经验方糖通饮对2型糖尿病合并非酒精性脂肪肝大鼠脂质代谢的影响及机制

Effect of tangtongyin formula on the lipid metabolism of type 2 diabetes mellitus complicated with rat non-alcoholic fatty liver disease and its mechanism

目的探讨经验方糖通饮对2型糖尿病(T2DM)合并非酒精性脂肪肝病(NAFLD)大鼠脂质代谢的影响及机制。方法选取8周龄(SD)雄性大鼠44只,随机取10只作为正常组(基础饲料喂养,注射等剂量柠檬酸-柠檬酸钠缓冲液),其余大鼠建立T2DM合并NAFLD模型(高脂高糖饲料喂养,低剂量链脲佐菌素腹腔注射),连续处理8周,于正常组和造模大鼠中各取2只验证造模成功与否;将余下造模成功的32只大鼠随机分为模型组及糖通饮低(12 g/kg)、中(24 g/kg)、高剂量(36 g/kg)组,各剂量组大鼠分别予相应浓度糖通饮灌胃,模型组和正常组大鼠予等容积双蒸水灌胃,连续8周;各组大鼠灌胃结束后,称取体质量,取尾静脉血检测空腹血糖(FBG)水平;10%水合氯醛腹腔注射麻醉各组大鼠,取腹主动脉血采用ELISA法检测血清胰岛素(FINS),分光光度计比色法检测血清游离脂肪酸(FFA),计算胰岛素抵抗指数(HOMA-IR)及胰岛素敏感指数(ISI);各组大鼠取血后迅速摘取肝组织计算肝脏指数、GPO-PAP法检测甘油三酯(TG)含量,苏木精-伊红(HE)染色法观察各组大鼠肝组织学特征,Western blot法检测磷酸化c-Jun氨基末端激酶-1(p-JNK1)/c-Jun氨基末端激酶-1(JNK1)、磷酸化胰岛素受体底物-1(p-IRS-1)/胰岛素受体底物-1(IRS-1)蛋白的表达。结果与正常组相比,模型组大鼠肝细胞内存在明显脂肪样变性,HOMA-IR、肝脏指数、血清FFA水平、肝组织TG含量及肝组织p-JNK1/JNK1、p-IRS-1/IRS-1蛋白表达水平均升高(P<0.05),ISI水平降低(P<0.05);与模型组比较,糖通饮各剂量组大鼠肝细胞脂肪样变性均有不同程度的改善,HOMA-IR、肝脏指数、血清FFA水平、肝组织TG含量、肝组织p-JNK1/JNK1及p-IRS-1/IRS-1蛋白表达水平均降低(P<0.05),ISI水平增高(P<0.05)。结论经验方糖通饮可有效调节T2DM合并NAFLD大鼠肝脏的脂质代谢,减轻肝细胞脂肪变性程度,其机制可能与抑制肝组织JNK信号通路相关蛋白表达有关。

Objective To explove the effect of Tangtongyin formula on the lipid metabolism of type 2 diabetes mellitus(T2DM)complicated with rat non-alcoholic fatty liver disease(NAFLD)and its mechanism.Methods Forty-four male SD rats aged 8 week old were selected,10 of them were randomly selected to be fed with basal diet and injected with equal dose of citric acid-sodium citrate buffer to establish a normal group,and the other rats were fed with high-fat and high-sugar diet and intraperitoneally injected with low-dose streptozotocin for 8 consecutive weeks to establish a T2DM with NAFLD model.Two rats were selected from normal group and T2DM with NAFLD model group,respectively to verify whether the modeling was successful.Based on Tangtongyin formula dose,32 rats in model group were randomly divided into a model group(0 g/kg),low-dose(12 g/kg),medium-dose(24 g/kg),and high-dose(36 g/kg)groups.The rats in each dose group were respectively given different doses of Tangtongyi formula by gavage,and the rats in model and normal groups were given the same volume of double distilled water by gavage administration for 8 consecutive weeks.After gavage administration,the rats in each group were weighed,and tail vein blood was collected to detect fasting blood glucose(FBG).Rats in each group were anesthetized by intraperitoneal injection of 10%chloral hydrate.Abdominal aortic blood was taken to detect serum insulin(INS)by ELISA assay,and serum free fatty acid(FFA)was detected by spectrophotometer colorimetric assay.HOMA-IR and insulin sensitivity index(ISI)were calculated.Rat liver tissues were quickly extracted immediately after blood collection to calculate liver index.GPO-PAP method was applied to detect triglyceride(TG)content.Hematoxylin-eosin(HE)staining was performed to observe the histological characteristics of rat liver tissues in each group.Western blot was used to detect the protein expression levels of phosphorylated c-Jun N-terminal Kinase-1(p-JNK1)/c-Jun N-terminal Kinase-1(JNK1),phosphorylated Insulin Receptor Substrate-1(p-IRS-1)/Insulin Receptor Substrate-1(IRS-1).Results When compared with normal group,there was obvious steatosis in rat hepatocytes in model group,and model group had increased HOMA-IR,liver index,serum FFA level,liver tissue TG content,and protein expression levels of liver p-JNK1/JNK1,p-IRS-1/IRS-1(P<0.05)but decreased ISI level(P<0.05).When compared to model group,the steatosis of rat hepatocytes in each dose group was improved to different degrees,HOMA-IR,liver index,serum FFA level,liver tissue TG content,the protein expression levels of p-JNK1/JNK1 and p-IRS-1/IRS-1 protein in liver tissues were decreased(P<0.05),while ISI level was increased(P<0.05).Conclusions Tangtongyin formula can effectively regulate liver lipid metabolism in rats with T2DM complicated with NAFLD and reduce steatosis in rat hepatocytes.Its mode of action may be related to the inhibition of JNK signaling pathway-associated protein expression in liver tissues.