丝胶对2型糖尿病大鼠肝脏胰岛素PI3K/Akt信号通路的调节作用及其机制

目的：观察丝胶对2型糖尿病大鼠肝脏胰岛素磷脂酰肌醇-3-激酶/蛋白激酶B（PI3K/Akt）信号通路中胰岛素受体（IR）、胰岛素受体底物1（IRS-1）、PI3K和Akt的调节作用，初步探讨丝胶降血糖的作用机制。方法：36只SPF级雄性SD大鼠随机分为正常组、模型组和实验组，每组12只。模型组和实验组大鼠采用高脂高糖饲料喂养联合腹腔注射链脲佐菌素（STZ，35 mg·kg^-1·d^-1，连续2 d）的方法制备2型糖尿病模型，以STZ注射结束后72 h的空腹血糖≥ 11.1 mmol·L^-1作为成模标准。模型建立成功后，实验组大鼠给予2.4·kg^-1·d^-1丝胶灌胃，正常组和模型组大鼠给予等体积生理盐水灌胃，连续用药35d。取各组大鼠血液标本，采用葡萄糖氧化酶法检测空腹血糖；取各组大鼠肝组织，采用免疫组织化学法和Western blotting法检测各组大鼠肝组织中IR、IRS-1、PI3K和Akt蛋白的表达，采用过碘酸-Schiff染色法检测各组大鼠肝糖原含量。结果：模型组大鼠血糖水平明显高于正常组（P<0.05），实验组大鼠血糖水平明显低于模型组（P<0.05）。正常组和实验组大鼠肝组织中IR、IRS-1、PI3K和Akt蛋白呈棕黄色和（或）棕褐色颗粒，小部分呈弥散状；模型组大鼠肝组织中IR、IRS-1、PI3K和Akt蛋白表达量明显减少，呈棕黄色和（或）棕褐色颗粒，其中IR、IRS-1和Akt蛋白主要位于肝细胞胞膜和胞质，PI3K蛋白主要位于肝细胞胞质。各组大鼠肝切片均可见肝糖原阳性染色的红色或紫红色颗粒，其中模型组大鼠肝糖原染色浅，面积较小。与正常组比较，模型组大鼠肝组织中IR、IRS-1、PI3K和Akt蛋白表达水平及肝糖原含量明显降低（P<0.05）；与模型组比较，实验组大鼠肝组织中IR、IRS-1、PI3K和Akt蛋白表达水平及肝糖原含量明显升高（P<0.05）。结论：丝胶可通过上调糖尿病大鼠肝组织中IR、IRS-1、PI3K和Akt的表达以增强肝脏胰岛素PI3K/Akt信号通路的转导效应，从而起到降低血糖的作用。

Regulatory effect of sericin on liver insulin PI3K/Akt signaling pathway in rats of type 2 diabetes mellitus and its mechanism

Objective: To observe the regulatory effects of sericin on the insulin receptor(IR), insulin receptor substrate-1(IRS-1), phosphatidylinositol-3-kinase(PI3K) and protein kinase B(Akt) in the insulin PI3K/Akt signaling pathway in the rats of type 2 diabetes mellitus,and to discuss the mechanism of sericin in lowering the blood glucose. Methods: Thirty-six SPF male SD rats were randomly divided into normal group, model group and experimental group; there were 12 rats in each group.The rats in model group and experimental group were given with high-fat, high-sugar feeding and intraperitoneal injection of streptozotocin (STZ) of 35 mg·kg^-1·d^-1 for 2 d to establish the type 2 diabetic rat models, and the standard for successfully establishing model was the fasting blood glucose ≥ 11.1 mmol·L^-1 after injecting STZ for 72 h. After successfully establishing the diabetes models,the rats in experimental group were given sericin(2.4 mg·kg^-1·d^-1) by gavage, and the rats in normal group and model group were lavaged with the equal volume normal saline; lasted for 35 d. The glucose oxidase method was used to detect the fasting blood glucose of the rats in various groups.Immunohistochemical staining and Western blotting methods were used to detect the expressions of IR, IRS-1, PI3K and Akt proteins in liver tissue of the rats in various groups and periodic acid-schiff staining was used to determine the contents of liver glycogen of the rats in various groups. Results: The blood glucose level of the rats in model group was significantly higher than that in normal group(P<0.05), and the blood glucose level of the rats in experimental group was obviously lower than that in model group(P<0.05).The expressions of IR, IRS-1,PI3K and Akt proteins were tan granules located in the rat liver slices in normal and experimental groups,and some of positive proteins were diffuse;the expression amounts of IR,IRS-1,PI3K and Akt proteins in liver tissue of the rats in model group were significantly decreased,and they were tan granules;the IR, IRS-1 and Akt proteins were mainly located in the membrane and cytoplasm of hepatocytes, while the PI3K protein was mainly located in the cytoplasm of hepatocytes.The staining of liver glycogen of the rats in various groups was red or purple granules; the staining of liver glycogen of the rats in model group was relative light,and the area was small. Compared with normal group, the expression levels of IR,IRS-1,PI3K and Akt proteins and the content of liver glycogen in liver tissue of the rats in model group were decreased significantly(P<0.05);compared with model group, the expression levels of IR,IRS-1,PI3K,and Akt proteins and the content of liver glycogen in liver tissue of the rats in experimental group were increased significantly(P<0.05). Conclusion: Sericin can enhance the transduction effect of liver insulin PI3K/Akt signaling pathway by up-regulating the expressions of IR, IRS-1, PI3K and Akt in the liver tissue of the rats of type 2 diabetes mellitus, thus reduce the blood glucose level.