Study On The Mechanism Of Apple Polyphenols’ Hypoglycemic Effect Based On The GLP-1 Receptor Pathway

Objective:Diabetes mellitus(diabetes mellitus, DM) is due to absolutely or relatively insufficient of insulin secretion, causes the glucose, fat and protein metabolism disordered. At the early stage, blood glucose and urine glucose increased. Then a series of heart, kidney and retinal microvascular diseases, neuropathy, infection and tuberculosis post-merged. In type 1 and type 2 diabetes, islet β-cell dysfunction is of the key pathological and physiological processes, wherein the islet β-cell apoptosis plays an important role. The clinical treatment of type 2 diabetes is with western medicine, for western medicine, the hypoglycemic effect is strong and rapid, but there are varying degrees of side effects, such as hypoglycemia, liver and kidney dysfunction and not preventing the apoptosis of islet cells. Therefore, searching for a good curative effect, little side effect of antidiabetic agents from natural resources has great significance. In recent years GLP-1 receptor based exendin have gradually accessed into people’s horizons and became a new research hotspot. Apple polyphenols(AP) are an important class of bioactive substances naturally in Apple, including: flavonoid, tannin, phenolic acids, anthocyanin and so on. In recent years, a large number of studies have shown that AP have a variety of activities, such as anti-oxidation, anti-tumor, anti-caries effects. AP could adjust a high expression of GFP report gene in islet β-cells via the RIP1-CRE transient transfection. In this study, observe the protective effects of AP on islet β-cell by H2O2-induced the apoptosis of cells. Establish diabetic mice by repeated low-dose injection of streptozotocin(STZ), and AP prophylactic administration to observe and analyze hypoglycemic effect of AP in diabetic mice and its mechanism.Method:1 Cell level in vitro1.1 RIP1-CRE transient transfection method to observe the effect of apple AP on the report gene GFP expression in islet β-cells, verify the role of GLP-1 agonists.1.2 MTT assay was used to observe the effect of AP on the proliferation in islet β-cell of RIN-m5 F.1.3 Immunofluorescence assay of AP on the effect of promoting insulin release in RIN-m5 F islet β-cell.1.4 MTT assay, AO/EB double staining method to observe the effect of AP on apoptosis and activity of RIN-m5 F islet β-cells under the action of H2O2. AP AP also could enhance antioxidant capacity of β-cells cell and the western blot method analysis changes in expression of Akt and Caspase-3 proteins closely related to apoptosis. Thus clearly identify the anti-apoptotic of AP by GLP-1 receptor pathway-based targets.2 The animal level:2.1 The mouse model of type 2 diabetes: selection of adult ICR mice and repeated low-dose STZ intraperitoneal injection to build diabetic mice model.2.2 Observation and recording the effects of AP prophylactic administration on the body weight, food intake, water intake, urine volume and other physiological metabolic indexes of diabetic mice.2.3 Determination the impact of AP prophylactic administration on fasting and non-fasting blood glucose, blood lipid and other biochemical indices in diabetic mice.2.4 Investigation the effects of AP prophylactic administration on the pancreas weight in diabetic mice.2.5 Observation of pancreatic tissue morphology by HE staining.2.6 Evaluation the protective and regulative effects of AP on islet β-cell by insulin immunohistochemical analysis of the number of cells, quality and its organizational structure in the pancreas.2.7 Exploring of the protective mechanisms of AP on islet β-cell via GLP-1 receptor pathway by Western Blot of PDX-1, the important GLP-1 receptordownstream.2.8 Semisolid paste to observe the effect of AP on the gastric emptying and intestinal propulsion in diabetic mice.Results:1 Cell level in vitro1.1 AP could adjust a high expression of GFP report gene in islet β-cells via the RIP1-CRE transient transfection.1.2 MTT assay showed that cell viability increased significantly(P<0.01), and showed a dose-dependent manner in AP treatment group.1.3 Immunofluorescence result showed that the positive of insulin results increased in AP treatment group(P <0.05).1.4 H2O2 induced the decreasing of cell viability significantly(P<0.01). AP treatment group could better improve cell viability(P<0.01) compared with the negative control group, and showed a dose-dependent manner. AO/EB staining found that in AP group cell morphology were more intact and apoptosis was significantly reduced than the negative control group. At the same time, the MDA content reduced and SOD content increased. The western blot analysis showed that, compared with the negative control group, AP group can enhance the expression of Akt(P<0.05), and decrease Caspase-3(P<0.05).2 The animal level:2.1 The comparison of weight, water intake, food intake, urine output of each model group: the body weight decreased(P<0.05) water intake, food intake, litter humidity increased significantly in model group compared with normal group. While no significant changes were found in body weight, but water intake, food intake and litter humidity well reduced for the AP administration group compared with the model group.2.2 The comparison of fasting and non-fasting blood glucose, serum lipid: the hyperglycemia was significantly higher in model mice than the control group(P<0.05). The blood glucose significantly reduced fasting and non-fasting in AP group diabetic mice(P<0.05), but blood triglycerides, total cholesterollevels had no significant effected.2.3 The quality comparison of pancreas in each group: the pancreas weight increased significantly the model group compared with the control group(P<0.05), AP prophylactic treatment group significantly lowered(P<0.05) the weight of the pancreas compared with the model group.2.4 Histological observation: the number of islet increased significantly in diabetic mice AP treatment group compared with diabetic model group, but still relatively less than the normal control group. The number of cells in the islets increased, pyknosis of nuclei and cytoplasmic vacuolization reduced compared with the diabetic model.2.5 Immunohistochemical analysis of insulin: Compared with normal group, the expression area of insulin and the number of pancreatic β-cell in diabetic mice reduced, AP treatment group could increase the positive expression and the number of pancreatic β-cell(P <0.05).2.6 Expression levels of PDX-1: Compared with normal group, PDX-1 expression in pancreas was significantly decreased(P<0.05) in MLDS mice, while in AP treatment group PDX-1 expression in pancreatic tissue was significantly enhanced(P <0.05).2.7 Gastric emptying and intestinal propulsion:Compared with control group, AP could slow down gastric emptying(P<0.05) in diabetic mice.Conclusion:AP could adjust a high expression of GFP report gene in islet β-cells via the RIP1-CRE transient transfection, and played a role of GLP-1 agonists.AP could promote the proliferation and secretion of RIN-m5 F islet β-cell. It had protective role in cell apoptosis of islet β-cell induced by H2O2, enhanced the antioxidant capacity of cells and its mechanism might be associated with up regulation of Akt expression and down-regulation of Caspase-3.Prophylactic administration of AP could improve the blood glucose levels of fasting and postprandial diabetic mice which were induced by repeated low-dose injection of STZ, improve the symptoms and insulin secretion, slow down gastric emptying, enhance sensitivity to insulin, protect β-cells, reducethe damage of pancreatic tissue and increase expression of PDX-1 protein in diabetic mice. It was clearly demonstrated that the AP protective effect on pancreatic β-cells was based on GLP-1 receptor pathway. Our research provided new targets and research directions for the development of new anti-diabetic drugs and hypoglycemic health products.