西格列汀对非酒精性脂肪性肝病大鼠肝脏IRS2/PI3K信号通路的影响

Influence of sitagliptin on IRS2 / PI3K signal pathway in liver tissues of NAFLD rats

目的探讨西格列汀对非酒精性脂肪性肝病(NAFLD)大鼠肝脏胰岛素受体底物2(IRS2)/磷脂酰肌醇3-激酶(PI3K)信号通路的影响。方法取35只大鼠,应用高脂饲料喂养8周构建NAFLD模型,取其中30只随机分为二甲双胍组、西格列汀组、模型组各10只,分别以二甲双胍500 mg/(kg·d)、西格列汀10 mg/(kg·d)、等体积生理盐水灌胃8周。另取5只未造模大鼠作为对照组,以等体积生理盐水灌胃8周。各组干预后,腹主动脉采血,检测血清空腹血糖(FBG)、肝功能指标(包括ALT、AST)、血脂指标(包括TC、TG)水平,采用酶联免疫吸附法检测血清FINS水平,计算胰岛素抵抗指数(HOMA-IR)。取出肝脏称重,计算肝指数(肝脏湿重/体质量)。制备肝脏匀浆,检测肝脏TG水平。取肝右叶组织行HE染色,观察肝细胞脂肪变性程度、炎性活动度及肝纤维化程度。采用RT-PCR法检测肝组织IRS2 mRNA表达,采用Western blot法检测肝组织PI3K-p85α蛋白表达。结果模型组肝指数、FBG、HOMA-IR、ALT、AST、TC、TG及肝脏TG水平均高于对照组(P均<0.05);二甲双胍组及西格列汀组肝指数、FBG、HOMA-IR、ALT、AST、TC、TG及肝脏TG水平均低于模型组(P均<0.05);西格列汀组HOMA-IR及肝脏TG水平均低于二甲双胍组(P均<0.05)。模型组肝组织见大量炎症细胞浸润,肝细胞出现明显的小泡性脂肪变性;二甲双胍组肝细胞空泡样变及细胞质疏松化较模型组减轻;西格列汀组以上病理改变较二甲双胍组减轻。与模型组相比,西格列汀组及二甲双胍组IRS2 mRNA表达增加、PI3K-p85α蛋白表达降低(P均<0.05)。西格列汀组IRS2 mRNA表达高于二甲双胍组、PI3K-p85α蛋白表达低于二甲双胍组(P均<0.01)。结论西格列汀可明显改善高脂诱导的NAFLD大鼠肝脏脂肪沉积及IR,其机制可能与调节肝脏IRS2/PI3K信号通路有关。

Objective To explore the effect of sitagliptin on liver insulin receptor substrate-2 / phosphatidylinositol 3-kinase（ IRS2 / PI3K） signal pathway in rats with nonalcoholic fatty liver disease（ NAFLD）. Methods Thirty-five SD rats were selected to establish NAFLD models by 8-week high-fat diet feeding,after that 30 NAFLD rats were chosen and were divide into 3 groups： the metformin group,sitagliptin group and model group which were treated with 500 mg /（ kg / d） metformin,10 mg /（ kg / d） sitagliptin and equal volume of saline by gavage for 8 weeks. Meanwhile,another 5 rats were treated with normal saline as the control group. Blood samples were obtained from abdominal aorta after 8-week treatment,the fasting blood-glucose（ FBG）,ALT,AST,TC,TG and insulin（ FINS） level were measured by ELISA method,and the homeostasis model assessment of insulin resistance（ HOMA-IR） index was calculated. We took out the liver,weighed and calculated the liver index（ liver wet weight / body mass）. Liver homogenate was prepared to detect TG level in liver. HE staining was carried out to observe the degree of liver fatty degeneration,inflammatory activity and degree of hepatic fibrosis. IRS2 mRNA was measured by using RT-PCR and the expression level of PI3K-p85α was determined by Western blotting. Results The liver index,FBG,HOMA-IR,ALT,AST,TC,TG and liver TG level were higher in the model group than those of the control group（ all P〈 0. 05）. The liver index,FBG,HOMA-IR,ALT,AST,TC,TG and liver TG content were reduced significantly in the metformin and sitagliptin groups as compared with those of the model group（ all P〈 0. 05）. What＇s more,the HOMA-IR and hepatic TG content of the sitagliptin group was lower than that of the metformin group（ all P〈 0. 05）. In the model group,there was a large amount of inflammatory cell infiltration in hepatic tissues and microvesicular fatty degeneration in the hepatocytes. The hepatic cell vacuolar degeneration and cytoplasm osteoporosis of the metformin group were alleviated as compared with thsoe of the model group. The pathological change was relieved in the sitagliptin group as compared with that of the metformin group. Compared with the model group,the expression of IRS2 mRNA was increased and PI3K-p85α protein was decreased significantly in the sitagliptin and metformin groups（ all P〈 0. 05）. IRS2 mRNA expression was higher and PI3K-p85α protein expression was lower in the sitagliptin group than that of the metformin group,respectively（ all P〈 0. 01）. Conclusion Sitagliptin could significantly improve the liver pathological changes and liver TG contents of NAFLD rats induced by high-fatty diet,and the mechanism could be related with adjusting the signal pathway of IRS2 / PI3 K in the liver.