| With the development of social economy and improvement of living standard, the incidence of diabetes increased year by year. The main feature of type 2 diabetes mellitus is insulin resistance, and one of the important reason of insulin resistance is dysfunction of insulin signaling pathway. One of the common chronic complications of type 2 diabetes mellitus is liver injury.So the earch for insulin signaling pathway in liver is of great significance to prevent 2 diabetes mellitus and its complications.Sericin is a kind of water-soluble protein, which is composed of 18 kinds amino acids and has biological compatibility. The molecular weight of sericin is 10-300 KD. The content of sericin is about 30% of cocoon silk, which is thrown away during silk reeling and refining.Our early study found that sericin can reduce blood glucose effectivly and can prevent increasing of blood glucose during diabetes. However, the mechanism is undefined. In this study, we will investigate the intervention effects of sericin on liver insulin PI3K/Akt signaling pathway using type 2 diabetes mellitus rats model induced by high sugar and high calorie diet with intraperitoneal injection of streptozotocin. The purpose of this study is to offer new ideas for preventing and treating diabetes complications.Objective:To investigate the roles and possible mechanisms of sericin on liver insulin PI3K/Akt signaling pathway by observing effects of sericin on expression of insulin receptor(IR), insulin receptor substrate-1(IRS-1), phosphatidylinositol 3-kinase(PI3K), protein kinase B(Akt/PKB), glucose transporter 4(GLUT4) and tribbles-related protein 3(TRB3) in liver of type 2 diabetes mellitus rats model.Methods:1. 60 male SPF SD rats were randomly divided into the following 5 groups with 12 rats in each group: normal control(NC) group, diabetes model(DM) group, high-dose sericin treatment(HS) group, low-dose sericin treatment(LS) group and positive control(PC) group. The type 2 diabetes mellitus rats model were induced by high sugar and high calorie diet with intraperitoneal injection of streptozotocin(35mg/kg,2d), and the standard for successfully establishing type 2 diabetes model was fasting blood-glucose ≥11.1mmol/L. Then, the rats in HS(2.4g/kg/d) group and LS(1.8g/kg/d) group were lavaged with sericin for 35 days; the rats in PC group were lavaged with metformin(55.33mmg/kg/d) for 35days; the rats in DM group and NC group were lavaged with normal saline in same dose and time as sericin group.2. Glucose oxidase method was used to detect the fasting blood glucose of rats in each group; ELISA was used to measure the insulin level in serum of rats in each group.3. HE staining was used to observe the changes of morphology and structure of liver.4. Periodic acid-schiff stain was used to detect the glucogen content in liver of rats.5. Immunohistochemical staining and Western Blotting were used to detect the expression of IR, IRS-1, PI3 K, Akt, GLUT4 and TRB3 protein in liver.6. Real Time Q-PCR was used to detect the expression of IR, IRS-1, PI3 K, Akt, GLUT4 and TRB3 m RNA in liver.Results:1.Blood glucoseThe blood glucose of rats in DM group was(29.45±4.82)mmol/L, which was obviously higher than that of rats in NC group(10.83±2.03)mmol/L(P<0.05). The blood glucose of rats in HS group,LS and PC group were respectively(13.20±4.09)mmol/L,(13.18±2.30)mmol/L,(10.04±2.29)mmol/L, which were all obviously lower than DM group(P<0.05); Moreover, there had no statistical significance between HS group, LS group and PC group(P>0.05).2.Insulin levelThe serum insulin level of rats in DM group was(7.28±0.58)μg/L, which was significantly lower than that of rats in NC group(11.45±1.72)μg/L(P<0.05). The serum insulin level rats in HS group, LS group and PC group were respectively(12.65±2.17)μg/L,(13.30±1.64)μg/L,(11.13±0.99)μg/L, which were all significantly higher than DM group(P<0.05); Moreover, there had no statistical significance between HS group, LS group and PC group(P>0.05).3. The changes of morphology and structure of liverNC group: The structure of hepatic lobule was clear; the liver cells arranged in funicular shape by taking central vein as center; the cell nucleus was large and round,and located in the center of liver cells; the dying of cytoplasm was even and hepatic sinusoid was clear. DM group: The liver cells nearly arranged in funicular shape by taking central vein as center; but liver cells swollen markely and vacuole like structure appeared in cytoplasm obviously; part of the liver cells lytic necrosis; the hepatic sinusoid became narrow or locked. HS group and LS groups: Compared with DM group, the pathological changes of liver obviously alleviated. PC group: Swollen of liver cells was not obvious; some liver cells lytic necrosis and hepatic sinusoid became narrow.4. Liver glucogen content in each groupLiver glucogen positive products were red or purple granules and mainly located in cytoplasm. Compared with rats in NC group, the liver glucogen content of rats in DM group obviously decreased(P<0.05); Compared with rats in DM group, the liver glucogen content of rats in HS, LS and PC group obviously increased(P<0.05); Moreover, there had no remarkable difference between HS group, LS group and PC group(P>0.05).5.IR expression in rat liverIR immunopositive products were brown or sepia granules and mainly located in membrane and cytoplasm of liver cells. Compared with rats in NC group, the expression of IR protein and m RNA in liver of rats in DM group obviously decreased(P < 0.05); Compared with rats in DM group, the expression of IR protein and m RNA in liver of rats in HS and PC group obviously increased(P<0.05); Moreover,there had no remarkable difference between HS group and PC group(P>0.05).6.IRS-1 expression in rat liverIRS-1 immunopositive products were brown or sepia granules and mainly located in membrane and cytoplasm of liver cells. Compared with rats in NC group, the expression of IRS-1 protein and m RNA in liver of rats in DM group obviously decreased(P < 0.05); Compared with rats in DM group, the expression of IRS-1 protein and m RNA in liver of rats in HS, LS and PC group obviously increased(P<0.05); Moreover, there had no remarkable difference between HS group, LS group and PC group(P>0.05).7.PI3 K expression in rat liverPI3K immunopositive products were brown or sepia granules and mainly located in membrane and cytoplasm of liver cells. Compared with rats in NC group, the expression of PI3 K protein and m RNA in liver of rats in DM group obviously decreased(P < 0.05); Compared with rats in DM group, the expression of PI3 K protein and m RNA in liver of rats in HS, LS and PC group obviously increased(P<0.05); Moreover, there had no remarkable difference between HS group and PC group(P>0.05).8.Akt expression in rat liverAkt immunopositive products were brown or sepia granules and mainly located in membrane and cytoplasm of liver cells. Compared with rats in NC group, the expression of Akt protein and m RNA in liver of rats in DM group obviously decreased(P < 0.05); Compared with rats in DM group, the expression of Akt protein and m RNA in liver of rats in HS, LS and PC group obviously increased(P<0.05); Moreover, there had no remarkable difference between HS group, LS group and PC group(P>0.05).9.GLUT4 expression in rat liverGLUT4 immunopositive products were brown or sepia granules and mainly located in membrane and cytoplasm of liver cells. Compared with rats in NC group, the expression of GLUT4 protein and m RNA in liver of rats in DM group obviously decreased(P<0.05); Compared with rats in DM group, the expression of GLUT4 protein and m RNA in liver of rats in HS, LS and PC group obviously increased(P<0.05); Moreover, there had no remarkable difference between HS group, LS group and PC group(P>0.05).10.TRB3 expression in rat liverTRB3 immunopositive products were brown or sepia granules and mainly located in membrane and cytoplasm. Compared with rats in NC group, the expression of TRB3 protein and m RNA in liver of rats in DM group obviously increased(P<0.05); Compared with rats in DM group, the expression of TRB3 protein and m RNA in liver of rats in HS, LS and PC group obviously decreased(P<0.05); Moreover, there had no remarkable difference between HS group, LS group and PC group(P>0.05).ConclusionsSericin can improve the abnormal changes of PI3K/Akt signaling pathway by up regulating IR, IRS-1, Akt, PI3 K, GLUT4 expression and down regulating TRB3 in liver of diabetes mellitus rats; Thereby, sericin play a role in lowering blood glucose. |
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