Medicine for treating non-alcoholic fatty liver disease

Abstract

The invention discloses a medicine for treating non-alcoholic fatty liver disease, the medicine comprises MIF-Anagonist V and p425, according to the medicine for treating the non-alcoholic fatty liver disease, by injecting the MIF-Anagonist V and the p425, the weight, blood sugar, blood fat and liver lipid deposition can be effectively improved, liver injury is avoided, and a novel medicine which is small in side effect and remarkable in curative effect is provided for NAFLD.

Description

Technical Field

[0001] The present invention relates to the technical field of drugs for treating fatty liver, and more particularly to a drug for treating non-alcoholic fatty liver. Background Art

[0002] Non-alcoholic fatty liver disease (NAFLD) refers to fatty infiltration of the liver that is not associated with alcohol overdose or other common causes of liver steatosis, such as viral or autoimmune hepatitis or drug-induced hepatitis, and includes multiple etiologies such as steatosis, steatohepatitis, fibrosis, and cirrhosis; it is now the most common cause of chronic liver disease worldwide. NAFLD-related non-alcoholic steatohepatitis (NASH) cirrhosis has surpassed all other causes of cirrhosis in developed countries, followed by the prevalence of obesity and type 2 diabetes. The prevalence of NAFLD is estimated to be 30%, of which nearly one-fifth of NAFLD will progress to NASH, and 20% of them will develop cirrhosis, subsequently leading to liver failure and hepatocellular carcinoma. Although most patients with NAFLD may not progress to advanced fibrosis or cirrhosis, due to its rapid increase in prevalence worldwide, together with metabolic syndrome and obesity, NAFLD is becoming a major cause of chronic liver disease and liver transplantation. Among the various phenotypes of NAFLD, nonalcoholic steatohepatitis (NASH) has the highest potential to progress to end-stage liver disease and cardiometabolic diseases, leading to liver-related and non-liver-related mortality.

[0003] Fatty liver disease (steatosis) is now very common, and is more common in association with obesity than with alcohol abuse. In more affluent areas, the community prevalence of nonalcoholic fatty liver disease (NAFLD) is approximately 15%. With the increasing prevalence of obesity, the prevalence of NAFLD has approximately doubled in the past decade. A striking feature of the NAFLD epidemic in Asia is the high prevalence of lean NAFLD (BMI <23) and non-obese NAFLD (BMI <25). Up to 19% of non-obese Asians have NAFLD, which may be attributed to the higher proportion of visceral obesity in Asians compared with other ethnic groups. Visceral obesity plays an important role in atherogenic dyslipidemia and insulin resistance. It is a major risk factor for type 2 diabetes and is associated with the development and progression of NAFLD. Lower BMI levels lead to longer disease duration and increased likelihood of complications. A variety of treatments have been used for NALFD. Current guidelines from the American Association for the Study of Liver Diseases indicate that pioglitazone can improve the histological appearance of patients with type 2 diabetes and NASH, while vitamin E may be beneficial in non-diabetic populations. MIF-Antagonist V, p425 is a symmetrical bis-(amino, hydroxynaphthalene disulfonic acid) compound that antagonizes the binding of macrophage migration inhibitory factor to its receptor. It is more effective than ISO-1 in inhibiting MIF-induced IL-6 and IL-8 production in human foreskin fibroblasts and MIF-induced MMP-3 production in human synovial fibroblasts. However, there are no reports on its use in the treatment of obesity and fatty liver. Summary of the invention

[0004] To this end, the technical problem to be solved by the present invention is to provide a drug for treating non-alcoholic fatty liver disease. By injecting MIF-Antagonist V, p425, the weight, blood sugar, blood lipids, and liver lipid deposition of mice can be effectively improved without liver damage, providing a new drug with few side effects and significant therapeutic effects for NAFLD.

[0005] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0006] A medicine for treating non-alcoholic fatty liver disease, comprising MIF-Antagonist V and p425.

[0007] The CAS number of MIF-Antagonist V, p425 is 2610-05-1, and its molecular formula is C 34 H 24 N6Na4O 16 S4.

[0008] MIF-Antagonist V, p425 is a symmetrical bis-(amino, hydroxynaphthalenedisulfonic acid) compound that antagonizes macrophage migration inhibitory factor binding to the receptor (IC50 = 810 nM for the interaction between hMIF and CD74). It is more effective than ISO-1 (Catalog No. 475837) in inhibiting MIF-induced IL-6 and IL-8 production in human foreskin fibroblasts (IC50 = 0.6-0.7 μM vs > 5 μM) and MIF-induced MMP-3 production in human synovial fibroblasts (IC50 = 0.1 μM vs > 10 μM). It shows little cytotoxicity in human foreskin fibroblast cultures even at concentrations as high as 100 μM. Unlike MIF antagonists I-IV, p425 targets a region at the interface of two MIF trimers distal to the active site.

[0009] Furthermore, the fatty liver is obese non-alcoholic fatty liver induced by a high-fat diet.

[0010] Furthermore, the content of MIF-Antagonist V, p425 in the drug is 2 mg / kg.

[0011] Furthermore, the drug improves non-alcoholic fatty liver disease by improving body weight, blood sugar, blood lipids, and liver lipid deposition.

[0012] Furthermore, the drug improves non-alcoholic fatty liver disease by reducing the expression of inflammatory factors.

[0013] The technical solution of the present invention achieves the following beneficial technical effects:

[0014] By injecting MIF-AntagonistV, p425 can effectively improve the body weight, blood sugar, blood lipids, and liver lipid deposition of mice without liver damage, providing a new drug with few side effects and significant efficacy for NAFLD. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 A schematic diagram of the weight change of a model mouse of a drug for treating non-alcoholic fatty liver disease of the present invention;

[0016] a is a schematic diagram of the weight changes of the three groups of experimental mice in the WTND group, WT HFD group and p425 group at 28 weeks and 4 weeks after the injection of p425; b is a comparison of the weights of the three groups of experimental mice in the WT ND group, WT HFD group and p425 group, * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, **** indicates P < 0.0001;

[0017] Figure 2A schematic diagram of changes in TC, TG, ALT, AST and blood sugar levels in model mice of a drug for treating non-alcoholic fatty liver disease of the present invention;

[0018] Among them, ad is the changes of glucose, TC, TG, ALT, and AST levels in the serum of mice in the WT ND group, WT HFD group, and p425 group; eh is the changes of TC, TG, ALT, and AST levels in the liver tissue of mice in the WT ND group, WT HFD group, and p425 group; i is the changes of blood glucose levels in the serum of mice in the WT ND group, WT HFD group, and p425 group;

[0019] Figure 3 A schematic diagram of the liver tissue structure of a model mouse of a drug for treating non-alcoholic fatty liver disease of the present invention;

[0020] A is HE staining, Oil Red O staining and immunohistochemical analysis of liver sections of mice in the WT ND group, WT HFD group and p425 group; a is lipid deposition analysis of mice in the WT ND group, WT HFD group and p425 group; b is F4 / 80 level analysis of mice in the WT ND group, WT HFD group and p425 group; c is MIF level analysis of mice in the WT ND group, WT HFD group and p425 group;

[0021] Figure 4 A schematic diagram of ELISA detection of a drug for treating non-alcoholic fatty liver disease in a model mouse according to the present invention;

[0022] ag are the analysis of serum MIF, TNF-a, IL-6, IL-1b, INS, HbA1C, and IL-10 levels in the three groups of mice: WT ND group, WT HFD group, and p425 group;

[0023] Figure 5 A schematic diagram of flow cytometry detection of liver tissue of model mice for a drug for treating non-alcoholic fatty liver disease of the present invention;

[0024] Among them, a is the type of polarized macrophages in the liver tissues of the three groups of mice: WT ND group, WT HFD group and p425 group; b is the comparison of macrophages in the liver tissues of the three groups of mice: WT ND group, WT HFD group and p425 group;

[0025] Figure 6 A schematic diagram of the liver tissue of a model mouse of a drug for treating non-alcoholic fatty liver disease of the present invention;

[0026] a is the western blot protein bands of the four groups of mice: WTND group, WT HFD group, p425 group and MIF-Ab group; b is the iNOS protein band analysis; c is the ARG1 protein band analysis; d is the MIF protein band analysis;

[0027] Figure 7 Schematic diagram of RNA levels of MIF and related inflammatory factors in liver tissues of three groups of mice using a drug for treating non-alcoholic fatty liver disease of the present invention. DETAILED DESCRIPTION

[0028] NAFLD model mouse preparation:

[0029] 4-6 weeks old SPF-grade immature male C57BL / 6 mice were used as model mice. The experiment was divided into four groups, WT ND group, WT HFD group, p425 group and MIF-Ab group. The body weight of each group was measured once a week during the experiment. Under the conditions of ambient temperature of 20-23℃, humidity of 40-60%, and light of 12h / d, the WT ND group, i.e., the control mice, were given normal feeding and free access to food and water for 28 weeks; the WT HFD group, i.e., the high-fat diet mice, were fed with 60% high-fat feed for 28 weeks; the p425 group, i.e., the high-fat diet mice, were fed with MIF-Antagonist injection after high-fat diet. V, p425 mice were fed with 60% high-fat diet for 28 weeks, and MIF-Antagonist V, p425 (2 mg / kg / ) was dissolved in normal saline and injected intraperitoneally (2 mg / kg / day) 28 weeks later for 30 consecutive days, during which time high-fat feeding was continued; the MIF-Ab group was fed with 60% fat / Kcal high-fat diet for 28 weeks, and then 5 mg / kg MIFAntibody (MIF-Ab, Yingmu Biotechnology (Shanghai) Co., Ltd.) was injected intraperitoneally (IP) for 4 consecutive weeks.

[0030] The above four groups of model mice were evaluated separately, and body weight, blood glucose, liver weight, triglyceride and cholesterol in blood, triglyceride and cholesterol in liver tissue, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum, H&E, Oil Red O, F4 / 80 in tissue sections, ELISA detection of MIF, IL-6, IL-10, TNF-a in blood, and detection of protein and RNA levels of MIF, iNOS, and ARG1 in liver were measured, as follows:

[0031] (1) Weight measurement

[0032] 1. Weight measurement method

[0033] Mouse weight is usually measured using a high-precision electronic balance or a dedicated mouse weighing scale. These tools have the advantages of accurate weighing and easy operation, and can meet the needs of mouse weight measurement.

[0034] 1) Prepare tools: Make sure the electronic balance or mouse weighing scale has been calibrated and is in accurate condition. Also, prepare a pen and paper or an electronic device to record the weight of the mouse;

[0035] 2) Fix the mouse: In order to accurately measure the weight of the mouse, it needs to be fixed in a stable position. You can use a special fixator or hold the mouse by hand, but be careful not to cause excessive pressure or injury to the mouse;

[0036] 3) Place the mouse: Gently place the mouse on the weighing pan of a balance or scale, making sure that its body is stable and will not slip;

[0037] 4) Reading data: After the balance or scale is stable, read and record the weight of the mouse. Be careful to read to one or two decimal places to ensure the accuracy of the data.

[0038] like Figure 1 As shown, from the behavioral observation of mice: the mice in the WT HFD group showed significant weight gain, messy and greasy hair, and reduced activity with the increase of feeding time. The mice in the p425 group had less weight, less messy and greasy hair, and more activity than those in the WT HFD group.

[0039] (2) Determination of TC, TG, ALT, and AST in blood

[0040] 1. Determination of ALT Concentration

[0041] 1) Select the sample from the -80℃ freezer and put it in an ice box to melt; add 20uL of matrix solution to each of the assay well and control well, and add 5μL of the sample to be tested to the assay well;

[0042] 2) Gently shake the 96-well plate to mix well, place in an incubator at 37°C, and incubate for 30 minutes;

[0043] 3) Add 20 μL of 2,4-dinitrophenylhydrazine solution to each sample well and control well, and add 5 μL of the sample to be tested to the control well;

[0044] 4) Place the 96-well plate on the table and shake gently to mix, then incubate at 37°C for 30 min;

[0045] 5) Add 200 μL of 0.4 mol / L sodium hydroxide to each well;

[0046] 6) The 96-well plate was gently shaken to mix and placed at room temperature for 15 min. The ELISA reader was set to a wavelength of 510 nm and the OD value of each well was measured.

[0047] 2. Determination of aspartate aminotransferase concentration

[0048] 1) Take the mouse plasma sample out of the -80°C freezer and place it in an ice box to melt;

[0049] 2) Add 20 μL of matrix solution to each of the assay well and control well, and add 5 μL of the sample to be tested to the assay well;

[0050] 3) Gently shake the 96-well plate to mix well, then place it in an incubator at 37°C and incubate for 30 minutes;

[0051] 4) Add 2,4-dinitrophenylhydrazine solution to the assay well and control well, respectively, and add 5 μL of the sample to be tested to the control well;

[0052] 5) Place the 96-well plate on the table and shake gently to mix, then incubate at 37°C for 30 min;

[0053] 6) Add 200 μL of 0.4 mol / L sodium hydroxide to each well;

[0054] 7) Mix well and place at room temperature for 15 minutes. Turn on the microplate reader in advance and measure the OD value of each well at a wavelength of 510 nm.

[0055] 3. Determination of total cholesterol concentration

[0056] 1) Take out the 96-well plate, add 2.5 μL of distilled water to the blank well, add 2.5 μL of sample to the sample well, and add 250 μL of working solution to each well, ensuring that all samples are accurately added to the bottom of the well to avoid bubbles;

[0057] 2) Incubate in a 37°C incubator for 10 min and set the microplate reader to measure at a wavelength of 510 nm.

[0058] 4. Determination of triglyceride concentration

[0059] 1) Take out the 96-well plate, add 2.5 μL of distilled water to the blank well, add 2.5 μL of calibrator to the calibration well, and add 2.5 μL of sample to the sample well;

[0060] 2) Add working solution: add 250 μL of working solution to each well;

[0061] 3) After gently shaking the 96-well plate to mix, incubate at 37°C for 10 min and measure the absorbance of each well.

[0062] The results are as follows Figure 2As shown, the TG, TC, and fasting blood glucose levels in the liver tissues of WT HFD mice were increased, while those in the p425 group were lower than those in the WT HFD group.

[0063] The levels of ALT and AST in the serum of WT HFD mice were increased, and those in the p425 group were lower than those in the WT HFD group.

[0064] (3) H&E staining, oil red staining, F4 / 80 staining

[0065] 1. H&E staining steps:

[0066] 1) Dewaxing and hydration: The sections were dewaxed by soaking in xylene twice for 10 minutes each, then hydrated in anhydrous ethanol, 95%, 80%, and 70% ethanol for 5 minutes each, and then rinsed with distilled water to remove the ethanol.

[0067] 2) Hematoxylin staining: Soak the sections in hematoxylin solution for 5-10 minutes, adjust the staining depth according to the intensity, and rinse with tap water to remove excess dye;

[0068] 3) Differentiation and blueing: The sections were differentiated in 1% hydrochloric acid ethanol differentiation solution for a few seconds to 1 minute, rinsed with tap water, blued with weak alkaline aqueous solution, and rinsed again with tap water;

[0069] 4) Eosin staining: Soak the sections in eosin stain for 1 to 2 minutes, then rinse with tap water to remove excess dye;

[0070] 5) Dehydration and transparency: The sections underwent a stepwise dehydration treatment from 70%, 80% to 95% ethanol and anhydrous ethanol, each step lasting 5 minutes, followed by two immersions in xylene for 10 minutes each to achieve transparency of the sections;

[0071] 6) Sealing: After the slices are dried, seal them with neutral gum to ensure that they are flat and free of bubbles;

[0072] 7) Observation and evaluation: All stained liver sections were observed using an optical microscope;

[0073] The fatty degeneration, inflammation and hepatocellular injury of liver tissue were evaluated according to the MAFLD activity score (NAS) criteria. The fatty degeneration was scored on a 0-3 scale according to the following criteria: 0 (<5%), 1 (5%-33%), 2 (33%-66%) or 3 (>66%). The lobular inflammation was scored on a 0-3 scale according to the following criteria: 0 (no lesions), 1 (<2 lesions per 20x field), 2 (2-4 lesions per 20x field) or 3 (>4 lesions per 20x field). The ballooning of hepatocellular degeneration was scored on a 0-2 scale according to the following criteria: 0 (none), 1 (mild, small amount) or 2 (moderate, large amount).

[0074] 2. Oil Red O Staining

[0075] 1) Preparation of Oil Red O working solution: Before use, mix the saturated Oil Red O stock solution and distilled water (or ddH2O) in a ratio of 3:2.

[0076] 2) For frozen sections, take out the sections from -20°C and let them stand at room temperature for 5-10 minutes to return to room temperature;

[0077] 3) Add the prepared Oil Red O working solution, cover the tissue sections, and stain at room temperature in the dark for 30 minutes (frozen sections). The specific time can be adjusted according to the depth of staining;

[0078] 4) Differentiation: discard the dye solution and immerse the sections in two cylinders of 60% isopropanol for 35 seconds (frozen sections) to remove excess dye solution;

[0079] 5) Washing: Immerse in two cylinders of pure water in turn (frozen sections) until the interstitium is clear and there is no excess stain;

[0080] 6) Re-staining: Add hematoxylin staining solution and immerse the slices in hematoxylin staining nuclei (frozen sections) to re-stain the cell nuclei. Wash, differentiate, wash, blue and then wash again (frozen sections) to remove excess hematoxylin staining solution;

[0081] 7) Sealing: After the water is slightly dried, add glycerol gelatin sealing agent to seal the slide;

[0082] 8) Observation: The staining results were observed under a microscope. Neutral fat appeared dark orange-red, and cell nuclei appeared blue or blue-purple. ImageJ software was used to quantify the degree of liver lipid deposition and the Oil Red O-positive area.

[0083] 3. Immunohistochemistry

[0084] 1) Dewaxing to water: The sections were dewaxed in environmentally friendly dewaxing solutions I, II, and III for 10 minutes each, and in anhydrous ethanol I, II, and III for 5 minutes each, and finally rinsed with distilled water;

[0085] 2) Antigen repair: After washing with PBS, select appropriate repair solution and conditions according to tissue characteristics, and cool naturally;

[0086] 3) Block endogenous enzymes: incubate in 3% hydrogen peroxide for 25 minutes in dark, then wash with PBS;

[0087] 4) Serum blocking: add 3% BSA to the tissue circle for blocking for 30 minutes;

[0088] 5) Add primary antibody: add KRT23 antibody and incubate overnight at 4°C;

[0089] 6) Add secondary antibody: After washing with PBS, add HRP-labeled secondary antibody and incubate at room temperature for 50 minutes;

[0090] 7) DAB color development: After washing with PBS, add DAB color development solution and control the color development time under a microscope;

[0091] 8) Nuclear counterstaining: counterstaining with hematoxylin, differentiation, blueing, and washing with running water;

[0092] 9) Dehydration and sealing: After gradient dehydration, n-butanol becomes transparent, and xylene finally becomes transparent, and the slides are sealed with sealing glue;

[0093] 10) Microscopic analysis: Digital scanning, Image J software analysis of AOD value to evaluate protein expression

[0094] The results are as follows Figure 3 As shown, H&E section observation showed that the liver tissue structure of the WT HFD group mice was significantly damaged, and the hepatic parenchymal cells showed vacuolar degeneration. The liver tissue structure of the p425 group was less damaged than that of the WT HFD group mice, and the hepatic parenchymal cells showed less vacuolar degeneration than that of the HFD group mice;

[0095] Oil-red results indicated that the WT HFD group had severe fat accumulation, while the p425 group had less fat accumulation than the HFD group mice;

[0096] The results of F4 / 80 showed that there was a certain amount of inflammatory cell infiltration in the WT HFD group, and the inflammatory cell infiltration in the p425 group was less than that in the WT HFD group. Compared with the WT HFD group, the level of F4 / 80 in p425 was lower (0.59±0.03 vs 0.35±0.02 ng / ml, P<0.0001), and the difference was statistically significant.

[0097] After 28 weeks of high-fat feeding, p425 was injected for 4 weeks, and the body weight changes were recorded weekly. p425 was compared with WT HFD, as shown in Figure 1 As shown, the body weight of mice decreased (40.18±7.17 vs 35.11±5.62 g, P<0.01); combined with Figure 2 i, p425 Compared with WTHFD, blood glucose was lower (11.54±1.62vs 6.22±0.97mmol / L, P<0.0001), and the difference was statistically significant.

[0098] Therefore, p425 intervention can significantly reduce the liver histological changes caused by high-fat diet feeding, and reduce the liver vacuolar degeneration and lipid droplet aggregation.

[0099] (4) Detection of inflammatory factors in blood

[0100] 1. Preparation before testing

[0101] 1) Select the sample from the -80℃ freezer, place it in an ice box to melt, and take the kit out of the freezer 20 minutes before the experiment and place it at room temperature for about 20 minutes;

[0102] 2) Dilute the washing solution: dilute the 20× concentrated washing solution with ddH2O at a volume ratio of 1:24;

[0103] 3) Standard working solution: First, centrifuge the standard at 10000g for 1 min, take 1mL of sample diluent to dissolve the standard to a concentration of 1000pg / mL, let it stand for 10min and then turn it upside down to mix thoroughly, taking care to avoid foaming, then use the method of multiple dilution as needed to prepare the following concentrations: 1000, 500, 250, 125, 62.5, 31.25, 15.63pg / mL;

[0104] 4) Before the experiment, calculate the amount needed for the experiment (calculated based on 100 μL per well). In actual operation, add 200 μL more to the calculated amount. Centrifuge the concentrated biotinylated antibody at 800g for 1 min, dilute it with biotinylated antibody diluent and concentrated biotinylated antibody at a volume ratio of 1:100, and set aside;

[0105] 5) Enzyme conjugate working solution: Before the experiment, calculate the amount needed for the experiment (calculated based on 100 μL per well). In actual operation, add 200 μL more to the calculated amount, centrifuge the concentrated HRP enzyme conjugate (800g, 1 min), dilute it with enzyme conjugate diluent and concentrated enzyme conjugate at a volume ratio of 1:100, and set aside.

[0106] 2. Operation steps

[0107] 1) Add 100 μL of sample diluent to the blank well, and add 100 μL / well of different concentrations of standard or sample to be tested to each of the remaining wells (each well has 2 replicates). When adding samples, add the samples to the bottom of the well. Then gently cover the ELISA plate with a transparent film and incubate it in a 37°C incubator for 90 minutes;

[0108] 2) Remove the film, pour out the liquid in the wells, add about 350 μL of washing solution to each well with a spray gun, shake off the liquid in the plate, and pat the liquid in the wells dry on absorbent paper. Repeat this operation 3 times. After washing the plate, add 100 μL of detection antibody working solution to each well, cover the ELISA plate with a sealing film, and incubate in a 37°C incubator for 60 minutes;

[0109] 3) Use a spray gun to add 100 μL of HRP conjugate working solution to each well, cover the ELISA plate with a sealing film, and incubate at 37°C in the dark for 30 minutes;

[0110] 4) Remove the film, pour out the liquid in the wells, and wash 4 times;

[0111] 5) Add 100 μL of substrate solution to each well, cover the ELISA plate with a sealing film, and incubate at 37°C in the dark for about 15 minutes (Note: If the color in the positive and negative control wells is too light, the color development time can be appropriately extended, but not more than 30 minutes), then terminate;

[0112] 6) Turn on the microplate reader in advance, set the wavelength to 450 nm and measure the OD of each well within 5 minutes.

[0113] The results are as follows Figure 4 As shown, ELISA assay showed that the levels of TNF-a, IL-6, IL-1b, INS, and HbA1C in the blood of mice in the WT HFD group were increased, while those in the p425 group were lower than those in the WT HFD group.

[0114] Inflammatory cytokines not only induce systemic insulin resistance, but also affect lipid metabolism. p425 alleviated HFD-induced obesity and significantly decreased the expression level of inflammatory factors in the blood of mice. Therefore, p425 reduces the level of chronic low-grade inflammation in the body by regulating the expression of inflammatory factors, thereby inhibiting obesity.

[0115] (5) Detection of protein and RNA levels of MIF, iNOS, and ARG1 in the liver

[0116] Flow cytometry was used to detect the types of macrophage polarization in liver tissue specimens of mice in the WTND group, WT HFD group, and p425 group. The results were as follows: Figure 5 As shown, the number of M1 macrophages in the WT HFD group mice was significantly increased, while the number of M1 macrophages in the p425 group mice was reduced compared with that in the WT HFD group mice.

[0117] from Figure 6 It can be seen that the protein and RNA levels of iNOS in the liver of the WT HFD group were significantly increased, and the protein and RNA levels of iNOS in the liver of the p425 group (0.76±0.22vs 0.45±0.22ng / ml, P<0.01) were significantly decreased;

[0118] The protein levels of ARG1 and RNA levels of CCR2 in the liver of the WT HFD group were significantly decreased, while the protein levels of ARG1 (0.56±0.24 vs 1.16±0.48 ng / ml) and RNA levels of CCR2 (1.18±0.18 vs 1.68±0.27, P<0.05) in the liver of the p425 group were significantly increased.

[0119] (6) Changes in MIF in each group of models

[0120] exist Figure 4In a, the serum MIF level in the WT HFD group was significantly increased, and the liver MIF level in the p425 group (30.41±3.12 vs 18.23±4.36 ng / ml, P<0.0001) was significantly decreased;

[0121] Combination Figure 6 and Figure 7 , the protein and RNA levels of MIF in the liver of the WT HFD group were significantly increased, and the protein and RNA levels of MIF in the liver of the p425 group (1.68±0.59 vs 0.61±0.18, P<0.05) were significantly decreased;

[0122] Combination Figure 3 c, The immunohistochemical results of liver tissues in the WT HFD group showed that the MIF level in the liver was significantly increased, while the MIF level in the p425 group was decreased (0.13±0.004 vs 0.12±0.007, P=0.0565).

[0123] In summary, injection of p425 can reduce the body weight of mice, lower blood lipids and blood sugar, and reduce inflammatory infiltration. Therefore, p425 can effectively improve the body weight, blood sugar, blood lipids, and liver lipid deposition of mice without liver damage.

[0124] Obviously, the above embodiments are merely examples for the purpose of clear explanation, and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived therefrom are still within the scope of protection of the claims of this patent application.

Claims

1. A drug for treating non-alcoholic fatty liver disease, characterized in that: The drugs include MIF-AntagonistV, p425.

2. The drug according to claim 1, characterized in that The fatty liver is obese non-alcoholic fatty liver induced by a high-fat diet.

3. The drug according to claim 1, characterized in that The content of MIF-Antagonist V, p425 in the medicine is 2 mg / kg.

4. The drug according to claim 1, characterized in that The drug improves non-alcoholic fatty liver by improving body weight, blood sugar, blood lipids, and liver lipid deposition.

5. The drug according to claim 1, characterized in that The drug improves non-alcoholic fatty liver disease by reducing the expression of inflammatory factors.

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