Application of isoquercitrin in preparation of medicine for improving insulin resistance and diabetes-related liver diseases

Abstract

The invention discloses an application of isoquercitrin in preparation of a medicine for improving insulin resistance and diabetes-related liver diseases, and belongs to the technical field of medicines. According to the application, a high-glucose and high-fat diet and streptozotocin combined method is adopted to successfully construct a type 2 diabetes mouse model; experiments find that the isoquercitrin can effectively relieve the symptoms of weight loss and water intake increase of T2DM mice, significantly reduce the fasting blood-glucose level and improve abnormal glucose tolerance; the isoquercitrin can reduce the liver index of a T2DM mouse, relieve fatty degeneration of the liver and repair a liver cell structure; by activating a PI3K / AKT signal channel, the isoquercitrin up-regulates the expression levels of PI3K, p-AKT, p-GSK3 and GLUT4 and down-regulates the expressions of GSK3 and PEPCK at the same time, so that the insulin resistance of T2DM mice is effectively improved, the fatty degeneration of the liver is improved, and the isoquercitrin has a protection or repair effect on liver injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and particularly relates to application of isoquercitrin in preparation of a medicine for improving insulin resistance and diabetes-related liver disease. BACKGROUND

[0002] Type 2 diabetes (T2DM) is an endocrine metabolic disorder characterized by abnormally elevated blood glucose levels, and has become one of the chronic diseases that seriously endanger human health. T2DM patients often have difficulty in effectively controlling blood glucose, resulting in high blood glucose, urine glucose and insulin levels, and typical symptoms including extreme thirst, frequent urination and weight loss. If not properly treated, it can cause complications such as kidney damage, poor circulation, foot numbness and cardiovascular disease.

[0003] Insulin resistance is a metabolic abnormality in which the body becomes less sensitive to insulin, reducing the efficiency of insulin in promoting glucose uptake and utilization. It is the core pathological basis of metabolic diseases such as type 2 diabetes and obesity, and is often associated with and synergistically progresses with non-alcoholic fatty liver disease (NAFLD). Diabetes-related liver disease is a series of liver diseases caused by insulin resistance (IR), hyperglycemia, lipid metabolism disorders and other core pathological changes of diabetes, leading to liver fat accumulation, inflammation and fibrosis. It is mainly characterized by excessive accumulation of liver fat, and can progress to non-alcoholic steatohepatitis (NASH), liver fibrosis, even cirrhosis and liver cancer. Its pathogenesis is closely related to lipid metabolism disorders, oxidative stress and inflammation caused by insulin resistance, and has become a major public health concern.

[0004] Current drugs for insulin resistance mainly include biguanides, thiazolidinedione insulin sensitizers, DPP-4 inhibitors, etc. Although these drugs can improve blood glucose control through different mechanisms, some have side effects such as gastrointestinal reactions and weight gain, and have limited effect on improving liver fat deposition. For non-alcoholic fatty liver disease, there is still a lack of specific drugs for clinical treatment. The first approved targeted drug, remogliflozin, is the first drug specifically approved for MASH, mainly improving liver steatosis and inflammation, but has limited effect on reversing liver fibrosis, and may have gastrointestinal adverse reactions such as diarrhea. It also faces challenges such as high price, limited effective rate and limited applicability. Existing drugs such as metformin can improve insulin resistance, but have no significant effect on reversing liver inflammation and fibrosis, and cannot effectively prevent or reverse the disease to a more severe stage (such as fibrosis and cirrhosis). Most candidate drugs are still in the clinical development stage. Therefore, developing new drugs that can improve insulin resistance and non-alcoholic fatty liver disease, have fewer side effects and high patient compliance has important clinical value and application prospects.

[0005] Isoquercitrin, also known as rubusoside, is a flavonol compound that exists widely in many medicinal plants such as Amomum villosum, mulberry leaves, rubus, Eclipta prostrata and so on, and can also be obtained by artificial synthesis. Isoquercitrin has the effects of antioxidant, anti-inflammatory and cardiovascular protection. At present, there is no report about the application of isoquercitrin in improving insulin resistance and non-alcoholic fatty liver disease, and the specific mechanism has not been clearly elucidated, so that the targeted drug application scheme cannot be formed. SUMMARY

[0006] The application provides an application of isoquercitrin in preparing a drug for improving insulin resistance and diabetes-related liver disease.

[0007] To achieve the above object, the technical scheme adopted by the application is as follows: The application of isoquercitrin in preparing a drug for improving insulin resistance and diabetes-related liver disease, and the structure formula is as follows: .

[0008] Further, the application of isoquercitrin in preparing a drug for slowing down the weight loss of a T2DM patient.

[0009] Further, the application of isoquercitrin in preparing a drug for relieving the polydipsia of a T2DM patient.

[0010] Further, the application of isoquercitrin in preparing a drug for reducing blood sugar and repairing impaired glucose tolerance.

[0011] Further, the application of isoquercitrin in preparing a drug for reducing liver steatosis and repairing liver cell structure.

[0012] Further, the isoquercitrin activates the PI3K / AKT signal pathway, up-regulates the expression levels of PI3K, p-AKT, p-GSK3 and GLUT4, and down-regulates the expression of GSK3 and PEPCK, so as to effectively improve insulin resistance and reduce liver steatosis and repair liver cell structure.

[0013] Further, in the above application, the dosage of isoquercitrin is 2.8-5.7 mg / kg.

[0014] Another object of the application is to provide a drug or health care product for improving insulin resistance.

[0015] Another object of the application is to provide a drug or health care product for improving diabetes-related liver disease.

[0016] Further, the effective component in the above drug or health care product includes isoquercitrin.

[0017] The medicine for preventing and treating insulin resistance and diabetes-related liver disease can be added to a medicine for preventing and treating insulin resistance and diabetes-related liver disease.

[0018] Further, the medicine or health product can be prepared into a tablet, granules, oral liquid, injection, etc. by using a conventional pharmaceutical excipient.

[0019] Compared with the prior art, the application has the following technical effects: The present application selects high-fat feed to induce insulin resistance in mice, and then injects STZ citrate solution to raise the blood glucose of mice, thereby successfully establishing a type 2 diabetes model (FBG≥11.1 mmol / L), with a model establishment rate of 75%. The effects of isoquercitrin on the body weight, water intake, FBG, IPGTT, FINS, HOMA-IR, liver index, liver tissue pathological structure, related protein expression in liver tissue, and related mRNA expression in liver tissue of T2DM mice are verified through experiments.

[0020] Through experiments, it is found that isoquercitrin can effectively alleviate the symptoms of weight loss and increased water intake in T2DM mice, significantly reduce the fasting blood glucose level (with a maximum reduction rate of 48.5%), and improve abnormal glucose tolerance.

[0021] Isoquercitrin can reduce the liver index of T2DM mice, reduce liver steatosis, and repair liver cell structure.

[0022] Isoquercitrin can activate the PI3K / AKT signaling pathway, up-regulate the expression levels of PI3K, p-AKT, p-GSK3, and GLUT4, and down-regulate the expression of GSK3 and PEPCK, thereby effectively improving the insulin resistance of T2DM mice.

[0023] Isoquercitrin can significantly improve the liver steatosis of T2DM mice and has a protective or repair effect on liver damage. BRIEF DESCRIPTION OF DRAWINGS

[0024] Figure 1 The flowchart of the experiment of the present application is shown in FIG. 1. Figure 2 FIG. 2 shows the effect of isoquercitrin on the body weight of T2DM mice (n=12). Figure 3 FIG. 3 shows the effect of isoquercitrin on the water intake of T2DM mice (n=12), compared with the blank group, ## p<0.01; **p<0.01, *p<0.05, compared with the model group; Figure 4 FIG. 4 shows the fasting blood glucose of mice before and after injection of STZ, compared with the blank group, ### p<0.001; Figure 5 Effect of isoquercitrin on FBG of T2DM mice (n=12), compared with the blank group, ## p<0.01; compared with the model group, **p<0.01, *p<0.05; Figure 6 Effect of isoquercitrin on IPGTT of T2DM mice (n=6), compared with the blank group, ## p<0.01; compared with the model group, **p<0.01, *p<0.05; Figure 7 Effect of isoquercitrin on IPGTT-AUC of T2DM mice (n=6); compared with the blank group, ### p<0.001; compared with the model group, **p<0.01, *p<0.05; Figure 8 Effect of isoquercitrin on FINS of T2DM mice (n=6); compared with the blank group, ### p<0.001; compared with the model group, ***p<0.001, **p<0.01; Figure 9 Effect of isoquercitrin on HOMA-IR of T2DM mice (n=6); compared with the blank group, ### p<0.001; compared with the model group, ***p<0.001; Figure 10 Effect of isoquercitrin on liver index of T2DM mice (n=12); compared with the blank group, #p<0.05; compared with the model group, **p<0.01, *p<0.05; Figure 11 Effect of isoquercitrin on the pathological structure of liver tissue of T2DM mice (40x); Figure 12 Expression levels of PI3K, p-AKT, AKT, p-GSK3β, GSK3β, GLUT4 proteins in the liver of experimental mice (n=3); (a): Effect of isoquercitrin on the expression of PI3K protein in the liver of T2DM mice; (b): Effect of isoquercitrin on the expression of p-AKT, AKT proteins in the liver of T2DM mice; (c): Effect of isoquercitrin on the expression of p-GSK3β, GSK3β proteins in the liver of T2DM mice; (d): Effect of isoquercitrin on the expression of GLUT4 protein in the liver of T2DM mice; compared with the blank group, ### p<0.001; compared with the model group, ***p<0.001, **p<0.01, *p<0.05; Figure 13 The relative expression of PI3K, AKT, PEPCK mRNA in the liver of experimental mice (n=3); (a) Effect of isoquercitrin on the expression of PI3K mRNA in the liver of T2DM mice; (b) Effect of isoquercitrin on the expression of AKT mRNA in the liver of T2DM mice; (c) Effect of isoquercitrin on the expression of PEPCK mRNA in the liver of T2DM mice; (d) Effect of isoquercitrin on the expression of PEPCK mRNA in the liver of T2DM mice; compared with the blank group, p<0.01; compared with the model group, **p<0.01. ## p<0.01; compared with the model group, **p<0.01. DETAILED DESCRIPTION

[0025] The conception, specific structure and technical effects achieved by the present application will be described in detail below with reference to the drawings, so as to understand the object, features and advantages of the present application more deeply.

[0026] The present application establishes a type 2 diabetes mouse model to study the improvement effect of isoquercitrin on type 2 diabetes mice in vivo and its mechanism.

[0027] 1. Experimental animals 140 SPF male healthy C57BL / 6J mice (body weight: 19±2 g) were purchased from Henan Zhongge Biotechnology Co., Ltd.

[0028] 2. Experimental instruments The instruments required for the experiment are shown in Table 1.

[0029] Table 1. Main instruments

[0030] 3. Experimental reagents The reagents and consumables used in the experiment are shown in Table 2.

[0031] Table 2. Experimental reagents and consumables

[0032] STZ citrate solution: concentration 1% (10 mg / ml), dissolve STZ powder in citric acid and sodium citrate buffer solution.

[0033] 0.5% carboxymethylcellulose sodium (CMC-Na) solution: weigh 0.25 g of CMC-Na powder into 50 ml of hot water under stirring (use a magnetic stirrer or a glass rod). Stir while pouring, until the powder is completely dispersed and dissolved, forming a translucent, uniform viscous colloidal solution. In the experiment, the oral gavage (i.g.) administration volume is 0.1 ml / 10 g body weight.

[0034] ​The isoquercitrin CMC-Na suspension is prepared by mixing a calculated amount of isoquercitrin with a calculated amount of 0.5% CMC-Na suspension.

[0035] Metformin CMC-Na suspension is prepared by mixing a calculated amount of metformin with a calculated amount of 0.5% CMC-Na suspension.

[0036] The dosage of drugs used in the experiment was calculated based on the weight of the experimental mice.

[0037] 4 Experimental Methods 4.1 Construction of animal models One hundred and forty male C57BL / 6J mice were subjected to a one-week acclimatization period, with the temperature maintained at 24±2℃, relative humidity controlled at 50%-70%, and a 12-hour light-dark cycle. During this period, the mice had free access to food and water. After one week, 12 mice were randomly selected as the blank control group and continued to be provided with maintenance diet, while the remaining 128 mice were fed a high-fat diet as the model group. After 6 weeks, the fasting blood glucose levels of all mice were measured. Subsequently, the mice in the high-fat diet group were injected intraperitoneally with 120 mg / kg STZ citrate solution after fasting. Fasting blood glucose (FBG) was measured in the mice using the tail clipping method on days 3, 7, and 14 after injection. Mice with two consecutive FBG ≥11.1 mmol / L were considered to have successfully established a diabetic model.

[0038] 4.2 Grouping and Dosing Regimen Mice that successfully modeled the disease were randomly divided into 8 groups using SPSS 21.0 software, with 12 mice in each group. The mouse groups were as follows: blank control group, model group, high-dose isoquercitrin group, medium-dose isoquercitrin group, low-dose isoquercitrin group, and metformin group. The specific drug administration regimens for each group were as follows: 1) Control group: 0.5% sodium carboxymethyl cellulose (CMC-Na) solution was administered by gavage daily, and feed and free access to water were maintained for 4 weeks.

[0039] 2) Model group: 0.5% CMC-Na solution was administered by gavage daily, and the group was fed a high-fat diet with free access to water for 4 weeks.

[0040] 3) High-dose isoquercitrin group (Is-H): 100 mg / kg isoquercitrin CMC-Na suspension was administered by gavage daily, fed on a high-fat diet with free access to water, for 4 weeks.

[0041] 4) Medium-dose group of isoquercitrin (Is-M): 50 mg / kg of isoquercitrin CMC-Na suspension was administered by gavage daily. The patient was fed a high-fat diet and had free access to water for 4 weeks.

[0042] 5) Isorhamnetin low-dose group (Is-L): 25 mg / kg of isorhamnetin CMC-Na suspension was administered intragastrically daily, high-fat feed was given, and free water was provided. The administration lasted for 4 weeks.

[0043] 6) Metformin group (Met): 97.5 mg / kg of metformin CMC-Na suspension was administered intragastrically daily, high-fat feed was given, and free water was provided. The administration lasted for 4 weeks.

[0044] 4.3 General observation, body weight, and water consumption measurement The mental state, fur color, food intake, water consumption, and urine output of mice in each group were observed at the same time every day, and the body weight and water consumption were recorded every week.

[0045] 4.4 Fasting blood glucose measurement The bedding was changed at 21:00 on Monday every week, and the mice were fasted. The next day at 9:00, the fasting blood glucose level of the mice was measured, and the differences in fasting blood glucose among the mice in different groups were compared.

[0046] 4.5 Intraperitoneal glucose tolerance test The bedding was changed at 19:00 the night before the experiment, and the mice were fasted. The next day at 9:00, the fasting blood glucose level of the mice was measured. Intraperitoneal glucose injection was performed at a dose of 2 g / kg, and the blood glucose concentration was measured at 15, 30, 60, and 120 min after injection. The area under the curve (AUC) of the blood glucose curve was calculated using GraphPad Prism software.

[0047] 4.6 Animal sacrifice and sample collection The mice were fasted for 12 h without water restriction, and the body weight of the mice was measured and recorded.

[0048] (1) Plasma: The blood was collected by enucleation, and the collected blood was placed in a centrifuge tube containing sodium heparin. After centrifugation, the supernatant was collected.

[0049] (2) Liver: The mouse abdominal cavity was opened using surgical scissors, and the liver was found using tissue forceps. The liver was completely removed. The liver was divided into three parts, one of which was placed in a 4% paraformaldehyde solution to fix the tissue; the other two were stored in a -80°C refrigerator for subsequent experiments.

[0050] 4.7 Liver index measurement During dissection, the mouse liver was removed and placed on a culture dish containing physiological saline to wash the surface blood. The liver weight was measured and recorded, and the mouse liver index was calculated according to Formula 1.

[0051] (1) 4.8 Plasma insulin content measurement and insulin resistance index calculation The fasting insulin (FINS) content of the mice was detected by enzyme-linked immunosorbent assay (ELISA). The operation was performed according to the method shown in the mouse insulin (INS) enzyme-linked immunodetection kit, and the experiment was repeated 3 times. The insulin resistance index (HOMA-IR) was calculated according to formula 2 to evaluate the degree of insulin resistance.

[0052] (2) 4.9 Liver histopathology observation Hematoxylin-eosin (H&E) staining: mouse liver → fixation → dehydration → wax immersion → embedding → sectioning → staining → observation of liver tissue morphology under an optical microscope.

[0053] 4.10 Western blotting detection of related protein expression in liver tissue Total protein extraction: Take an appropriate amount of liver tissue from the -80°C refrigerator, weigh, cut, and then put into a tissue homogenizer. Add tissue lysis solution containing protease inhibitors (RIPA:PMSF:protein phosphatase inhibitor = 100:1:1, v / v / v), grind on ice, and after grinding, transfer the tissue homogenate into a centrifuge tube and continue to lyse on ice for 30 min, then centrifuge for 15 min, and take the supernatant.

[0054] Protein quantification (BCA method): Transfer the centrifuged supernatant into a new centrifuge tube, add known concentration of standard protein sample into the 96-well plate, and dilute with PBS. Prepare BCA reaction solution according to the ratio of 50:1 (A liquid:B liquid) and add it into the centrifuge tube. After incubation at room temperature for 30 min, use the enzyme label instrument to detect the absorbance value, and draw the standard curve to calculate the protein concentration in the sample.

[0055] Protein denaturation: Add an appropriate amount of loading buffer to the sample for dilution, and then heat in boiling water at 100°C for 10 minutes to denature the protein.

[0056] SDS-PAGE gel electrophoresis: Prepare the separation gel and concentration gel according to the formula provided in the kit, place the polymerized gel into the electrophoresis tank, and add the electrophoresis buffer. After protein denaturation, add the sample to the loading well for electrophoresis.

[0057] Membrane transfer: Cut the PVDF membrane to an appropriate size, activate it with methanol, and then place it in the membrane transfer buffer. The sponge pad and filter paper need to be soaked in the membrane transfer buffer for 10 min in advance. According to the order of sponge pad-filter paper-PVDF membrane-gel-filter paper-sponge pad, stack them and place them in the membrane transfer device, add the membrane transfer solution, and transfer the membrane at a voltage of 100 V for 1 hour under ice bath conditions.

[0058] Blocking: Place the PVDF membrane in the TBST blocking solution containing 5% skimmed milk powder, and incubate on a shaking table at room temperature for 1 h.

[0059] Primary antibody incubation: add diluted primary antibody (1:1000), incubate overnight at 4°C. After incubation, wash the membrane with TBST three times, 10 min each time.

[0060] Secondary antibody incubation: add diluted secondary antibody (1:5000), incubate for 2 h at room temperature on a shaker. After incubation, wash the membrane with TBST three times, 10 min each time.

[0061] ECL luminescence method development: add pre-prepared ECL luminescence working solution, incubate for 3 min at room temperature on a shaker, then take out the membrane and expose it in a dark room.

[0062] Gray scale analysis: use Image J software to analyze the gray scale of the protein image and calculate the results.

[0063] 4.11 Real-time fluorescent quantitative PCR detection of related mRNA expression in liver tissue (1) Total RNA extraction Take 15 mg of liver tissue and put it into a grinding tube containing RNA extraction solution. Use a grinder to fully break up the sample, then centrifuge and collect the supernatant. Add chloroform substitute, centrifuge, then transfer the supernatant to a new centrifuge tube. Add isopropanol, centrifuge after standing at -20°C for 15 min, and the white precipitate at the bottom of the centrifuge tube is the RNA. Remove the supernatant and wash the RNA precipitate with 75% ethanol twice. Absorb the remaining liquid, add RNA dissolving solution, and detect the RNA concentration and purity. If the concentration is too high, dilute it appropriately.

[0064] (2) Reverse transcription The reverse transcription reaction system is shown in Table 3.

[0065] Table 3 PCR reaction system

[0066] Mix the above reagents, centrifuge, and use a PCR instrument to react at 25°C for 5 min, 42°C for 30 min, and 85°C for 5 s. Reverse transcribe the RNA into cDNA.

[0067] (3) Quantitative PCR The PCR reaction system is shown in Table 4.

[0068] Table 4 PCR reaction system

[0069] Add the reagents in Table 4 to the PCR reaction plate according to the volume, seal the membrane, centrifuge, and complete the amplification on a fluorescent quantitative PCR instrument. Use the ΔΔCT method to process the results.

[0070] 4.12 Statistical analysis The experimental results were plotted and statistically analyzed using GraphPad Prism 10.1.2 software. The data were expressed as mean ± standard deviation (mean ± SD), and the T test was used for comparison between two groups, and the One-Way ANOVA was used for comparison among multiple groups, with p<0.05 being statistically significant.

[0071] 5 Results and analysis Type 2 diabetes mellitus (T2DM) has become a chronic disease that seriously endangers human life and safety, but its pathogenesis is still unclear, so it is particularly important to establish an animal model that simulates the pathogenesis of human T2DM. The most widely used is the high-sugar high-fat diet combined with streptozotocin-induced induced T2DM mouse model. This animal model not only has low cost and short preparation period, but also has insulin resistance characteristics, and can be used to study the pathogenesis of T2DM and evaluate the hypoglycemic effect of drugs, which has important research value. Therefore, this application chooses to feed mice with high-fat feed to induce insulin resistance, and then inject STZ citrate solution to raise the blood glucose of mice (FBG≥11.1mmol / L) to successfully establish a type 2 diabetes mellitus model, with a success rate of 75%.

[0072] 5.1 General situation Before modeling, the fur of mice in each group was black and shiny, and the behavior was active, and the water intake, food intake and urine volume were all at normal levels. After successful modeling, in addition to the normal group, the rest of the mice showed varying degrees of listlessness, dull and disheveled fur, and symptoms of polydipsia, polyphagia, polyuria and weight loss (i.e. "three more and one less"). After intervention with different doses of metformin and isoquercitrin, the "three more and one less" symptoms of mice in the drug groups were relieved to some extent, and the hair glossiness was also improved compared with the model group.

[0073] 5.2 Effect of isoquercitrin on body weight of T2DM mice The effect of isoquercitrin on body weight of T2DM mice is shown in Table 5, Figure 2 During the experiment, the body weight of mice in the Control group increased steadily, with a growth rate of 37.0%; after injection of STZ, the average body weight of mice in the Model group decreased from 27.0g to 25.0g. After 4 weeks of drug intervention, compared with the Control group, the body weight of mice in the Model group continued to decrease (P<0.001), which was consistent with the symptoms of type 2 diabetes; the body weight of mice in the Is-H group, Is-M group, Is-L group and Met group increased significantly (P<0.05), among which the body weight of mice in the Is group showed a positive growth trend with the increase of the dose of the drug, and there was no significant difference in the body weight of mice between the Is group and the Met group. p <0.05), among which the body weight of mice in the Is group showed a positive growth trend with the increase of the dose of the drug, and there was no significant difference in the body weight of mice between the Is group and the Met group. p <0.05), among which the body weight of mice in the Is group showed a positive growth trend with the increase of the dose of the drug, and there was no significant difference in the body weight of mice between the Is group and the Met group.

[0074] Table 5 Effect of isoquercitrin on body weight of T2DM mice (unit g,

[0075] Compared with the blank group, ### p <0.001; compared with the model group, ** p <0.01, * p <0.05 The study showed that the body weight of mice fed with high-fat diet was significantly higher than that of mice fed with ordinary diet after 4 weeks; during the process of inducing insulin resistance of type 2 diabetes by diet combined with STZ, the body weight of mice was significantly reduced after receiving STZ injection for two weeks compared with before injection. Table 5 and Figure 2 showed that the body weight of mice in the model group was gradually higher than that in the blank control group during the stage of inducing insulin resistance in mice by high-sugar high-fat diet (1-7w), because high-sugar high-fat diet was more likely to cause obesity in mice. After injection of STZ citrate solution, the body weight of mice in the model group was significantly lower than that in the blank group, because the function of pancreatic islets in T2DM mice was impaired, which led to the body unable to effectively use glucose as an energy source, and instead relied on the decomposition of fat and protein to obtain energy, resulting in the decrease of body weight of mice. The body weight of T2DM mice after intervention of isoquercitrin and metformin was significantly higher than that of the model group (p<0.05), indicating that isoquercitrin could effectively slow down the decrease of body weight of T2DM mice to a certain extent. p <0.05), indicating that isoquercitrin could effectively slow down the decrease of body weight of T2DM mice to a certain extent.

[0076] 5.3 Effect of isoquercitrin on water intake of T2DM mice The effect of isoquercitrin on water intake of T2DM mice is shown in Figure 3 During the experiment, the Control group of mice drank water normally, with an average daily water intake of 7.8 mL per mouse. After injection of STZ, the water intake of mice in the remaining groups increased sharply compared with the Control group, with an average daily water intake of 24.2 mL per mouse. After 4 weeks of drug intervention, compared with the Model group, the water intake of mice in the Is-H, Is-M, Is-L and Met groups was significantly decreased (p<0.01), with a decrease rate of 33.5%, 24.4%, 19.8% and 31.5%, respectively; there was no significant difference in water intake between the Is-H and Is-M groups and the Met group.

[0077] Figure 4 , 5 showed that after the T2DM mice were successfully modeled, the daily water intake was significantly higher than that of the blank group (p<0.01). p ​<0.01%, because excessively high blood glucose concentration prevents the renal tubules from completely reabsorbing filtered glucose. Excess glucose is then excreted in the urine, increasing urine osmotic pressure and causing osmotic diuresis. Water loss leads to thirst and frequent drinking. Water intake in T2DM mice significantly decreased after intervention with isoquercitrin and metformin. p The result was <0.01, indicating that isoquercitrin can improve the polydipsia symptoms in T2DM mice.

[0078] 5.4 Effects of isoquercitrin on FBG in T2DM mice After 6 weeks of high-fat feeding, fasting blood glucose levels in mice were measured. The average blood glucose concentration in the Model group was 8.2 mmol / L, which was higher than that in the Control group. At week 7, mice were intraperitoneally injected with 120 mg / kg STZ citrate solution. Fasting blood glucose levels were measured at weeks 8 and 9. The results showed that the average blood glucose concentration in the Model group was 20.8 mmol / L, significantly higher than that in the Control group (p<0.001). Figure 4 A total of 96 mice were successfully modeled, with a modeling success rate of 75%.

[0079] The effect of isoquercitrin on fasting blood glucose in T2DM mice is as follows: Figure 5 As shown: After 4 weeks of drug intervention, compared with the control group, the fasting blood glucose level of mice in the model group remained elevated (p<0.01); compared with the model group, the fasting blood glucose levels of mice in the Is-H group, Is-M group, Is-L group and Met group were significantly reduced (p<0.01), with reduction rates of 48.5%, 40.3%, 25.4% and 31.3%, respectively.

[0080] STZ can specifically destroy mouse pancreatic islets Cellular dysfunction leads to a significant reduction in insulin secretion, thereby raising blood sugar levels. Figure 4 The results showed that the average blood glucose concentration in the model group mice was 20.8 mmol / L two weeks after injection of STZ citrate solution, which was significantly higher than that in the control group mice. p <0.001), successfully establishing a type 2 diabetes model. Figure 5 The results showed that after 4 weeks of intervention with isoquercitrin and metformin, fasting blood glucose levels in T2DM mice were significantly reduced. p <0.01), among which, the fasting blood glucose of mice in the high-dose isoquercitrin group decreased most significantly, with a decrease rate of 48.5%.

[0081] 5.5 Effects of isoquercitrin on IPGTT in T2DM mice Mice underwent the IPGTT test at week 14, and the results were as follows: Figure 6As shown: the fasting blood glucose level of Model group mice was significantly higher than that of Control group and drug groups (p<0.01) before injection of glucose; the blood glucose values of mice in each group rapidly increased and reached the peak value after 30 min of injection of glucose; the blood glucose value of Control group mice showed a significant downward trend (p<0.01) after 60 min of injection of glucose; the blood glucose of Is-H group, Is-M group, Is-L group and Met group mice all significantly decreased (p<0.05) after 120 min of injection of glucose.

[0082] According to the IPGTT curve, the area under the curve (AUC) can be calculated Figure 7 Results: Compared with Control group, the AUC of Model group mice significantly increased (p<0.01); compared with Model group, the AUC of Is-H group, Is-M group, Is-L group and Met group all significantly decreased (p<0.01).

[0083] IPGTT can judge the body's ability to regulate blood glucose by measuring the blood glucose level at fasting and at a certain time point after injection of glucose, and it is an important basis for diagnosing diabetes, and can also be used to evaluate insulin secretion function and glucose metabolism status. IPGTT-AUC refers to the area enclosed by IPGTT curve and time axis, which can reflect the overall change of blood glucose of an individual within a certain time, and is an important indicator for evaluating insulin secretion function and glucose metabolism status. Higher AUC value usually indicates an increased risk of abnormal glucose tolerance or diabetes. In normal glucose tolerance test, the fasting blood glucose before injection of glucose should not be higher than 6.1 mmol / L, and the blood glucose should be reduced to below 7.8 mmol / L after 2 h of injection of glucose. In this experiment, only the mice in the blank group met the standard requirements; the fasting blood glucose of Model group mice was significantly higher than that of the blank group and drug groups (p<0.001), reached the peak value of blood glucose meter after 30 min of injection of glucose, and the blood glucose did not decrease significantly after 120 min, indicating that the mice had hyperglycemia and severe insulin resistance; the blood glucose of drug groups mice decreased significantly (p<0.05) after 120 min of injection of glucose compared with Model group, indicating that isorhamnetin and metformin can reduce the blood glucose of T2DM mice, repair impaired glucose tolerance, and alleviate insulin resistance in mice. p <0.001), reached the peak value of blood glucose meter after 30 min of injection of glucose, and the blood glucose did not decrease significantly after 120 min, indicating that the mice had hyperglycemia and severe insulin resistance; the blood glucose of drug groups mice decreased significantly (p<0.05) after 120 min of injection of glucose compared with Model group, p <0.05), indicating that isorhamnetin and metformin can reduce the blood glucose of T2DM mice, repair impaired glucose tolerance, and alleviate insulin resistance in mice. Figure 6 5.6 Effect of isorhamnetin on FINS and HOMA-IR of T2DM mice

[0084] The effect of isorhamnetin on FINS and HOMA-IR of T2DM mice is as follows , Figure 8 , 9As shown: Compared with the control group, the FINS and HOMA-IR of mice in the Model group were significantly increased (p<0.001); compared with the Model group, the FINS of mice in the Is-H group and Is-M group were significantly decreased (p<0.05); and the HOMA-IR of mice in the Is-H group, Is-M group, Is-L group and Met group were all significantly decreased (p<0.001).

[0085] Abnormal insulin secretion by pancreatic β cells is a typical pathological feature of type 2 diabetes. This is because insulin resistance reduces the body's sensitivity to insulin. Cells need to compensate by secreting more insulin to maintain stable blood sugar levels. Figure 8 The results showed that the fasting plasma insulin level in the model group was significantly higher than that in the blank group. p <0.001); After 4 weeks of intervention with isoquercitrin and metformin, plasma insulin levels in diabetic mice were significantly lower than those in the model group. p <0.05). HOMA-IR is an indicator for assessing the degree of insulin resistance; a higher HOMA-IR indicates more severe insulin resistance. Figure 9 The results showed that, compared with the model group, the HOMA-IR levels in the drug-treated mice were significantly reduced ( p The result was <0.001, indicating that isoquercitrin can effectively reduce fasting insulin levels in T2DM mice and improve insulin resistance.

[0086] 5.7 Effects of isoquercitrin on liver index in T2DM mice The liver index was calculated based on the ratio of mouse liver weight to pre-sacrifice body weight. The results are shown in Table 6 and... Figure 10 The mice in the Model group had a decreased body weight but an increased liver weight, so their liver index was significantly higher than that of the Control group (p < 0.05); compared with the Model group, the liver index of mice in the Is-H group, Is-M group and Met group was significantly lower (p < 0.05).

[0087] Table 6. Effects of isoquercitrin on liver index in T2DM mice (unit: g, )

[0088] Compared with the blank group, # p < 0.05; compared with the model group, **p < 0.01, *p < 0.05 5.8 Effects of isoquercitrin on the pathological structure of liver tissue in T2DM mice H&E staining results showed ( Figure 11In the Control group, hepatocytes were neatly arranged and morphologically normal, with clear nuclei and no fatty degeneration observed. In contrast, hepatocytes in the Model group were disordered, exhibiting significant fatty degeneration accompanied by numerous vacuoles. After 4 weeks of drug treatment, compared with the Model group, the degree of hepatic fatty degeneration in both the Is and Met groups was alleviated to varying degrees.

[0089] Diabetic patients often experience lipid metabolism disorders, which can lead to fat accumulation in the liver, causing varying degrees of liver enlargement and ultimately resulting in pathological damage to liver tissue. (Table 6 and...) Figure 9 The results showed that the liver index of the model group mice was significantly higher than that of the normal group. p <0.05%, because diabetes leads to weight loss and increased liver weight; isoquercitrin and metformin can slow down weight loss and alleviate liver enlargement in T2DM mice, and reduce the liver index in mice. H&E staining showed that isoquercitrin can significantly improve steatosis in the liver of T2DM mice and has a protective or repairing effect on liver damage. Figure 11 ).

[0090] 5.9 Effects of isoquercitrin on the expression of related proteins in liver tissue of T2DM mice The effects of isoquercitrin on the expression of PI3K, p-AKT, AKT, p-GSK3β, GSK3β, and GLUT4 in liver tissue were detected by Western blotting. The results are as follows: Figure 12 As shown: Compared with the Control group, the expression level of PI3K protein in the Model group mice was significantly decreased (p < 0.001); compared with the Model group, the expression level of PI3K protein in the drug-treated groups was significantly increased (p < 0.05). Compared with the Control group, the expression level of p-AKT protein in the Model group mice was significantly decreased (p < 0.001); compared with the Model group, the expression level of p-AKT protein in the drug-treated groups was significantly increased (p < 0.05). Compared with the Control group, the expression level of p-GSK3β protein in the Model group mice was decreased (p < 0.001); compared with the Model group, the expression level of p-GSK3β protein in the Is-H, Is-M, and Met groups was increased (p < 0.01). Compared with the Control group, the expression level of GLUT4 protein in the Model group mice was significantly downregulated (p < 0.001); compared with the Model group, the expression level of GLUT4 protein in the Is-H, Is-M, and Met groups was significantly upregulated (p < 0.05).

[0091] 5.10 Effects of isoquercitrin on the expression of related mRNAs in liver tissue of T2DM mice The effects of isoquercitrin on the mRNA expressions of PI3K, AKT, GSK3β, PEPCK and GLUT4 in liver tissues were detected by RT-qPCR technology, and the results are shown in Figure 13 As compared with the Control group, the PI3K mRNA expression in the Model group was significantly reduced (p<0.01); as compared with the Model group, the PI3K mRNA expression in the drug groups was significantly increased (p<0.01). As compared with the Control group, the AKT mRNA expression in the Model group was significantly reduced (p<0.01); as compared with the Model group, the AKT mRNA expression in the Is-H, Is-M and Met groups was significantly increased (p<0.05). As compared with the Control group, the GSK3β mRNA expression in the Model group was increased, but there was no significant difference; as compared with the Model group, the GSK3β mRNA expression in the Is-H, Is-M and Met groups was significantly reduced (p<0.05). As compared with the Control group, the PEPCK mRNA expression in the Model group was significantly increased (p<0.05); as compared with the Model group, the PEPCK mRNA expression in the drug groups was significantly reduced (p<0.01).

[0092] In the present application, the protein and mRNA expressions related to the PI3K / AKT signal transduction pathway in the liver tissues of mice were detected by Western Blot and RT-qPCR. The Western Blot results show that, as compared with the Model group, the PI3K and p-AKT protein expressions in the isoquercitrin groups were significantly increased (p<0.01); as compared with the Model group, the p-GSK3β and GLUT4 protein expressions in the isoquercitrin high and medium dose groups were significantly increased (p<0.05). p <0.01). p <0.01). Figure 12 The RT-qPCR results show that, as compared with the Model group, the PI3K mRNA expression in the isoquercitrin groups was significantly increased (p<0.01); as compared with the Model group, the AKT mRNA expression in the isoquercitrin high and medium dose groups was significantly increased (p<0.05); as compared with the Model group, the GSK3β mRNA expression in the isoquercitrin high and medium dose groups was significantly reduced (p<0.05); the PEPCK mRNA expression in the isoquercitrin groups was significantly reduced (p<0.01). p <0.01). p <0.05). p <0.01). p <0.01). Figure 13 .

Claims

1. Application of isoquercitrin in the preparation of drugs to improve insulin resistance and diabetes-related liver disease.

2. The application of isoquercitrin according to claim 1 in the preparation of drugs for improving insulin resistance and diabetes-related liver disease, characterized in that: The application of isoquercitrin in the preparation of drugs to slow down weight loss in patients with type 2 diabetes mellitus (T2DM).

3. The application of isoquercitrin according to claim 1 in the preparation of drugs for improving insulin resistance and diabetes-related liver disease, characterized in that: The application of isoquercitrin in the preparation of drugs to relieve thirst in patients with type 2 diabetes mellitus (T2DM).

4. The application of isoquercitrin according to claim 1 in the preparation of drugs for improving insulin resistance and diabetes-related liver disease, characterized in that: The application of isoquercitrin in the preparation of drugs that lower blood sugar and repair impaired glucose tolerance.

5. The application of isoquercitrin according to claim 1 in the preparation of drugs for improving insulin resistance and diabetes-related liver disease, characterized in that: Application of isoquercitrin in the preparation of drugs that reduce hepatic steatosis and repair hepatocyte structure.

6. The application of isoquercitrin according to claim 1 in the preparation of drugs for improving insulin resistance and diabetes-related liver disease, characterized in that: Isoquercitrin activates the PI3K / AKT signaling pathway, upregulating the expression levels of PI3K, p-AKT, p-GSK3, and GLUT4, while downregulating the expression of GSK3 and PEPCK, thereby effectively improving insulin resistance, alleviating hepatic steatosis, and repairing hepatocyte structure.

7. The use of the active ingredient of Prunus mume according to any one of claims 1-6 in the preparation of drugs for improving insulin resistance and diabetes-related liver disease, characterized in that: The dosage of isoquercitrin is 2.8~5.7 mg / kg.

8. A drug or health product for improving insulin resistance, characterized in that: Its active ingredient includes isoquercitrin.

9. A drug or health product for improving diabetes-related liver disease, characterized in that: Its active ingredient includes isoquercitrin.

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