| ObjectiveType 2 diabetes mellitus (T2DM) is generally identified by reduced insulin secretion, dysfunction of pancreatic islets. Insulin resistance is the typical characteristic of T2DM, and it is also the basis of T2DM. The incidence of T2DM has increased steadily, and T2DM and its complications caused by the mortality are also increasing. Nowadays, the main measure for the treatment of T2DM involves diet control, exercise, the use of oral hypoglycemic drugs and injection of insulin. Most of the patients with T2DM were treated with chemical hypoglycemic drugs, such as biguanides, sulfonylureas, Thiazolidinediones. Benefits of pharmaceutical factors to treat the disease aggressively early have been recommended, but medications may have unwanted side effects, such as white blood cell reduction, hemolytic anemia, damage to the digestive tract. Thus, there has been a growing interest in herbal remedies that can be introduced into the general population with least side effects and the maximal preventive outcome.Jin Nan Compound (JNC), as a folk prescription, plays an important role in the prevention and treatment of diabetes, high blood pressure and so on. Therefore, the aim of the present study is to evaluate the effects of JNC on insulin resistance, glucose metabolism and the expression of GLUT-4, p-IRS-1 Ser307 and IRS-1 through vivo and vitro, explore the mechanism of JNC improving insulin resistance. The cells were set up in the control group, model group, JNC high, middle and low dose group.MethodsPart one:HepG2 cells were incubated in 10% CS DMEM medium containing glucose (1 g/L) in 5% CO2 incubator at 37 C. The subculture of HepG2 cells were plated into 96-well plates, incubated in 10% CS DMEM medium containing glucose (1g/L) in 5% CO2 incubator at 37 C for 24 h. Removing the culture medium and washing one time with PBS. The addition of 10 containing insulin was added to the culture medium with 200μl in 5% CO2 incubator at37 C for 24 h to establish the IR cell model. The insulin resistant cells were treated with different concentrations of JNC and insulin for 24 hours and the non insulin resistant cells were used as a control. The glucose concentration in culture medium was detected by the method of glucose oxidase-peroxidase (GOD-POD). According to the glucose concentrations in blank medium and those in the medium of culturing cells 24 hours later, the rate of absorption glucose by the cells was calculated. We performed an MTT assay to determine cytotoxicity effects of JNC on HepG2 cells. The expression of p-IRS-1 Ser307, PI3K and GLUT-4 were detected by Western blot.Part two:Male Sprague-Dawley rats were acclimated with free access to regular rodent chow and water for a week, the rats were injected with STZ dissolved in citrate buffer at a dose of 50 mg/kg body weight and tested for fasting blood glucose levels (FBG) 3days post-injection. The rats with FBG value above 11.1 mmol/1 were randomly divided into 5 groups (n=10 each). One group was used as diabetic control, and the other 4 were orally gavaged with JNC at doses of 300’, 150,75 mg/kg and Shenqi granule 300 mg/kg body weight per day. Rats in control and diabetic groups were with saline. Oral glucose tolerance testing was performed during the last week of treatment after a 12 h fasting. All rats were sacrificed and tissues and organ were harvested and weighted.Part three:Male Sprague-Dawley rats were divided into six groups of ten rats each. Normal control group was maintained on normal rat chow diet. The remaining five groups were rendered diabetic with low dose STZ (45 mg/kg) after which they were fed a high-fat diet (HFD) for 4 weeks to induce insulin resistance. The diabetic rats were confirmed 3 days later by a fasting blood glucose level above 11.1 mmol/1 after which diabetic rats received HFD along with the treatment regimens for 4 weeks. Diabetic rats were received JNC (160,80,40 mg/kg) or metfoemin (200 mg/kg) under study orally and once daily for 4 weeks. At the end of study, animals were fasted overnight and blood samples obtained from removalling eyeball. After centrifugation, the serum was collected. The serum samples were analyzed for estimation of blood glucose, blood lipid levels and insulin. The liver and pancreatic tissues were isolated and fixed in 10% formalin for histopathological examination. GLUT-4 and p-IRS-1 Ser307 expression in liver was deteced by immunohistochemical method.ResultsPart one:Incubated with 10-7 mol/L insulin for 24 hours, the insulin resistance cell model had been built. The rate of glucose absorption of the model cell treated with high concentration JNC (120μg/ml) was significantly improved. According to the model cells, the expression of GLUT-4 and PI3K decreased significantly compared to non insulin resistance cells. While the expression of p-IRS-1 Ser 307 was inhibited and GLUT-4 and PI3K expression were increased in the insulin resistance cells after treated with different dose JNC.Part two:After 3 weeks treatment, levels of FBG and PBG were significantly reduced in JNC 150,75 mg/kg rats as compared to diabetic rats. Glucose challenge increased the blood glucose levels in diabetic rats, whereas JNC 150,75 mg/kg treatment groups significantly prevented the blood glucose levels. During the treatment, impaired movement were not observed in the rats of JNC groups.Part three:The JNC possessed anti-diabetic activities as shown by the decreased serum levels of fasting blood glucose (FBG), postprandial blood glucose (PBG), glycosylated hemoglobulin Alc (HbAlc), total cholesterol (TC), triglyceride (TG), low density lipoprotein-cholesterol (LDL-C) levels and free fatty acid (FFA), fasting Insulin (FINS), malondialdehyde (MDA) content and insulin resistance (HOMA-IR), as well as increased serum levels of hepatic glycogen and protein expression of GLUT-4 and reduction of IRS-1 in liver. On the other hand, JNC improved the glucose metabolism to a certain degree.Conclusions(1) JNC exert beneficial effects on hyperglycosemia in IR rats and cells possibly through regulating the levels of GLUT-4 and p-IRS-1 Ser307 in hepatic tissues and levels of PI3K in HepG2 cell.(2) JNC had the potential to attenuate the glucose metabolism disorder and nearly normalized the lipid metabolism. These changes may be related to the reduction of the levels of FFA in serum. The beneficial effect of JNC on FFA could be related to its antioxidant property. |
PDF Full Text Request