石斛合剂对2型糖尿病合并非酒精性脂肪肝大鼠AMPK/TFEB信号通路自噬蛋白的影响

Effect of Shihu Mixture on Autophagy Protein of AMPK/TFEB Signaling Pathway in Rats with T2DM-NAFLD

目的:观察石斛合剂干预后,2型糖尿病(T2DM)合并非酒精性脂肪肝(NAFLD)大鼠腺苷酸活化蛋白激酶(AMPK)/转录因子EB(TFEB)自噬信号通路蛋白表达。方法:40只雄性SD大鼠,根据体质量随机选择10只作为正常组。其余30只大鼠高脂高糖饮食喂养6周,然后腹腔注射链脲佐菌素建立2型糖尿病合并非酒精性脂肪肝模型。分为正常组(10 mL·kg^-1·d^-1),模型组(10 mL·kg^-1·d^-1),二甲双胍组(100 mg·kg^-1·d^-1),石斛合剂组(11.3 g·kg^-1·d^-1),每组大鼠灌胃4周。灌胃结束后,对大鼠实施安乐死。取腹主动脉血、肝组织,检测各组大鼠空腹血糖(FBG),血清中糖化血清蛋白(GSP),胰岛素(INS),甘油三酯(TG),总胆固醇(TC),高密度脂蛋白胆固醇(HDL-C),低密度脂蛋白胆固醇(LDL-C)含量;苏木素-伊红(HE)染色观察肝脏组织形态变化;蛋白免疫印迹法(Western blot)检测AMPK/TFEB信号通路相关蛋白的表达。结果:与模型组比较,石斛合剂组与二甲双胍组大鼠的FBG,GSP下降(P<0.05),INS升高(P<0.05);与模型组比较,石斛合剂组与二甲双胍组大鼠HDL升高(P<0.05),TC,TG,LDL含量明显降低(P<0.05)。石斛合剂与二甲双胍均能一定程度上改善T2DM合并NAFLD大鼠肝脏形态。石斛合剂与二甲双胍p-AMPK/AMPK升高(P<0.05),TFEB,LC3Ⅱ表达升高(P<0.05)。结论:石斛合剂可通过激活AMPK/TFEB自噬信号通路来改善糖脂代谢,改善肝脏病理形态。

Objective:To observe the expression of adenosine monophosphate activated protein kinase(AMPK)/transcription factor EB(TFEB)autophagy signaling pathway protein in type 2 diabetes(T2DM)complicated with nonalcoholic fatty liver disease(NAFLD)rats after intervention with Shihu mixture(SHM).Method: Among 40 male SD rats,10 rats were randomly selected as normal group according to body weight.The remaining 30 rats were fed with high-fat and high-sugar diet for 6 weeks,and then intraperitoneally injected with streptozotocin(STZ)to establish a T2DM NAFLD model. They were divided into normal control group(10 mL·kg^-1·d^-1),model control group(10 mL·kg^-1·d^-1),metformin group(100 mg·kg^-1·d^-1),SHM group(11.3 g·kg^-1·d^-1). The rats in each group were gavaged for 4 weeks. After gavage,the rats were euthanized.Abdominal aortic blood and liver tissue were collected to detect fasting blood glucose(FBS),glycated serum protein(GSP),insulin(INS),triglyceride(TG),total cholesterol(TC),high density lipoprotein cholesterol(HDL-C),low-density lipoprotein cholesterol(LDL-C)content. Htoxylin eosin(HE)staining was performed to observe changes in liver tissue morphology. Western blot was used to detect AMPK/TFEB signaling pathwayrelated protein expression. Result: Compared with the model control group,FBS and GSP of the SHM group and the DMBG group decreased(P<0.05),while INS increased(P<0.05). Compared with the model group,HDL increased in the SHM group and the DMBG group(P<0.05),whereas TC,TG and LDL contents decreased(P<0.05). Liver HE staining results showed that both SHM and Metformin could improve the liver morphology of T2DM and NAFLD rats to some extent. Western blot results showed that p-AMPK/AMPK of SHM and metformin increased(P<0.05),while the expressions of TFEB and LC3 Ⅱ increased(P<0.05).Conclusion: SHM can improve glucose and lipid metabolism by activating AMPK/TFEB autophagy signaling pathway,so as to improve liver pathological morphology.