益气解毒活络中药通过上调自噬发挥对糖尿病肾病大鼠保护作用的机制研究

Mechanism of Protective Effect of Yiqi Jiedu Huoluo Formula(益气解毒活络方)on Diabetes Nephropathy Rats by Upregulating Autophagy

目的探讨益气解毒活络中药通过上调自噬水平减轻糖尿病肾病大鼠肾组织损伤的机制。方法采用高糖高脂饲料联合链脲佐菌素(streptozotocin,STZ)建立糖尿病肾病(diabetic kidney disease,DKD)大鼠模型,并将DKD大鼠模型按体质量随机分为以下7组:模型组,白藜芦醇组(resveratrol,RES组),尼克酰胺组(nictotinamide,NAM组),益气解毒活络方高剂量组(中药高剂量组),益气解毒活络方中剂量组(中药中剂量组),益气解毒活络方低剂量组(中药低剂量组),洛汀新组。连续给药8周后检测各组大鼠肾脏指数、血糖(blood glucose,BG)、血肌酐(serum creatinine,Scr)、血尿素氮(blood urea nitrogen,BUN)及24 h尿蛋白水平(24 h-UAlb);采用逆转录—聚合酶链反应(RT-PCR)检测各组大鼠肾脏组织中纤维连接蛋白(fibronectin,FN)、四型胶原蛋白(typeⅣcollagen,COLIV)、人沉默信息调节因1(silent infor-mation regulator 1,Sirt1)、叉头状转录因子O1(forkhead transcription factor O1,FoxO1)、自噬蛋白5(autophagy Protein 5,Atg5)mRNA的表达水平;WesternBlot法分析肾组织LC3-Ⅱ/I及Atg5蛋白表达。HE染色观察肾组织形态变化。结果与正常组比较,模型组及NAM组大鼠肾脏指数、血糖、血肌酐、尿素氮、24 h尿蛋白水平明显升高,Sirt1和FoxO1 mRNA表达下降,LC3-Ⅱ/I比值下降,FN、COLIV mRNA表达升高,Atg5蛋白及mRNA表达均明显降低。与模型组相比较,除NAM组外,其余各治疗组大鼠肾脏指数、血糖、血肌酐、尿素氮、24 h尿蛋白水平均有不同程度的下降,Sirt1、FoxO1 mRNA表达升高,LC3-Ⅱ/I蛋白表达均有不同程度的升高,Atg5蛋白及mRNA表达均明显升高,其中以RES组、中药高剂量及洛汀新组效果明显。HE染色显示各治疗组肾组织病理损伤有不同程度的改善。结论益气解毒活络中药减缓了糖尿病肾病的发病进程,其机制可能与上调Sirt1-FoxO介导的肾脏自噬有关。

Objective To explore the mechanism of Yiqi Jiedu Huoluo Formula(益气解毒活络方)in alleviating renal tissue damage in diabetes nephropathy rats by up regulating the autophagy level.Methods The DKD rat model was established using a combination of high sugar and high fat feed and streptozotocin(STZ).The diabetic kidney disease(DKD)rat model was randomly divided into the following 7 groups according to body weight:model group:resveratrol(RES)group,nicotinamide(NAM)group,high-dose group of Yiqi Jiedu Huoluo Formula,medium dose group of Yiqi Jiedu Huoluo Formula,low dose group of Yiqi Jiedu Huoluo Formula and lotensin group.After continuous administration for 8 weeks,renal index,blood glucose(BG),serum creatinine(Scr),blood urea nitrogen(BUN)and 24-hour urine protein level(24-hour UAlb)were measured in each group.Reverse transcription polymerase chain reaction(RT-PCR)was used to detect the expression levels of FN,typeⅣcollagen(COLIV),silent information regulator 1(Sirt1),forkhead transcription factor O1(FoxO1)and autophagy protein 5(Atg5)mR-NA in the kidney tissues of rats in each group.Western Blot method was used to analyze the expressions of LC3-Ⅱ/I and Atg5 proteins in renal tissue.HE staining was used to observe the morphological changes of renal tissue.Result Compared with those of the normal group,the levels of kidney index,blood glucose,creatinine,urea nitrogen and 24-hour urine protein of the model group and NAM group rats were significantly increased,the expressions of Sirt1 and FoxO1 mRNA decreased,LC3-Ⅱ/I ratio de creased,the expressions of FN and COLIV mRNA increased,and the expressions of Atg5 protein and mRNA were significantly reduced.Compared with those in the model group,except the NAM group,the levels of kidney index,blood glucose,creatinine,urea nitrogen and 24-hour urine protein of rats in the other treatment groups decreased to varying degrees.The expressions of Sirt1 and FoxO1 mRNA increased,the expression of LC3-Ⅱ/I protein increased to varying degrees,and the expressions of Atg5 protein and mRNA were significantly increased.Among them,the RES group,Yiqi Jiedu Huoluo Formula high-dose group and Lotensin group showed significant effects.HE staining showed varying degrees of improvement in renal tissue pathological damage in each treatment group.Conclusion Yiqi Jiedu Huoluo Formula can slow down the pathogenesis of diabetes nephropathy,and its mechanism may be related to the up regulation of Sirt1 FoxO mediated renal autophagy.