBS buffer solution, and permeabilized.
2) Permeabilization
The fixed slide was immersed in a permeabilizing solution (PBS buffer containing 0.5% Triton-X100 and 0.05% SDS), permeabilized at room temperature for 30 minutes, washed with PBS buffer, and blocked.
3) Closure
Surrounding the tissue with an immunohistochemical pen, 10. Mu.L of blocking solution (PBS buffer containing 5% goat serum and 1% BSA) was added dropwise to the surrounding tissue, and the mixture was placed in a wet box, blocked at room temperature for 2 hours, washed with PBS buffer, and subjected to primary antibody.
4) First antibody
Ki-67 (1:200, CST, D385) was diluted with blocking solution in a certain ratio, 10. Mu.L of the prepared primary antibody was added dropwise in circles, placed in a wet box, incubated overnight at 4℃and washed 3 times with PBS buffer for 5 minutes each.
5) Secondary antibody
Diluting the fluorescent secondary antibody (1:200, FITC: ABclonal, AS011, DAPI: solaro, C0060) with a blocking solution according to a certain proportion, dropwise adding 10 mu L of the mixed secondary antibody into a circle in a dark place, placing the circle in a wet box, incubating for 1 hour at room temperature, washing with PBS buffer solution for 3 times, and sealing the plate each time for 5 minutes.
6) Sealing sheet
The washed slide was spin-dried, anti-fluorescence quenchant (BOSTER, AR 1109) was added dropwise, cover slip was covered, nail polish was smeared four weeks, air-dried, and photographed with an inverted fluorescence microscope.
See fig. 21 for results:
[ result]: ALKBH5 induced with DEN flox/flox Compared with mouse liver cancer, DEN-induced ALKBH5-HKO mouse liver cancer Ki-67 positive signals are obviously reduced, and cell proliferation capacity is obviously reduced.
Example 22: effects of ALKBH5-HKO on Diethylnitrosamine (DEN) induced liver TG in liver cancer in mice.
DEN-induced ALKBH5-HKO and control ALKBH5 were obtained as in example 17 flox/flox Liver cancer mice were sacrificed, livers were collected, and photographed.
[ materials and methods ]:
DEN-induced ALKBH5-HKO and control ALKBH5 flox/flox Liver cancer mice
Liver TG detection method is described in example 12
See fig. 22 for results:
[ result]: ALKBH5 induced with DEN flox/flox Compared with mouse liver cancer, the DEN-induced ALKBH5-HKO mouse liver cancer liver TG content is obviously reduced.
Example 23: ALKBH5-HKO on mouse liver
EGFR/PI3K/AKT/mTOR/S6K/ULK1/LC3/FASN/SCD1 signaling pathway.
The ALKBH5-HKO mice obtained in example 4 and the control ALKBH5 were fed with normal feed flox/flox Mice were starved for 6 hours after 8 weeks (8:00-14:00 early), sacrificed, livers were harvested and Western Blot tested for EGFR/PI3K/AKT/mTOR/S6K/ULK1/LC3/FASN/SCD1 signaling pathway protein expression.
[ materials and methods ]:
normally raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
The Western Blot procedure is detailed in example 1.
The antibody details are shown below:
EGF Receptor (CST, 4267) and its phosphorylated form Phospho-EGF Receptor (Y1068) (CST, 3777), PI3 Kinase P55 (CST, 11889) and its phosphorylated form Phospho-PI3 Kinase P85 (Thr 458)/P55 (Tyr 199) (CST, 4228), AKT (CST, 4691) and its phosphorylated form Phospho-Akt (S473) (CST, 3787) and Phospho-Akt (T308) (CST, 4056), mT (mT 2949) and its phosphorylated form Phospho-mTOR (2448) (Sigma, SAB 4504476), S6K (Proteintech, 14485-1-AP) and its phosphorylated form Phospho-P70S 6 Kise (Thr 389) (Thr 384) ULK1 (Proteinth, 20986-1-AP) and its phosphorylated form Phospho-Akt (S473) (CST, 3787) and its phosphorylated form Phospho-mTOR (Ser 2448) (Sigma, SAB 4504476), and its phosphorylated form Phospho-mTOR (Phospho-1-P6) and its phosphorylated form Phospho-Pi-P (35) (CST 38) (6).
See fig. 23 for results:
results (results): ALKBH5-HKO mice showed significantly reduced liver levels of p-EGFR, EGFR, p-PI3K-p55, pAKT (S473), pAKT (T308), p-mTOR, p-S6K, p-ULK1, significantly increased LC3II/LC3I, significantly reduced FASN, SCD 1. ALKBH5-HKO mice reduce mTorrC 1 activity by reducing EGFR and its phosphorylated form, promote autophagy of liver, reduce accumulation of lipid droplets, and reduce lipid synthesis.
Example 24: effect of ALKBH5-HKO on PI3K kinase activity.
The ALKBH5-HKO mice obtained in example 4 and the control ALKBH5 were fed with normal feed flox/flox Mice were starved for 6 hours after 8 weeks (8:00-14:00 a earlier), sacrificed, livers were harvested and the PI 3-kinase activity ELISA kit was used to detect mouse liver PI3K kinase activity.
[ materials and methods ]:
normally raised ALKBH5-HKO mice and control ALKBH5 flox/flox And (3) a mouse.
The PI3K kinase activity ELISA (Echelon Biosciences, K-1000 s) assay was as follows:
1) Buffer preparation:
KBZ Reaction Buffer: 1 bottle of 4mL of 5x KBZ Reaction Buffer was added to 15.76mL dH 2 0, and 40. Mu.l of 1M DTT and 200. Mu.l of 10mM ATP are added for use.
PI (4, 5) P2 subtropie: 230. Mu.L of dH was added to a tube of PI (4, 5) P2 subset 2 O, mixing evenly, and instantly separating to obtain 100 mu M solution for later use, and diluting to 10 mu M by KBZ Reaction Buffer when in use.
Kinase Stop Solution: EDTA was added as needed to KBZ Reaction Buffer at a final concentration of 4mM for use.
Standard Curve Buffer: PI (4, 5) P2 subtropine and EDTA were added to KBZ Reaction Buffer on the day of the reaction at final concentrations of 2 μm, 2.4mM, respectively.
PI (3, 4, 5) P3 Standard 250. Mu.L dH was added to a tube of PI (3, 4, 5) P3 Standard on the day of the reaction 2 O, mixing uniformly, and performing instantaneous separation to obtain 3.6 mu M PI (3, 4, 5) P3 Standard solution for later use.
PI (3, 4, 5) P3 Detector: the day of the reaction was diluted as desired with PI (3, 4, 5) P3 Detector using a Detection Buffer at a ratio of 1:800.
TBS-T Buffer: 1 bottle of 20mL 10 XTBS-T Buffer was added to 180mL dH 2 0, for standby.
Secondary Detector: on the day of the reaction, the reaction was diluted Secondary Detector as required using TBS-T Buffer in a ratio of 1:800 for use.
2) Sample preparation:
taking 20mg liver tissue, adding 200uL of Lysis Buffer, grinding, cracking for 20min on ice, centrifuging for 10min at 4 ℃ and 10000g, transferring supernatant to a new EP tube, measuring protein concentration by a BSA method, taking 20 mu g of protein to the new EP tube, adding KBZ Reaction Buffer to 30 mu l, and placing on ice for standby.
3)Kinase reaction:
To the sample in 2) was added 30. Mu.l 10. Mu.M PI (4, 5) P2 substrere and reacted at 37℃for 2h. And adding 90 mu l Kinase Stop Solution into the reacted solution, and uniformly mixing for later use.
4)ELISA:
Taking 90 mu l of 3.6 mu M PI (3, 4, 5) P3 Standard solution, adding 210 mu l Standard Curve Buffer, uniformly mixing, and sequentially diluting three times based on the mixture to obtain 360nm, 120nm, 40nm, 13.3nm and 4.4nm Standard solution for later use.
Transfer 60 μl of the post-reaction sample, standard, and add 60ul PI (3, 4, 5) P3 Detector thereto and incubate at room temperature for 60min.
Transfer 100 μl of the post-reaction solution to ELISA assay plate and incubate at room temperature for 60min.
The solution was discarded, the residual liquid was dried, 200. Mu.l of TBS-T solution was added to each well, and repeated 3 times.
100 μ l Secondary Detector was added to each well and reacted at room temperature for 30min.
The solution was discarded, the residual liquid was dried, 200. Mu.l of TBS-T solution was added to each well, and repeated 3 times.
To each well, 100. Mu.l of TMB solution was added, and the reaction was allowed to proceed at room temperature for 30 minutes in the dark, and if the reaction was weak, the time was prolonged appropriately.
To each well 50 μl of Stop solution was added and the absorbance at 450nm was read immediately.
5) And (3) calculating:
and calculating standard curve through the absorbance value of the standard substance, and bringing the absorbance value of the sample into the sample absorbance value to obtain the PI 3-kinase activity.
See fig. 24 for results:
results (results): compared with a control mouse, the liver PI3K kinase activity of the ALKBH5-HKO mouse is obviously reduced. PI3K directly affects mTorrC 1 activity, and this result suggests that ALKBH5-HKO reduces EGFR/PI 3K/mTorrC 1 signaling pathway.
Example 25: galNAc-siAlkbh5 targets db/db BKS mouse liver Alkbh5.
[ materials and methods ]:
GalNAc-coupled modified siRNA has been widely used in clinic and proved to be a safe and effective method for inhibiting liver targets. GalNAc-coupled siRNA is capable of promoting hepatic parenchymal cell uptake by interaction with ASGPR specifically expressed by hepatic parenchymal cells, enabling hepatic parenchymal cell-specific delivery. In addition, siRNA can be stabilized by making different modifications at different positions of the siRNA, and the silencing durability can be obviously improved, which is shown in published literature (Brown, christopher R et al nucleic Acids Res.2020;48 (21): 11827-11844.). We designed siRNA against Alkbh5 with different modifications at different positions and coupled N-acetylgalactosamine (GalNAc). GalNAc-siAlkbh5 sense and antisense strands are c-c-acccaAgCUAugcucagaul (SEQ ID NO: 1) and, respectively
a-U-cugAagCauagCuGggugg-U-a (SEQ ID NO: 2); the sense and antisense strands of the control GalNAc-SiCon were u-u-cuccGaACGaguacguuL (SEQ ID NO: 8) and a-C-gugAcuCguucGgGgaa-u-u (SEQ ID NO: 9), respectively. Capital letters indicate 2-oxygen-2-fluoro (2-F) modifications, lowercase letters indicate 2-O-methyl (2-OMe) modifications, -PS linkages, L galNAc linkages.
db/db BKS mice have metabolic diseases such as obesity, type 2 diabetes, hyperlipidemia, and metabolic fatty liver. In order to determine whether liver ALKBH5 can be used as a drug target of metabolic diseases, we have made a targeted study of siRNA in db/db BKS metabolic disease model. We injected rat tail with GalNAc-siAlkbh5 RNA (1 mg/kg body weight) at 8 weeks old, expressed as GalNAc-siAlkbh5 mice, to knock down Alkbh5 expression in the liver, control rat db/db BKS injected with GalNAc-siCon RNA (1 mg/kg body weight), expressed as GalNAc-siCon mice, and examined liver ALKBH5 knockdown by Western Blot 2 weeks after injection, as described in example 1.
See fig. 25 for results:
results (results): galNAc-siAlkbh5 can significantly knock down db/db BKS mouse liver ALKBH5.
Example 26: effect of GalNAc-siAlkbh5 on blood glucose in db/db BKS mice.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were prepared as in example 25 and starved for 14 hours (18:00 late-8:00 early) after 1 week of injection, and blood glucose was measured.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
Blood glucose meter (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury type Qiangsheng)
See fig. 26 for results:
results (results): the GalNAc-siCon db/db BKS mice had significantly lower fasting blood glucose than the control GalNAc-siCon db/db BKS mice.
Example 27: effect of GalNAc-siAlkbh5 on glucose tolerance in db/db BKS mice.
GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice were prepared as in example 25 and starved for 15 hours (17:00 late-8:00 early) after 1 week of injection, and glucose tolerance experiments were performed.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice.
Glucometer (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury, qiangsheng).
Glucose (G116302, aladin), sodium chloride injection (Harbin triple pharmaceutical Co., ltd.) and sterile syringes (1 mL, jiangsu medical devices Co., ltd.).
The detailed method for glucose tolerance test is as follows:
1) 0 point blood glucose test
After the mice starved for 15 hours, the mouse tail tips were gently sheared, a drop of full blood was gently squeezed out, and blood glucose and body weight were recorded by blood glucose meter detection.
5) Tolerance test
Glucose solution (0.05 g/ml) was prepared with physiological saline, and the solution was injected at a dose of 0.5g/kg (e.g., 200. Mu.L for 20g mice) according to the weight of mice, and blood glucose was measured 15min, 30min, 60min, and 120min after the injection, respectively, and recorded. See fig. 27 for results:
Results (results): the GalNAc-siAlkbh5db/db BKS mice had significantly improved glucose tolerance compared to the control GalNAc-siCon db/db BKS mice.
Example 28: effect of GalNAc-siAlkbh5 on lactate tolerance in db/db BKS mice.
GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice were prepared as in example 25 and starved for 15 hours (17:00 late-8:00 early) after 1 week of injection, and lactic acid tolerance experiments were performed.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice.
Glucometer (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury, qiangsheng).
Sodium lactate (71718, sigma), sodium chloride injection (haerbin trigeminy pharmaceutical products Co., ltd.) and sterile syringes (1 mL, jiangsu medical devices Co., ltd.).
1) 0 point blood glucose test
After the mice starved for 15 hours, the mouse tail tips were gently sheared, a drop of full blood was gently squeezed out, and blood glucose and body weight were recorded by blood glucose meter detection.
6) Tolerance test
A sodium lactate solution of 0.05g/ml was prepared with physiological saline, and the solution was injected at a dose of 0.5g/kg according to the body weight of the mice (e.g., 200. Mu.L for 20g mice), and blood glucose was measured 15min, 30min, 60min, 120min after the injection, respectively, and recorded.
See fig. 28 for results:
results (results): galNAc-siCon db/db BKS mice had significantly reduced lactate-induced glucose production compared to control GalNAc-siCon db/db BKS mice.
Example 29: effect of GalNAc-siAlkbh5 on glucagon tolerance in db/db BKS mice.
Glucagon tolerance experiments were performed by preparing GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice as in example 25, starved for 6 hours (8:00-14:00 earlier) after 1 week of injection.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice.
Glucometer (ONETOUCH UltraEasy, qiangsheng) and blood glucose test paper (ONETOUCH Ultra-stable luxury, qiangsheng).
Glucagon (GP 21258, GLPBIO), sodium chloride injection (halbine trigeminy pharmaceutical products inc.) and sterile syringes (1 mL, jiangsu medical devices inc.).
The detailed method for glucagon tolerance experiments is as follows:
1) 0 point blood glucose test
After 6 hours of starvation, the mouse was gently sheared at the tip of the tail, a drop of filled blood was gently squeezed out, and blood glucose and body weight were recorded by blood glucose meter.
7) Tolerance test
A glucagon solution of 0.6. Mu.g/ml was prepared with physiological saline and injected at a dose of 6. Mu.g/kg according to the body weight of the mice (e.g., 200. Mu.L for 20g mice), and blood glucose was measured 15min, 30min, 60min, 120min after the injection, respectively, and recorded. See fig. 29 for results:
Results (results): galNAc-siCon db/db BKS mice were significantly less glucagon sensitive than the control GalNAc-siCon db/db BKS mice.
Example 30: effect of GalNAc-siAlkbh5 on db/db BKS mice GCGR/PKA/CREB signaling pathway.
GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 25, starved for 20 hours, sacrificed, livers were collected, and WesternBlots detected changes in the GCGR/PKA/CREB signaling pathway.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice.
Western Blot detects expression of GCGR, p-PKA Sub, p-CREB, as detailed in example 1.
See fig. 30 for results:
results (results): galNAc-siCon db/db BKS mice had significantly lower liver GCGR, p-PKA Sub and p-CREB levels than the control GalNAc-siCon db/db BKS mice. This suggests that GCGR signal pathway lowering is a significant cause of GalNAc-siAlkbh5 hypoglycemic.
Example 31: effect of GalNAc-siAlkbh5 on db/db BKS mouse cAMP.
GalNAc-siAlkbh5db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 25, starved for 20 hours, sacrificed, livers collected and assayed by cAMP ELISA kit.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
The detection method of the cAMP ELISA kit is shown in example 10.
See fig. 31 for results:
results (results): galNAc-siCon db/db BKS mice had significantly lower liver cAMP levels than the control GalNAc-siCon db/db BKS mice. This suggests that the reduction of GCGR/cAMP signaling pathway is a significant cause of the reduction of GalNAc-siAlkbh5 blood glucose.
Example 32: effect of GalNAc-siAlkbh5 on serum triglyceride content in db/db BKS mice.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 25, starved for 20 hours, sacrificed, blood was taken, centrifuged at 4000rpm/min at 4℃for 10min, and the supernatant, i.e., serum, was transferred and frozen at-80℃for later use.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
Triglyceride (TG) test box (F001-1-1, built in Nanjing) and detection method are described in example 11.
See fig. 32 for results:
results (results): the serum triglyceride content of the GalNAc-siAlkbh5 db/db BKS mice was significantly reduced compared to the control GalNAc-siCon db/db BKS mice.
Example 33: effect of GalNAc-siAlkbh5 on db/db BKS mice serum total cholesterol content.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 29, and serum was collected for use.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
Total cholesterol (T-CHO) test kit (A111-1-1, built in Nanjing) and the detection method is described in example 12.
See fig. 33 for results:
results (results): the serum total cholesterol content of the GalNAc-siAlkbh5 db/db BKS mice was significantly reduced compared to the control GalNAc-siCon db/db BKS mice.
Example 34: effect of GalNAc-siAlkbh5 on serum low density lipoprotein cholesterol content in db/db BKS mice.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 29, and serum was collected for use.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
A kit for measuring low density lipoprotein cholesterol (LDL-C) (A113-1-1, built in Nanjing) and a detection method are shown in example 13.
See fig. 34 for results:
results (results): the serum low density lipoprotein cholesterol content of the GalNAc-siAlkbh5 db/db BKS mice was significantly reduced compared to the control GalNAc-siCon db/db BKS mice.
Example 35: effect of GalNAc-siAlkbh5 on db/db BKS mouse serum ALT.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 29, and serum was collected for use.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
GPT kit (C009-2-1, built in Nanjing) and detection method are described in example 14.
See fig. 35 for results:
results (results): serum ALT levels were significantly reduced in GalNAc-siAlkbh5 db/db BKS mice compared to control GalNAc-siCon db/db BKS mice.
Example 36: effect of GalNAc-siAlkbh5 on db/db BKS mouse liver TG.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 25, starved for 20 hours, sacrificed, livers were collected, and changes in liver TG were detected.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
Glycerol reagent (F6428, sigma), liver TG assay is described in example 15.
See fig. 36 for results:
results (results): galNAc-siCon db/db BKS mice had significantly lower liver TG than control GalNAc-siCon db/db BKS mice.
Example 37: effect of GalNAc-siAlkbh5 on liver lipid droplet accumulation in db/db BKS mice.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 25, starved for 20 hours, sacrificed, livers were collected, 4% paraformaldehyde solution was fixed for 4 hours, HE stained, and photographed.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
Study grade inverted fluorescence microscope imager (IX 71+DP74, olympus) was photographed.
See fig. 37 for results:
results (results): galNAc-siCon db/db BKS mice showed significantly reduced liver lipid droplets compared to control GalNAc-siCon db/db BKS mice.
Example 38: galNAc-siAlkbh5 versus db/db BKS mouse liver
EGFR/PI3K/AKT/mTOR/S6K/ULK1/LC3/FASN/SCD1 signaling pathway.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 25, starved for 20 hours, sacrificed, livers were harvested, and Western Blot examined EGFR/PI3K/AKT/mTOR/S6K/ULK1/LC3/FASN/SCD1 signaling pathway-related protein expression.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
The Western Blot procedure is detailed in example 1.
See fig. 38 for results:
results (results): compared with the control GalNAc-siCon db/db BKS mice, the liver p-EGFR, EGFR, p-PI3K-p55, pAKT (S473), pAKT (T308), p-mTOR, p-S6K, p-ULK1 levels of GalNAc-siAlkbh5 db/db BKS mice were significantly reduced, LC3II/LC3I was significantly increased, FASN, SCD1 was significantly reduced. This suggests that EGFR/AKT/mTorrC 1 signaling pathway is decreased, lipid synthesis is decreased, and autophagy is increased, which are important causes of GalNAc-siAlkbh5 for reducing fatty liver and blood lipid.
Example 39: effect of GalNAc-siAlkbh5 on db/db BKS mouse PI3K kinase activity.
GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice were obtained as in example 25, starved for 20 hours, sacrificed, and livers were collected and assayed for liver PI3K kinase activity by ELISA kit.
[ materials and methods ]:
normally raised GalNAc-siAlkbh5 db/db BKS and its control GalNAc-siCon db/db BKS mice.
The ELISA method for detecting PI3K kinase activity is shown in example 22.
See fig. 39 for results:
results (results): compared with a control GalNAc-siCon db/db BKS mouse, the liver PI3K kinase activity of the GalNAc-siAlkbh5 db/db BKS mouse is obviously reduced. This suggests that EGFR/PI 3K/mORC 1 signaling pathway reduction is a significant cause of GalNAc-siAlkbh5 reduction in fatty liver and blood lipids.
It will be appreciated by persons skilled in the art that although the invention has been specifically described with reference to the above embodiments, the invention is not limited to these specific embodiments. Based on the methods and technical solutions taught by the present invention, those skilled in the art can make appropriate modifications or improvements without departing from the spirit of the present invention, and the equivalent embodiments thus obtained are within the scope of the present invention.
Claims (10)
1. The application of the reagent for detecting ALKBH5 expression level in preparing a diagnostic agent or a diagnostic kit for metabolic diseases.
2. The use according to claim 1, wherein the reagent is a specific probe, a gene chip, PCR primers or an antibody or Western Blotting reagent for detecting the albh 5 protein of the albh 5 gene.
3. The use of claim 1, wherein the metabolic disease is selected from obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver, liver cancer (hepatocellular carcinoma), or a combination thereof, and/or the agent detects an alk bh5 expression level in the liver of a subject.
4. Use of an agent that reduces or inhibits expression of ALKBH5 in the manufacture of a medicament for preventing or treating a metabolic disorder.
5. The use of claim 4, wherein the agent that reduces or inhibits expression of albh 5 is selected from the group consisting of: gapmer, antisense RNA, siRNA, esiRNA, shRNA, miRNA, RNA aptamer, TALEN, CRISPR, zinc finger nuclease, small molecule compound, alk bh 5-specific antibody, and (liver) alk bh5 knockout or knockdown reagent.
6. The use according to claim 4 or 5, wherein the metabolic disease is selected from obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver, liver cancer (hepatocellular carcinoma) or a combination thereof.
7. The use of claim 4, wherein the agent reduces or inhibits the expression level of alk bh5 in the liver of the subject.
8. Use according to claim 7, wherein the agent is an N-acetylgalactosamine (GalNAc) coupled modified siRNA, preferably GalNAc-siAlkbh5, the sense and antisense strands (5 '→3') of which are c-c-accagaaucucuavaul (SEQ ID NO: 1) and a-U-cugAagCauagCuGggugg-U-a (SEQ ID NO: 2), respectively, wherein the capital letters represent 2-deoxy-2-fluoro (2-F) modifications, the lowercase letters represent 2-O-methyl (2-OMe) modifications, -PS (phosphothioase, phosphorothioate) linkages, L represents GalNAc linkages.
9. The use according to claim 3, 7 or 8, wherein the subject is a mammal, preferably a human.
10. A method of screening for a drug for preventing or treating a metabolic disease, the method comprising the steps of:
1) Determining the expression level of ALKBH5 in the cell over-expressing ALKBH 5;
2) Contacting the candidate compound with the cells of step 1);
3) Determining the expression level of alk bh5 in the cell after step 2); and
4) Comparing the expression levels of ALKBH5 determined in step 1) and step 3), wherein a decrease in the expression level of ALKBH5 indicates that the candidate compound has the potential to prevent or treat metabolic disorders,
preferably the cells are liver cells, more preferably liver cells from a subject suffering from a metabolic disorder, still more preferably the metabolic disorder is selected from obesity, hyperglycemia (type 2 diabetes), impaired glucose tolerance, hyperlipidemia, metabolic fatty liver, liver cancer (hepatocellular carcinoma) or a combination thereof, more preferably the subject is a mammal, most preferably a human.
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