CN115006516A

CN115006516A - Application of CXCL13 in preparation of medicine for treating NAFLD-related insulin resistance and hepatic fibrosis

Info

Publication number: CN115006516A
Application number: CN202210672224.1A
Authority: CN
Inventors: 张南南
Original assignee: West China Second University Hospital of Sichuan University
Current assignee: West China Second University Hospital of Sichuan University
Priority date: 2022-06-14
Filing date: 2022-06-14
Publication date: 2022-09-06

Abstract

The invention relates to the technical field of non-alcoholic fatty liver disease medicines, in particular to the application of chemokine CXCL13 in the preparation of medicines for treating non-alcoholic fatty liver disease-related insulin resistance and liver fibrosis. The inventors used the CXCL13 gene knockout technology to construct CXCL13 knockout mice, and used high-fat and high-sugar diet to induce non-alcoholic fatty liver disease. The body weight of the mice was monitored weekly. After 16 weeks of induction, the body weight, liver function, blood lipids, blood sugar, glucose tolerance, insulin resistance and liver fibrosis levels of the mice were detected, proving that CXCL13 is an effective agent for non-alcoholic fatty liver disease. therapeutic target. This solution provides an effective regulatory factor for non-alcoholic fatty liver disease, solves the technical problem of the lack of drugs that can comprehensively treat non-alcoholic steatohepatitis without causing significant side effects in the prior art, and has an ideal application prospects.

Description

Application of CXCL13 in preparation of medicine for treating NAFLD-related insulin resistance and hepatic fibrosis

Technical Field

The invention relates to the technical field of non-alcoholic fatty liver disease drugs, in particular to application of a chemotactic factor CXCL13 in preparation of drugs for treating insulin resistance and hepatic fibrosis related to non-alcoholic fatty liver disease.

Background

Non-alcoholic fatty liver disease (NAFLD) is closely related to metabolic disorder syndromes such as obesity, type 2 diabetes mellitus, hyperlipidemia and the like, and is mainly characterized by fatty lesion of liver, and severe patients can develop cirrhosis of liver and even liver cancer. According to statistics, the incidence rate of NAFLD is about 25%; in China, the incidence rate is as high as 32.9 percent, and the incidence rate is on the trend of rising year by year. NAFLD has replaced chronic hepatitis B, and is the first chronic liver disease in China. Nonalcoholic steatohepatitis (NASH) is a key turning point in the process of developing NAFLD diseases, accounts for about one third of NAFLD patients, can cause poor fate of hepatic fibrosis, liver cirrhosis, even liver cancer and the like, can cause complications such as diabetes, pancreatic hyperglycemia, cardiovascular diseases and the like, and seriously harms human health. At present, the clinically common medicament for NASH has better treatment effect, but may show certain adverse reaction after long-term administration. For example, pioglitazone, as an agonist of the peroxisome proliferator-activated receptor gamma (PPAR γ), improves carbohydrate and lipid metabolism in the body, but can cause osteoporosis and even increase the risk of bladder cancer after long-term use. Vitamin E, an antioxidant, significantly ameliorates hepatic steatosis and inflammatory infiltrate in NASH patients, but does not alleviate further liver fibrosis, and high doses of vitamin E (> 400 units per day) may increase the risk of patient mortality. Therefore, intensive research on the pathogenesis of NASH and the discovery of more key and effective regulatory factors remain an urgent need and development direction for NASH treatment.

Disclosure of Invention

The invention aims to provide application of a chemotactic factor CXCL13 in preparation of a medicine for treating insulin resistance or hepatic fibrosis related to non-alcoholic steatohepatitis, so as to solve the technical problem that the prior art lacks a medicine which can comprehensively treat the non-alcoholic steatohepatitis and does not cause obvious side effects.

In order to achieve the purpose, the invention adopts the following technical scheme:

the application of the chemotactic factor CXCL13 in preparing the medicine for treating the insulin resistance and the hepatic fibrosis related to the non-alcoholic fatty liver disease.

The scheme also provides an application of the chemotactic factor CXCL13 in drug screening.

The scheme also provides an application of the chemokine CXCL13 in constructing an animal model.

The principle and the advantages of the scheme are as follows:

the B lymphocyte can regulate liver insulin resistance, liver inflammation infiltration related to non-alcoholic fatty liver disease, liver cell damage necrosis and even liver cancer. However, it is not clear that B cells affect the signal pathways associated with hepatic insulin resistance and hepatic fibrosis, and their effects and associated mechanisms on non-alcoholic fatty liver disease. The inventor conducts intensive research on the action mechanism of the B cell and finds that the chemokine CXCL13 related to the B cell can be used as a drug action target of insulin resistance and hepatic fibrosis related to the non-alcoholic fatty liver disease. The inventor utilizes CXCL13 gene knockout technology to construct a CXCL13 knockout mouse, and utilizes high-fat high-sugar feed to induce non-alcoholic fatty liver disease. The body weight of the mice is monitored weekly, and after 16 weeks of induction, the body weight, liver function, blood fat, blood sugar, glucose tolerance, insulin tolerance and liver fibrosis level of the mice are detected, so that the CXCL13 is proved to be an effective treatment target point of the nonalcoholic fatty liver disease.

Among them, the Chemokine CXCL13(Chemokine C-X-C motif ligand 13, CXCL13) is a regulator of B lymphocyte signaling pathway and plays a very important role in the growth, differentiation and maturation of B cells. CXCL13 can regulate the maturation and development of B lymphocyte, participate in the differentiation and migration of Th cell and B lymphocyte, promote the homing of immature B lymphocyte and regulate the formation of lymph organ and germinal center. The CXCL13 signaling pathway also regulates the differentiation of B lymphocytes into mature plasma cells, regulating the secretion of antibodies IgM, IgA, and IgG. After antigen stimulation, CXCL13 can be combined with a specific receptor CXCR5 thereof to activate downstream PI3K, PLC, MEK and Paxillin signals and further activate various downstream signal proteins such as NF-kB, JNK, AKT, Ca2+ signals and the like to play specific physiological or pathological functions. Abnormal expression or sustained activation of the CXCL13/CXCR5 signaling pathway can lead to disorders of the lymphatic system, resulting in a variety of diseases such as tumors, autoimmune arthritis, asthma, etc. However, whether the CXCL13/CXCR5 signal pathway and B lymphocytes have a regulation relationship between insulin resistance and hepatic fibrosis related to non-alcoholic fatty liver disease is not reported in the prior art. The technical scheme discovers the relation between CXCL13 and insulin resistance and hepatic fibrosis related to non-alcoholic fatty liver disease for the first time, and applies the relation to preparation and screening of related medicaments and construction of animal models for researching pathogenic mechanisms of the non-alcoholic fatty liver disease.

After confirming that CXCL13 is an effective therapeutic target for nonalcoholic fatty liver disease, we can use an inhibitor (inhibitor or shRNA) of CXCL13 to inhibit the expression level of CXCL13 gene or protein, or to inhibit its protein activity, thereby treating insulin resistance or liver fibrosis due to nonalcoholic steatohepatitis. In addition, the function of CXCL13 as a drug action target can be used for evaluating the action effect of the drug which takes CXCL13 as the target and is used for treating the non-alcoholic steatohepatitis. In addition, by regulating the expression level of the CXCL13 gene in an animal body, various animal models can be constructed, including a CXCL13 gene overexpression animal model and a CXCL13 gene knockout animal model, so that the disease occurrence and development mechanism of non-alcoholic steatohepatitis animals can be researched, and related drug screening can be carried out.

Further, the application of the chemokine CXCL13 in preparing the medicine for treating the insulin resistance and the hepatic fibrosis related to the nonalcoholic fatty liver disease comprises the step of inhibiting the expression level of the CXCL13 gene. Experiments prove that after the CXCL13 gene of a mouse is knocked out or the expression level of the CXCL13 gene is inhibited, the weight, the liver weight and the fat content of the mouse are obviously improved; serum AST level and serum ALT level are also obviously reduced; the insulin resistance and the hepatic fibrosis of the mice are also obviously improved; shows that the insulin resistance and the hepatic fibrosis related to the non-alcoholic fatty liver disease can be treated by inhibiting the expression level of the CXCL13 gene.

Further, expression of CXCL13 gene was inhibited using at least one of R788, SKLB-852 and metformin.

R788 and SKLB-852 are spleen tyrosine kinase inhibitors, and are used for inhibiting obesity and further reducing the expression level of CXCL13 genes. The inventors previously constructed spleen tyrosine kinase knockout mice in which the degree of hepatic steatosis was reduced and the expression level of CXCL13 gene was suppressed after high fat and high sugar diet in the mice. The inventors further treated wild-type mice after induction of high-glucose high-lipid with a spleen tyrosine kinase inhibitor and found that the expression level of CXCL13 gene in the mice was significantly reduced compared to the control group. This suggests that a spleen tyrosine kinase inhibitor can be used for the inhibition of the CXCL13 gene, and the CXCL13 gene can regulate the occurrence and development of nonalcoholic fatty liver disease caused by high-sugar and high-fat diet. In addition to spleen tyrosine kinase inhibitors, the inventors also treated mice with metformin, a first-line drug for the treatment of type 2 diabetes, to inhibit obesity and reduce the expression level of CXCL13 gene (example 8).

The technical scheme takes CXCL13 gene as a target spot, screens out spleen tyrosine kinase inhibitors R788 and SKLB-852 and type 2 diabetes mellitus drug metformin, and realizes the treatment of NASH by regulating and controlling the expression of CXCL13 gene. In addition, R788, SKLB-852 and metformin can also be used as CXCL13 gene indirect inhibitors and applied to practical operation of research on CXCL13 signal paths and NASH pathogenic mechanisms.

Further, the expression of CXCL13 gene was suppressed by knocking out CXCL13 gene.

In addition to the use of related inhibitors, means to directly knock out the CXCL13 gene can be used to mitigate the effect of this gene on NASH and related insulin resistance and liver fibrosis.

Further, the application of the chemokine CXCL13 in drug screening is used for treating insulin resistance caused by non-alcoholic steatohepatitis or treating insulin resistance caused by non-alcoholic steatohepatitis. By using the chemotactic factor CXCL13 target, the effective degree of the medicine for treating the non-alcoholic steatohepatitis can be evaluated according to the inhibition degree of the medicine to CXCL 13.

Experiments prove that (example 5), the CXCL13 can effectively reduce the insulin resistance degree of the liver of a non-alcoholic steatohepatitis model mouse by inhibiting the level of CXCL13 due to insulin resistance caused by non-alcoholic steatohepatitis, and the glucose tolerance and the insulin tolerance of the model mouse are obviously improved.

Experiments prove that (example 6), CXCL13 is related to hepatic fibrosis caused by non-alcoholic steatohepatitis, the level of CXCL13 is inhibited, the fibrosis degree of the liver of a non-alcoholic steatohepatitis model mouse can be effectively reduced, and the occurrence of cirrhosis and cancer caused by the non-alcoholic steatohepatitis is further avoided.

Further, the application of the chemokine CXCL13 in constructing an animal model comprises the following steps which are sequentially carried out: knocking out CXCL13 gene in animal body; the animal model is then obtained by feeding the animal with a high fat and high sugar diet. By adopting the method, an animal model for researching the NASH related signal path can be constructed so as to clarify the occurrence and development mechanism of the disease.

Further, the high-fat high-sugar feed is Research Diets D12492.

Experiments prove that Research Diets D12492 high-fat feed (60% fat) can better simulate non-alcoholic fatty liver disease caused by metabolism. While the commonly used MCD feed (choline methionine deficient feed) induces a model principle that choline methionine deficiency results in a decrease in Very Low Density Lipoprotein (VLDL) synthesis, impairing triglyceride secretion. Although MCD feed can cause liver fibrosis lesion, the weight of the model mouse is obviously reduced, and the weight of the model mouse is only less than 15g on average after 8 weeks of induction, so that the model mouse cannot well simulate the nonalcoholic fatty liver disease of a human. The Research Diets D12492 of the technical scheme can successfully induce the NASH model without causing the weight loss of the mice.

Further, the application of the chemokine CXCL13 in constructing an animal model comprises the following steps which are sequentially carried out: the animal model was obtained by feeding animals with a high-fat high-sugar diet and treating the animals with a spleen tyrosine kinase inhibitor. By adopting the method, an animal model for researching the NASH related signal path can be constructed so as to clarify the occurrence and development mechanism of the disease.

Drawings

FIG. 1 shows the liver tissue gene sequencing and Elisa test results of CXCL13 expression level after induction of high lipid and high sugar in wild-type mice in example 1.

FIG. 2 shows the weight, liver weight and fat content of NASH mice after the CXCL13 knockout of example 2.

FIG. 3 shows the results of the oil red staining experiment and the statistics of liver fat in the NASH mice after CXCL13 knockout in example 2.

Fig. 4 is a graph of improvement in serum liver function following CXCL13 knockout of example 3.

FIG. 5 shows the changes in serum insulin, blood glucose, total cholesterol and LDL in NASH mice after the CXCL13 knockout of example 4.

FIG. 6 is a graph of the change in glucose tolerance and insulin resistance in mice of the NASH model after the CXCL13 knockout of example 5.

FIG. 7 is a graph showing the changes in the expression levels of Collagen1 α 1, Collagen1 α 2 and TGF-. beta.in the NASH model after CXCL13 knockout in example 6.

Fig. 8 is an image of the level of liver fibrosis detected in polarized light after sirius red staining of example 6.

FIG. 9 shows the results of expression analysis of liver metabolism-related molecules LXR, ChREBP and SREBP-1c of example 7.

FIG. 10 is a graph of liver fat deposition in liver-specific knockout Syk mice of example 8.

Fig. 11 is a heat map of chemokine expression of liver-specific knockout Syk mice of example 8.

FIG. 12 is a volcanic plot of gene expression of liver-specific knockout Syk mice of example 8.

FIG. 13 is liver fat infiltration in NASH mice under the action of Syk small molecule inhibitor of example 8.

FIG. 14 is a statistical graph of the expression of CXCL13 in the NASH mice of example 8 under the action of Syk small molecule inhibitors.

FIG. 15 is a graph showing the weight change of mice fed with different feeds according to comparative example 1.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the following examples and experimental examples are conventional means well known to those skilled in the art, and the materials, reagents and the like used therein are commercially available. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art.

Example 1: expression of CXCL13 in mouse model of nonalcoholic fatty liver disease

The genome data of CXCL13 in nonalcoholic fatty liver disease mice are detected, and the expression level of related proteins is detected. After wild type mice (C57BL/6J) were induced by high-fat high-sugar diet to non-alcoholic steatohepatitis (NASH), expression of CXCL13 gene was analyzed by transcriptome sequencing.

The specific construction method of the non-alcoholic steatohepatitis (NASH) mouse model is as follows: wild type mice were fed with high fat diet (60% fat) of Research diet D12492, and given high fat diet (Research diet D12492), for 12-16 weeks, and liver function and pathological changes of liver tissues were examined, and non-alcoholic steatohepatitis (NASH) mouse models were successfully constructed (see WT-NCD and WT-HFD data in fig. 3 and fig. 4).

Wherein, the raw materials of Research Diets D12492 high-fat feed (60% fat) comprise: casein (Casein lactic), Cystine (L-Cystine), Corn Starch (Corn Starch), Maltodextrin (Maltodextrin), Sucrose (Sucrose), Cellulose (Cellulose BW200), Soybean Oil (Soybean Oil), Lard (Lard), complex minerals (Mineral Mix S10026), Calcium hydrogen Phosphate (Dicalcium Phosphonate), Calcium Carbonate (Calcium Carbonate), Potassium Citrate (1H) ₂ O)), Vitamin complex (Vitamin Mix V10001), Choline Bitartrate (Choline Bitartrate).

The experimental result shows that the CXCL13 expression level is obviously increased. Specific experimental data referring to fig. 1, fig. 1A is an experimental result of liver tissue gene sequencing detection of CXCL13 expression level after wild-type mouse high-fat high-sugar induction; FIG. 1B shows that Elisa measures the CXCL13 expression level of mouse liver tissue after induction of wild type mouse with high fat and high sugar. The results show that the mouse is induced to form NAFLD, and the mouse CXCL13 obviously increases the expression level at both gene level and protein level.

Example 2: preparation of CXCL13 knockout mice

Construction of CXCL13 knockout mice (human CXCL 13: Gene ID: 10563; mouse CXCL 13: Gene ID: 55985) using conventional means of the prior art can be entrusted to biotech companies for completion (Jackson lab) roughly as follows: first constructs for transgenics were constructed, including sequences in which 18-116 base pairs of exon 2 of the endogenous gene were replaced with an in-frame stop codon, an endosome entry site, an enhanced green fluorescent protein gene (EGFP), and flanking loxp-type neomycin resistance genes. This construct was electroporated into 129X 1/SvJ-derived JM-1 embryonic stem cells (ES). Correctly targeted ES cells were injected into mouse C57BL/6J blastocysts, and the resulting chimeric male embryos were backcrossed to transfer the germline to the C57BL/6J female embryo. The progeny were crossed with C57BL/6J expressed by C57BL/6J gene, removing neomycin resistance gene. Backcrossing the newly-cut heterozygote with C57BL/6J for 10 generations to obtain homozygote, and obtaining CXCL13 complete knockout mouse (CXCL 13) ^-/- )。

High-fat high-sugar diet induction of NASH was performed on CXCL13 knockout mice (the procedure was the same as that of example 1 for wild mice), and 16 weeks after induction, the effects of CXCL13 knockout on body weight, liver weight, and fat content were examined. Experimental data referring to FIG. 2, FIG. 2 shows the changes in body weight, liver weight and fat content of NASH mice after CXCL13 knock-out (WT denotes wild type mice, CXCL13) ^-/- Represents CXCL13 knock-out mice; NCD Normal diet (common feed for mice), HFD high-fat high-sugar diet using D12492, CXCL13 ^-/- ). FIG. 2A is a graph of the change in body weight of mice after CXCL13 knock-out; fig. 2B shows liver weight in mice 16 weeks after CXCL13 knockout; fig. 2C is a body weight statistical graph of mice after CXCL13 knockout; FIG. 2D is a graph of the change in white fat content of NASH mice after CXCL13 knock-out (statistical data are shown by X + -SD;. P < 0.01 or. P < 0.05, with significant statistical significance), and experiments were repeated three times with 6/H/EGroups). Experimental data show that after CXCL13 is knocked out, the weight, the liver weight and the fat content are obviously improved. Wild type mice were used as controls in the experiment, and after CXCL13 knockout, the mice lost 38.03% of body weight, 63.92% of liver weight, and 166.26% of white fat content.

The oil red staining analysis of the liver tissue was performed on the experimental mice, and the experimental results are shown in fig. 3 (the statistical data are represented by X ± SD;. P < 0.001 or. P < 0.05, which have significant statistical significance, the experiment was repeated three times, 6/group each time), and the oil red staining results show that after CXCL13 was knocked out, the number of lipid droplets formed in the liver was significantly reduced, and the infiltration of the liver tissue fat was significantly reduced.

Example 3: inhibition of the effect of CXCL13 expression on the NASH phenotype

To verify the remission of NASH phenotype after inhibition of CXCL13 expression, serum liver function of knockout mice was examined using a fully automated biochemical analyzer and the results of the experiment are shown in fig. 4. FIG. 4 shows improvement of serum liver function following CXCL13 knockout, wherein FIG. 4A is a graph of the change in serum ALT levels in high fat diet-induced NASH mice following CXCL13 knockout; FIG. 4B is a graph of the change in serum AST levels in mice after induction of NASH by high fat diet after CXCL13 knock-out (statistics are shown by X + -SD;. P < 0.01 or. P < 0.05, with significant statistical significance, experiments were repeated three times, 6/group each). Compared with wild model mice, after CXCL13 is knocked out, ALT is downregulated from 488.8U to 61.33U, AST is downregulated from 399.20U to 240U, and the liver function of the mice is obviously relieved.

Example 4: inhibition of the effects of CXCL13 expression on blood glucose and blood lipid in non-alcoholic fatty liver disease

We induced a mouse non-alcoholic fatty liver disease model with high-fat high-sugar feed. Blood glucose, insulin and blood lipid levels of the knockout mice were tested for 16 weeks of induction and the results are shown in figure 5. Figure 5 shows the variation of insulin, blood glucose, total cholesterol and low density lipoprotein in serum of NASH mice after CXCL13 knock-out (statistical data are expressed as X ± SD;. P < 0.001,. P < 0.01 or P < 0.05, with significant statistical significance, experiments were repeated three times, 6/group each). The results show that compared with wild model mice, after CXCL13 knockout, NASH mice have 54.50% blood glucose reduction, insulin reduction from 11.97ng/mL to 3.86ng/mL, 64.49% serum total cholesterol reduction and 201.61% low density lipoprotein reduction.

Example 5: study of the relationship of CXCL13 to NASH-associated insulin resistance

To further validate CXCL13 as a target for treatment of NASH insulin resistance, knockout mice were tested for induction of NASH processes, glucose tolerance and insulin resistance. After the mice were starved overnight (8 hours) in the fasting state, fasting blood glucose was measured, and then glucose or insulin was intraperitoneally injected, and blood glucose was measured every half hour, and the experimental results are shown in fig. 6. FIG. 6 shows a graph of the NASH model mouse glucose tolerance and insulin resistance changes following CXCL13 knock-out, wherein the results of FIG. 6A are shown for mouse glucose tolerance; FIG. 6B shows insulin resistance in mice (statistical data are shown by X + -SD;. P < 0.001 or. P < 0.05, with significant statistical significance, experiments were repeated three times, 6/group at a time). The experimental results show that after CXCL13 is knocked out, the glucose tolerance and the insulin tolerance of the NASH model mouse are obviously improved.

Example 6: relationship between CXCL13 and NASH-associated hepatic fibrosis

To verify that CXCL13 can be used as a therapeutic target for liver fibrosis in non-alcoholic fatty liver disease, we tested the degree of fibrosis and fibrosis-related indicators of liver tissues of a knockout CXCL13 mouse, and the experimental results are shown in fig. 7 and fig. 8. FIGS. 7A-C show graphs of the Real-time PCR assay for the NASH model after the CXCL13 knock-out of Collagen1 α 1, Collagen1 α 2, and TGF-. beta.expression changes (statistical data are shown by X. + -. SD;. P < 0.0001, significant statistical significance, experiments were repeated three times, 6/group each); FIG. 8 shows the level of liver fibrosis measured in polarized light after sirius red staining. After NASH mice are induced by high-fat feed, compared with wild mice, CXCL13 knockout mice obviously improve the progression level of liver from NASH to hepatic fibrosis.

Example 7: expression analysis of liver metabolism related molecules LXR, ChREBP and SREBP-1c

After NASH mice are induced by high-fat feed, compared with wild type mice, the CXCL13 knockout mice improve the expression of liver metabolism related molecules LXRa, ChREBP and SREBP-1c, and the CXCL13 knockout mice obviously improve the liver lipid metabolism level. Figure 9 shows experimental data for Real-time PCR detection of the expression levels of mouse LXRa, ChREBP, SREBP-1c after CXCL13 knock-out (statistics are expressed as X ± SD;. P < 0.0001,. P < 0.001,. P < 0.01 are statistically significant, experiments were repeated three times, 6/group each).

Example 8: molecular mechanism study

The inventors constructed a mouse model of liver-specific knockout of Syk (spleen tyrosine kinase) at a previous stage and induced obesity and NASH generation in Syk knockout mice using the manner of example 1. Knockout of Syk was found to significantly reduce fat deposition in the liver (fig. 10). And transcriptome analysis results showed that CXCL13 expression was significantly reduced in a liver-specific Syk knockout mouse model (fig. 11 and 12). The inventors further considered the use of related inhibitors of Syk to modulate the expression of CXCL13 for the treatment of NASH in mice. The heat map of fig. 11 shows a significant reduction in CXCL13 expression. In the volcano plot of FIG. 12, Syk knockout mice (SC-HFD, Syk) are shown ^loxp/loxp Alb Cre group) genes up-and down-regulated on a high fat diet relative to wild type mice (WT-HFD).

After the mouse is intervened by using Syk small molecule inhibitor R788 (CAS: 901119-35-5), SKLB-852 (abbreviated as 852, see patent CN201410174964.8, CN105017159B 5-fluoro-2, 4-disubstituted aminopyrimidine derivatives, and preparation methods and applications thereof) and metformin (CAS: 657-24-9), the liver fat infiltration condition of the mouse is obviously relieved (figure 13), the CXCL13 gene expression is obviously reduced (figure 14, the protein level of CXCL13 is shown, statistical data are expressed by X +/-SD), CXCL < 0.0001, CXCL < 0.001, CXCL < 0.01 has obvious statistical significance, and experiments are repeated three times, 6 individuals per group, so that CXCL13 can regulate the occurrence and development of nonalcoholic fatty liver disease caused by high-sugar high-fat diet, and the expression of CXCL13 can be regulated and controlled by Syk small molecule inhibitors.

The experiment was carried out in such a manner that the mice were fed in the same manner as in example 1, and after 16 weeks, the average body weight of the mice reached 40g or more, and drug administration was started. R788, SKLB-852 and metformin are orally administered once a day in amounts of R78880. mu.g/kg, 85250. mu.g/kg, 100. mu.g/kg and metformin 100mg/kg, respectively, and the detection is carried out after 12 weeks of intervention. Among them, Vehicle is a solvent for the drug (placebo), consisting of 20% DMSO, 10% PEG, 70% sterile water. NS was a non-NASH model group and administered placebo.

Comparative example 1:

this comparative example is essentially the same as example 1, except that choline methionine deficiency (MCD) feed was used instead of Research Diets D12492 high fat feed (60% fat) induced NASH model. Experiments prove that Research Diets D12492 high-fat feed (60% fat) can better simulate non-alcoholic fatty liver disease caused by metabolism. While MCD feed, a choline methionine-deficient feed, induces a model principle that choline methionine deficiency results in a decrease in Very Low Density Lipoprotein (VLDL) synthesis, impairing triglyceride secretion. Although MCD feed can cause liver fibrosis lesion, the weight of the model mouse is obviously reduced, and the weight of the model mouse is only less than 15g on average after 8 weeks of induction, so that the model mouse cannot well simulate the nonalcoholic fatty liver disease of a human. Mice were fed MCD diet +20g high fat diet (Research Diets D12492 high fat diet) for 8 weeks and tested for body weight, and it was found that MCD feeding caused the mice to be too light (fig. 15).

The foregoing is merely an example of the present invention and common general knowledge in the art of designing and/or characterizing particular aspects and/or features is not described in any greater detail herein. It should be noted that, for those skilled in the art, without departing from the technical solution of the present invention, several variations and modifications can be made, which should also be regarded as the protection scope of the present invention, and these will not affect the effect of the implementation of the present invention and the practicability of the patent. The scope of the claims of the present application shall be determined by the contents of the claims, and the description of the embodiments and the like in the specification shall be used to explain the contents of the claims.

Claims

1. The application of the chemotactic factor CXCL13 in preparing the medicine for treating the insulin resistance and the hepatic fibrosis related to the non-alcoholic fatty liver disease.

2. The use of the chemokine CXCL13 as claimed in claim 1, for the preparation of a medicament for the treatment of insulin resistance and liver fibrosis associated with non-alcoholic fatty liver disease, comprising the step of inhibiting the expression level of CXCL13 gene.

3. The use of the chemokine CXCL13 of claim 2 for the preparation of a medicament for the treatment of non-alcoholic fatty liver disease-associated insulin resistance and liver fibrosis, wherein at least one of R788, SKLB-852 and metformin is used to inhibit the expression of the CXCL13 gene.

4. The use of the chemokine CXCL13 in the preparation of a medicament for the treatment of insulin resistance and liver fibrosis associated with non-alcoholic fatty liver disease according to claim 2, wherein the expression of CXCL13 gene is inhibited by knocking out CXCL13 gene.

5. The application of the chemokine CXCL13 in drug screening.

6. The use of the chemokine CXCL13 in drug screening according to claim 5, wherein the drug is used for treating insulin resistance caused by non-alcoholic steatohepatitis or insulin resistance caused by non-alcoholic steatohepatitis.

7. The application of the chemokine CXCL13 in constructing animal models.

8. The use of the chemokine CXCL13 of claim 7 for the construction of an animal model, comprising the following sequential steps: knocking out CXCL13 gene in animal body; the animal model is then obtained by feeding the animal with a high fat and high sugar diet.

9. The use of the chemokine CXCL13 in the construction of an animal model according to claim 8, wherein the high fat and high sugar diet is Research Diets D12492.

10. The use of the chemokine CXCL13 of claim 7 for the construction of an animal model, comprising the following sequential steps: the animal model was obtained by feeding animals with a high-fat high-sugar diet and treating the animals with a spleen tyrosine kinase inhibitor.