CN112494487A

CN112494487A - Application of hirsutine in preparation of medicines for improving insulin resistance, diabetes and complications thereof

Info

Publication number: CN112494487A
Application number: CN202011306445.4A
Authority: CN
Inventors: 朱依谆; 胡葳; 朱依纯
Original assignee: Macau University of Science and Technology
Current assignee: Macau University of Science and Technology
Priority date: 2020-11-19
Filing date: 2020-11-19
Publication date: 2021-03-16

Abstract

The invention discloses application of hirsutine in preparation of medicines for improving insulin resistance, diabetes and complications thereof, and relates to the technical field of medicines. The hirsutine has certain treatment effect on insulin resistance, diabetes and complications thereof, can exert drug effect at low concentration, has no cytotoxicity, can effectively improve insulin resistance, treat type II diabetes and reverse liver steatosis, and provides a new treatment method and a new treatment medicine for clinically treating type II diabetes.

Description

Application of hirsutine in preparation of medicines for improving insulin resistance, diabetes and complications thereof

Technical Field

The invention relates to the technical field of medicines, in particular to application of hirsutine in preparing medicines for improving insulin resistance, diabetes and complications thereof.

Background

Diabetes is a metabolic disease with hyperglycemia as a main characteristic, wherein the percentage of type II diabetes is more than 90 percent, insulin resistance is taken as a central factor, and cardiovascular disease complications such as fatty liver, dyslipidemia, hypertension, atherosclerosis and the like are mostly accompanied to different degrees, so that the physical and psychological health of human beings is seriously threatened, heavy economic burden is caused to countries and individuals, and the public health is greatly threatened. Type II diabetes is more frequent among older people and has a great tendency to be younger.

As a common traditional Chinese medicine in the application history of traditional Chinese medicines, uncaria has been proved by clinical experience and modern research to have the function of resisting hypertension, and the extract of uncaria has obvious curative effect on cardiovascular and central nervous system diseases. The monomer extract hirsutine is proved to have the effects and activities of reducing blood pressure, resisting arrhythmia, resisting myocardial ischemia and the like.

The antidiabetic drugs which are generally used clinically at present mainly comprise insulin, metformin, sulfonylurea drugs, thiazolidinedione drugs which are recently marketed and the like, and although the curative effect is good, the drugs still have the problems of easy generation of hepatotoxicity, weight gain, incomplete control of glycosylated hemoglobin level and hypoglycemia and the like after long-term use. Therefore, the extraction of more effective natural compounds from traditional Chinese medicines to develop new therapeutic drugs becomes a focus of attention, and the development of safer and more effective antidiabetic drugs for clinical treatment has important significance. At present, the application of hirsutine to type II diabetes has not been reported.

Disclosure of Invention

The present invention is directed to solving at least one of the problems in the prior art. Therefore, the invention provides the application of hirsutine in preparing the medicines for improving insulin resistance, diabetes and complications thereof, and the medicines have good treatment effect on the diabetes and related diseases.

The invention also provides the application of the hirsutine in preparing an activator for improving insulin resistance, diabetes and related pharmacological approaches of the medicaments for complicating diseases of the diabetes and the diabetes.

Use of hirsutine according to an embodiment of the first aspect of the invention for the manufacture of a medicament for improving insulin resistance, diabetes and complications thereof.

According to some embodiments of the invention, the Hirsutine (Hirsutine) has the structural formula:

according to some embodiments of the invention, the hirsutine has a molecular weight of 368.47.

According to some embodiments of the invention, the hirsutine is extracted from the traditional Chinese medicine uncaria.

According to some embodiments of the invention, the diabetes is type II diabetes.

According to some embodiments of the invention, the complication comprises a disorder of lipid metabolism and/or nephropathy; preferably, the disorder of lipid metabolism disease is nonalcoholic fatty liver disease.

According to some embodiments of the invention, the agent for treating insulin resistance, diabetes and complications thereof is an agent that inhibits the expression of gluconeogenesis-associated genes G6Pase, PEPCK or PGC-1 α of cells in an insulin resistant state.

According to some embodiments of the invention, the medicament further comprises a pharmaceutically acceptable carrier or excipient.

According to some embodiments of the present invention, the dosage form of the drug is various dosage forms conventional in the art, preferably in solid, semi-solid or liquid form, and may be an aqueous solution, a non-aqueous solution or a suspension, more preferably a tablet, a capsule, a soft capsule, a granule, a pill, an oral liquid, a dry suspension, a drop pill, a dry extract, an injection or an infusion.

According to some embodiments of the present invention, the mode of administration of the drug may be a mode of administration conventional in the art, including but not limited to injection or oral administration. The injection can be intravenous injection, intramuscular injection, intraperitoneal injection, intradermal injection or subcutaneous injection.

Use of hirsutine according to an embodiment of the second aspect of the invention for the preparation of an activator for improving pharmacological pathways related to insulin resistance, diabetes and complications thereof.

According to some embodiments of the invention, the pharmacological pathway is the PI3K/Akt/GSK3 β pathway.

According to some embodiments of the invention, the use of hirsutine for the preparation of activators of the PI3K/Akt/GSK3 β signaling pathway.

According to some embodiments of the invention, the application is activation of Akt, PDK1 and GSK3 β protein expression.

The application of hirsutine in preparing the medicines for improving insulin resistance, diabetes and complications thereof according to the embodiment of the invention at least has the following beneficial effects: the hirsutine provided by the scheme of the invention can be used for preparing medicines for improving insulin resistance, diabetes and fatty liver or PI3K/Akt/GSK3 beta signal pathway activators, can exert drug effects at low concentration, has no cytotoxicity, can effectively improve insulin resistance, treat type II diabetes and reverse liver steatosis combined with diabetes.

Drawings

FIG. 1 is a graph showing the results of glucose consumption in HepG2 model of insulin resistance induced by hirsutine in the state of high glucose and high insulin (high glucose and high pancreas for short in Chinese) at different times in example 2 of the present invention, wherein, &, &isp <0.05 and p <0.01 compared with the con group, respectively, and p <0.05 and p <0.01 compared with the HGHI model group, respectively;

FIG. 2 is a graph showing the results of glucose uptake by HepG2 cells under the conditions of increased insulin resistance by hirsutine at various concentrations in example 2 of the present invention, wherein p <0.05 compared to the con group, and p <0.05 and p <0.01 compared to the HGHI model group, respectively;

FIG. 3 is a graph showing the results of the effect of hirsutine at different concentrations on the hepatic glucose output of HepG2 cells under conditions of decreased insulin resistance in example 2 of the present invention, wherein & & is p <0.01 as compared to the con group, and p <0.05 and p <0.01 as compared to the HGHI model group, respectively;

FIG. 4 is a graph showing the results of increasing glycogen content in HepG2 cells under insulin resistance conditions by hirsutine at various concentrations in example 2 of the present invention, wherein p <0.01 in comparison with the con group, and p <0.05 and p <0.01 in comparison with the HGHI model group, respectively;

FIG. 5 is a graph showing the results of gene expression related to gluconeogenesis in the inhibition of hirsutine in example 2 of the present invention, wherein Panel A is a G6Pase gene expression graph, Panel B is a PEPCK gene expression graph, and the & representation is p <0.01 as compared with the con group, and p <0.05, p <0.01 and p <0.001 as compared with the HGHI model group, respectively;

fig. 6 is a graph showing the results of the inhibition of hirsutine-associated gene expression with gluconeogenesis in example 2 of the present invention, wherein a is a PGC-1 α gene expression map, B is a FoxO1 gene expression map, and & & represents p <0.01 as compared with the con group, and p <0.05, p <0.01 and p <0.001 as compared with the HGHI model group, respectively;

FIG. 7 is a graph showing the effect of hirsutine in example 2 on the expression level of glucose transporter GLUT2 in HepG2 cells in an insulin resistant state;

FIG. 8 is a graph showing the effect of hirsutine in example 2 on glycolytic capacity of HepG2 cells in insulin resistant state, wherein graph A is extracellular acidification rate at different times, graph B is extracellular acidification rate at different degrees of glycolysis, representing p <0.05 compared to HGHI model group;

FIG. 9 is a graph showing the effect of hirsutine in example 3 on the phosphorylation levels of Akt, PDK1 and GSK3 β in HepG2 cells under insulin resistance;

fig. 10 is a graph showing the results of the effect of PI3K inhibitor on glucose uptake by HepG2 cells under conditions of inhibition of hirsutine-enhanced insulin resistance in example 3 of the present invention, wherein, & indicates p <0.05 compared to the con group, & indicates p <0.01 and p <0.001 compared to the HGHI model group, respectively, & # and # & indicates p <0.01 and p <0.001 compared to the respective concentrations of the hirsutine-administered group;

FIG. 11 is a graph showing the results of the expression and phosphorylation levels of PI3K, Akt and GSK3 beta proteins in HepG2 cells under the condition that the PI3K inhibitor of example 3 of the present invention inhibits the stimulation of insulin resistance by hirsutine;

FIG. 12 is a graph showing the effect of hirsutine on body weight of db/db diabetic mice in example 4 of the present invention;

FIG. 13 is a graph of the effect of various concentrations of hirsutine in example 4 of the present invention on fasting plasma glucose levels in db/db diabetic mice, where p <0.01 and p <0.001, respectively, compared to the HGHI model group;

fig. 14 is a graph showing the effect of hirsutine on the oral glucose tolerance (1mg/kg gavage concentration of glucose) of db/db diabetic mice in example 4 of the present invention, wherein a is a graph showing the change in blood glucose level of diabetic mice at different times after gavage, and B is a graph showing the change in oral glucose tolerance of diabetic mice. # is p <0.001 compared to WT group, and # are p <0.01 and 0.001 compared to db/db group, respectively;

FIG. 15 shows the effect of hirsutine on insulin (1 Ukg) in db/db diabetic mice according to example 4 of the present invention^-1Insulin intraperitoneal injection concentration), wherein, the graph A is a graph of the change of blood glucose value at different time after the injection of insulin, the graph B is a graph of the result of the influence of the insulin resistance of db/db diabetic mice, and # is p compared with WT group<0.001, and p are compared to db/db group<0.01 and 0.001;

FIG. 16 is a graph showing the effect of hirsutine in example 4 of the present invention on the appearance of liver in db/db diabetic mice;

FIG. 17 is a graph showing the results of the effect of hirsutine in example 4 of the present invention on hepatic coefficients of db/db diabetic mice, # # is p <0.001 compared to the WT group, # and # are p <0.05 compared to db/db group, p <0.01 and 0.001, respectively;

FIG. 18 is a graph showing the effect of hirsutine on lipid deposition in liver of diabetic mice in example 4 of the present invention (upper scale: 300. mu.M, lower scale: 100. mu.M);

FIG. 19 is a graph showing the effect of hirsutine on the degree of fatty liver pathology, degree of cell swelling and degree of inflammation in the liver of diabetic mice (upper scale: 300. mu.M, lower scale: 100. mu.M) in example 4 of the present invention;

FIG. 20 is a graph showing the results of the effect of hirsutine on the activity score of nonalcoholic fatty liver in diabetic mice according to example 4 of the present invention, wherein, # # is p <0.01 in the con group, and x are p <0.05 and 0.01 in the model group, respectively;

FIG. 21 is a graph showing the effect of hirsutine on islet cells of db/db diabetic mice in example 4 of the present invention (upper scale: 300. mu.M, lower scale: 50. mu.M);

FIG. 22 is a graph showing the effect of hirsutine in example 4 of the present invention on the kidney of diabetic mice (upper scale: 300. mu.M, lower scale: 50. mu.M).

Detailed Description

In order to explain the technical content, the objects and the effects of the present invention in detail, the following description will be given with reference to the embodiments. The test methods used in the examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are commercially available reagents and materials unless otherwise specified. The hirsutine used in examples 1-3 below was purchased from Douglas Biotech, Inc. under CAS number 7729-23-9. Example 4 the use of hirsutine was purchased from Shanghai Shidande Standard technical services, Inc.

Example 1: high-sugar and high-insulin induction HepG2 cells to generate insulin resistance

The HepG2 cell is a human-derived hepatoblastoma cell, retains many characteristics of the hepatocyte, has a phenotype very similar to that of the hepatocyte, and is an ideal cell line for researching the pathogenesis of insulin resistance.

The HepG2 cells are induced for 36h by high-sugar high-insulin stimulation, so that the number of insulin receptors on the cell surface is reduced, and insulin resistance is formed, namely the glucose consumption and the glucose uptake of the cells are obviously inhibited compared with the cells in a normal state. And the insulin resistance model can be stable for 48 hours.

Example 2: effect of hirsutine on insulin resistant HepG2 intracellular carbohydrate homeostasis

1. The experimental steps are as follows: HepG2 cells were divided into 6 groups, as follows: (1) a control group, (2) a high-sugar high-insulin resistance model group, (3) a high-sugar high-insulin resistance model +0.01 mu M hirsutine, (4) a high-sugar high-insulin resistance model +0.1 mu M hirsutine, (5) a high-sugar high-insulin resistance model +1 mu M hirsutine, and (6) a high-sugar high-insulin resistance model +1mM metformin. Culture medium supernatants are collected respectively in 3h, 6h, 12h and 24h after administration, and glucose content of the culture medium supernatants is detected by a glucose oxidase kit (MO10) purchased from Nanjing to indirectly calculate the amount of glucose consumed by cells. The glucose uptake was expressed as the fluorescence of 2-NBDG, a fluorescent glucose analog. HepG2 cells were collected, disrupted using ultrasound, and then assayed for hepatic glycogen content at various concentrations of hirsutine by using a glycogen content assay kit (BC0345) purchased from Beijing Solebao technologies, Inc. After molding and administration, the culture solution is changed into sugar-free glucose output buffer solution (without phenol red, containing 2mM sodium pyruvate and 20mM sodium lactate) to culture the cells for 3h, and glucose oxidase kit is used for detecting the glucose content in the buffer solution to detect the glucose output of the hepatic cells so as to correspond to the gluconeogenesis degree. Then, the expression quantity of gluconeogenesis related genes in HepG2 cells, such as G6Pase, PEPCK and PGC-1 alpha, is detected through real-time quantitative PCR (qPCR), the protein expression of a glucose transporter GLUT2 in the cells is detected through a cellular immunofluorescence method, the glycolysis capability of the cells is detected through a seahorse technology, and the glycolysis level of the cells is represented by the extracellular acid production rate.

Real-time quantitative PCR: to verify the differential expression of gluconeogenesis associated genes in HepG2 cells, real-time quantitative PCR was used to detect the expression levels of G6Pase, PEPCK, PGC-1 α. Total mRNA in HepG2 cells was extracted with TRIzol reagent and the concentration thereof was measured. It was reverse transcribed to cDNA using the Biotool cDNA kit. The water used in the operation is diethyl pyrocarbonate (DEPC) treated for use so as to prevent RNA enzyme pollution. Q-PCR reaction conditions: using SYBR Green qPCR Mix kit, the amplification cycle condition is pre-denaturation at 95 ℃ for 10min, and then 40 cycles of denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s and extension at 72 ℃ for 30s are carried out. Primers are designed by using Premier 5.0 software according to related gene sequences of G6Pase, PEPCK and PGC-1 alpha published by NCBI. Beta-actin is used as an internal reference gene. Primer design is shown in table 1 below:

TABLE 1

Cell immunofluorescence: soaking 24mm × 24mm cover glass, and cleaningFine cleaning, washing with tap water, soaking with dilute hydrochloric acid for 8h, washing with running water, soaking with chromic acid overnight, washing with running water, washing with distilled water and triple distilled water for three times, placing in a glass culture dish, sterilizing under high pressure, and oven drying for use; dripping a small amount of cell culture medium into a 6-hole cell culture plate, clamping a cover glass by aseptic operation, and putting the cover glass into a hole of the culture plate to attach the cover glass to the bottom of the hole; taking HepG2 cells with good growth state, washing the HepG2 cells once by PBS, digesting the cells by trypsin, collecting the cells, counting the cells, inoculating the cells on a cover glass in a hole of a 6-hole cell culture plate, and carrying out 4 multiplied by 10 plating⁵cells/ml, 2ml/well, at 37 ℃ with 5% CO₂And (5) culturing the cells in a cell culture box for 24 hours. After HepG2 cells are cultured to grow a monolayer, an insulin resistance model is established, and after administration is finished, the culture medium is sucked away, precooled PBS is washed for 3 times, residual liquid is sucked away, the cells are fixed by 4% paraformaldehyde at room temperature, and the time is 30 min; discarding and washing 3 times with PBS; dropwise adding 3% BSA, and sealing at room temperature for 2 h; removing the confining liquid, dropwise adding GLUT2(sc-518022, working concentration 1: 100) primary-antibody diluent to the cell surface, and incubating at room temperature for 2 h; standing overnight at 4 deg.C, taking out the next day, rewarming, and washing with PBS for 3 times; adding FITC labeled mouse anti-goat IgG secondary antibody (working concentration is 1: 100) dropwise, and incubating for 2h at 37 ℃; the process is protected from light to prevent quenching of the fluorescent group, and washed for 3 times by PBS; dyeing nuclei with DAPI, keeping out of the sun, adding a little DAPI dye solution to cover the sample, incubating at room temperature for 5min, removing the dye solution, and washing with PBS solution for 3 times; sealing 90% buffer glycerol, adding an anti-fluorescence quenching liquid, avoiding bubbles as much as possible, and performing microscopic examination and photographing recording.

seahorse technology: HepG2 cells were seeded 8000 cells/well into eight-well seahorse XFP cell culture dishes (80. mu.l of medium per well) and were model-administered. The measurement probe card was lifted up and 200. mu.l of the calibration solution was added, and then the probe card was placed at 37 ℃ without CO₂Incubate overnight. After the drug effect was completed, the medium was discarded, and the cells in the eight well plates were washed 3 times with seahorse bioenergy analysis medium, 180. mu.l each time, without washing off the cells. The eight well plates were then placed at 37 ℃ CO-free₂Incubate in dressing box for 1 h. Adding prepared oligomycin, carbonyl cyanide-p-trifluoromethoxyzone (FCCP), antimycin A and rotenone working solution with concentrations of 10 μ M, 5 μ M and 10 μ M respectivelyThe probe plate was inserted into A, B, C wells corresponding to the probe plate, and then the eight well plate with the probe plate placed thereon was placed in a seahorse bio-energy analyzer while detecting Oxygen Consumption (OCR) and extracellular acid production rate (ECAR).

2. The experimental results are as follows: the effect of hirsutine on intracellular glucose homeostasis of insulin resistant HepG2 in this example is shown in fig. 1 and 2, where it can be seen that hirsutine significantly increased glucose consumption and uptake compared to the control HGHI group. The results of the hepatic glycogen synthesis and hepatic glucose output tests are shown in FIGS. 3 and 4, from which it can be seen that hirsutine increases glycogen content and inhibits gluconeogenesis in insulin resistant HepG2 cells. The quantitative PCR of the gluconeogenesis-associated gene expression in the insulin resistance state is shown in FIGS. 5 and 6, and it can be seen from the figure that the expression level of the control group-associated gene is abnormally increased, and the increase can be inhibited by hirsutine, which indicates that the hirsutine can maintain the glucose homeostasis by inhibiting gluconeogenesis. GLUT2, a glucose transporter widely present in hepatocytes, is inhibited in the insulin-resistant state, and as shown in FIG. 7, hirsutine increases the expression of GLUT2 and increases the glucose transport amount. The glycolysis results of HepG2 cells are shown in FIG. 8, and it can be seen that hirsutine can improve glycolysis ability of insulin resistant HepG2 cells, thereby improving glycometabolism.

Example 3: action path of hirsutine in preparation of insulin resistance-related pharmacological approaches

1. The experimental steps are as follows: western Blot is used for detecting protein expression and phosphorylation levels of Akt, PDK1 and GSK3 beta, the phosphorylation levels of Akt, PDK1 and GSK3 beta are used for reflecting the activity of the channel, then the glucose uptake of HepG2 cells under the condition that the PI3K inhibitor LY294002 improves insulin resistance of hirsutine is detected, and then the activation of PI3K/Akt/GSK3 beta channels of HepG2 cells under the condition that the insulin resistance is stimulated by hirsutine is obviously inhibited through detecting the PI3K inhibitor LY294002 through Western Blot, so that the increase of the glucose uptake by activating insulin-related PI3K/Akt/GSK3 beta signal channels is reversely verified.

Western Blot: extracting total protein of cultured cells by using RIPA protein lysate, detecting the protein concentration by using BCA, adjusting the concentration, adding protein loading buffer solution, boiling and denaturing. The samples were transferred to NC membranes after SDS-PAGE. Blocking with 5% milk-TBST blocking solution for 1h, washing membrane with TBST three times after one-time reaction at 4 ℃ overnight, each time for 5min, incubating the second antibody at room temperature for 1h, and performing imaging detection after membrane washing.

2. The experimental results are as follows: the Western Blot results are shown in FIG. 9, and it can be seen from the figure that insulin resistance can cause key protein Akt phosphorylation disorder in insulin signaling pathway PI3K/Akt/GSK3 beta, and hirsutine can activate Akt, PDK1 and GSK3 beta protein under the pathway to phosphorylate. As shown in FIGS. 10 and 11, it can be seen from the graphs that after adding PI3K inhibitor LY294002 of PI3K/Akt/GSK3 beta pathway, glucose uptake and phosphorylation of PI3K/Akt/GSK3 beta pathway are both inhibited, indicating that hirsutine improves insulin resistance in connection with activation of PI3K/Akt/GSK3 beta pathway.

Example 4: harpagine test for improving fasting blood glucose, glucose tolerance and liver lipid deposition of db/db diabetic mice

1. The experimental steps are as follows: 6 groups of mice were selected, 12 per group. The mice are divided into wild type C57BL/6 mice (con), db/db model diabetes mellitus mice, db/db +5mg/kg hirsutine groups, db/db +10mg/kg hirsutine groups, db/db +20mg/kg hirsutine groups and db/db +200mg/kg metformin groups. Gavage administration was started from 6 weeks of age for six weeks. Body weight and fasting plasma glucose were measured once a week, and insulin resistance and oral glucose tolerance tests were performed for the last week. The mouse liver, pancreas, and kidney were removed, the weight was recorded, fixed with 4% paraformaldehyde solution, and dehydrated as follows: 70% ethanol for 4 h; 80% ethanol for 2 h; 90% ethanol for 1 h; 95% ethanol for 2 times, each for 2 hr; 100% ethanol for 2 times, each for 1 hr; xylene for 3 times, each time for 1 h; hard wax for 2 times, each time for 2 h; dehydrating, embedding in paraffin, and slicing (4 μm thick). And then carrying out subsequent H & E staining or oil red O staining tests.

H & E staining: placing the paraffin section on a baking sheet machine for 15min at 60-65 ℃, and carrying out conventional dewaxing and dyeing according to the following steps: xylene for 3 times, each for 7 min; 100% ethanol for 2 times, each for 5 min; 95% ethanol for 2 times, each for 5 min; 80% ethanol for 5min, 70% ethanol for 5min, and flowing water for 2 min; and (3) hematoxylin staining, namely placing a staining rack carrying the slide in distilled water I, distilled water II, draining, hematoxylin for 2min, washing with a large amount of tap water, standing in tap water for 30min, carrying out anti-blue for 30min, observing by a microscope, and dehydrating: 50% ethanol-70% ethanol-80% ethanol-95% ethanol (0.5-1 min per pass); eosin staining for 2min, then dehydrating and clearing, namely 80% ethanol for 15 s; 95% ethanol for 2 times, each for 1 min; 100% ethanol for 2 times, each for 1 min; xylene for 3 times, each for 3 min; the neutral gum is encapsulated during which air bubbles are avoided.

Dyeing with oil red O: the liver tissue after paraffin-embedded section was subjected to OCT embedding, sectioned by a cryomicrotome (4 μm thick), and stored in a refrigerator at-80 ℃. Taking out the frozen section at-80 deg.C, drying for 10min, washing with 50% ethanol, soaking in 60% isopropanol dissolved oil red O staining solution for 10min, differentiating with 60% ethanol for 10s, washing with flowing water for 2min, counterstaining with hematoxylin for 2s, differentiating with 1% hydrochloric acid ethanol for 15s, washing with flowing water for 5min, and sealing with glycerol gelatin to avoid bubble generation.

2. The experimental results are as follows: as shown in FIG. 12, it can be seen that hirsutine had no significant effect on the body weight of db/db diabetic mice. On the premise of not obviously affecting the body weight, the hirsutine can improve the insulin resistance state of db/db diabetic mice, as shown in figures 13, 14 and 15, and as can be seen from the figures, the hirsutine obviously reduces the fasting blood glucose, the oral glucose tolerance and the insulin tolerance of the db/db diabetic mice. As shown in FIGS. 16 and 17, it can be seen that hirsutine can reverse the liver hypertrophy and other problems caused by fatty liver in db/db diabetic mice, and can improve the yellowish liver color in diabetic state to make it close to the brownish red color of normal liver. The results of liver H & E, oil red O and NAS are shown in figures 18, 19 and 20, and it can be seen from the figures that hirsutine can significantly reduce lipid deposition in the liver of db/db diabetic mice, reduce macrovesicular and vesicular lipogenesis, restore normal arrangement and shape of liver cells, reduce infiltration of inflammatory cells, reduce activity of non-alcoholic fatty liver, and indicate that hirsutine can target the liver, and improve diabetic fatty liver. The pancreatic H & E results are shown in FIG. 21, where it can be seen that hirsutine improved the irregular islet cell morphology and vacuolike degeneration to near normal levels in db/db diabetic mice. The kidney results are shown in fig. 22, and it can be seen that the glomerulus hypertrophy and hyperplasia of glomerular mesangial basement membrane in early stage of diabetes mellitus can be seen from the figure, and the hirsutine can improve the disease, so that the shape and volume of the glomerulus tend to be normal, and the thickening of the glomerular mesangial basement membrane is reduced, which also indicates that the hirsutine can relieve the diabetic nephropathy symptoms to a certain extent.

In conclusion, the hirsutine provided by the invention has certain treatment effect on insulin resistance, diabetes and complications thereof, can achieve curative effect at low concentration, and can reverse liver steatosis, and meanwhile, the experiments show that the pharmacological action path of the hirsutine for treating the insulin resistance, the diabetes and the complications thereof improves the glucose uptake by activating the inhibited PI3K/Akt/GSK3 beta pathway under the disease state. Provides a new treatment method and a new treatment medicine for clinically treating the type II diabetes.

The above description is only an embodiment of the present invention, and not intended to limit the scope of the present invention, and all equivalent changes made by using the contents of the present specification and the drawings, or applied directly or indirectly to the related technical fields, are included in the scope of the present invention.

Sequence listing

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Claims

1. Application of hirsutine in preparing medicine for improving insulin resistance, diabetes and its complications is provided.

2. The use as claimed in claim 1, wherein the hirsutine is extracted from uncaria rhynchophylla.

3. The use according to claim 1, wherein the diabetes is type II diabetes.

4. The use according to claim 1, wherein the complications comprise disorders of lipid metabolism and/or renal disease; preferably, the disorder of lipid metabolism disease is nonalcoholic fatty liver disease.

5. The use according to claim 1, wherein the medicament for the treatment of insulin resistance, diabetes mellitus and complications thereof is a medicament for inhibiting the expression of gluconeogenesis associated genes G6Pase, PEPCK or PGC-1 α in cells in an insulin resistant state.

6. The use according to any one of claims 1 to 5, wherein the medicament further comprises a pharmaceutically acceptable carrier or adjuvant.

7. The use according to any one of claims 1 to 5, wherein the medicament is in a solid, semi-solid or liquid form; preferably tablets, capsules, soft capsules, granules, pills, oral liquid, dry suspensions, dropping pills, dry extracts, injections or infusions.

8. The use according to any one of claims 1 to 5, wherein the medicament is administered by injection or orally; preferably, the administration by injection is intravenous, intramuscular, intraperitoneal, intradermal or subcutaneous injection.

9. Application of hirsutine in preparing PI3K/Akt/GSK3 beta signal pathway activator is provided.

10. The use as claimed in claim 9 wherein the activator is for activating Akt, PDK1 and GSK3 β protein expression.