番泻叶苷A对糖尿病肾病大鼠足细胞自噬的影响

Influence of sennoside A on podocyte autophagy in diabetic nephropathy rats

目的探究番泻叶苷A(sennoside A,SA)对糖尿病肾病(diabetic nephropathy,DN)大鼠足细胞自噬及上皮-间质转化(EMT)的影响及可能的作用机制。方法随机选择10只雄性Wistar大鼠作为正常组,剩余大鼠尾静脉注射55 mg·kg^(−1)链脲佐菌素(streptozotocin,STZ)诱导DN大鼠模型。将成模大鼠随机分为模型组、SA低剂量组、SA高剂量组及贝那普利组,每组10只。SA低剂量组、SA高剂量组大鼠每日分别灌胃给予SA 40、80 mg·kg^(−1),贝那普利组大鼠每日灌服给予盐酸贝那普利10 mg·kg^(−1),模型组及正常组大鼠每日灌服等量生理盐水,连续给药21 d。检测24 h尿白蛋白(albumin,Alb)含量、大鼠空腹血糖(fasting blood glucose,FBG)、血肌酐(serum creatinine,Scr)和血尿素氮(blood urea nitrogen,BUN)水平;染色观察肾组织学变化;透射电镜观察肾脏组织自噬体形成情况;蛋白印迹法(Western blotting)检测肾组织中微管相关蛋白1轻链3(icrotubule associated protein 1 light chain 3,LC3)、Bcl-2相互作用蛋白1(Bcl-2 interacting protein 1,Beclin-1)、E-钙黏蛋白(E-cadherin,E-cad)、波形蛋白(vimentin,Vim)、沉默信息调节因子1(silencing information regulator 1,Sirt1)和肾病蛋白nephrin蛋白的相对表达水平。结果与正常组比较,其他4组大鼠24 h Alb含量、FBG及Scr、BUN水平升高,肾小球和肾小管发生病理损伤,胶原纤维及糖原明显沉积,肾小球系膜及基底膜增厚,肾组织中自噬体数量减少,LC3Ⅱ/LC3Ⅰ比值、Beclin-1、E-cad、nephrin及Sirt1蛋白的相对表达量降低,Vim蛋白的相对表达量升高(P<0.05);与模型组比较,3个给药组大鼠24 h Alb含量、FBG及Scr、BUN水平降低,肾小球和肾小管病理损伤情况呈不同程度减轻,胶原纤维及糖原沉积减弱,肾小球系膜及基底膜增厚情况改善,自噬体数量增加,LC3Ⅱ/LC3Ⅰ比值、Beclin-1、E-cad、nephrin及Sirt1蛋白的相对表达量升高,Vim蛋白的相对表达量降低(P<0.05);SA低剂量组、SA高剂量组及贝那普利组对DN大鼠的作用效果逐渐增强(P<0.05)。结论SA可改善DN大鼠肾功能障碍,其作用机制可能与激活Sirt通路进而促进足细胞自噬并减轻EMT有关。

Objective To investigate the effects of sennoside A(SA)on autophagy and epithelial-mesenchymal transformation(EMT)of popocytes in diabetic nephropathy(DN)rats and its possible mechanism.Methods Ten male Wistar rats were randomly selected as normal group,and the remaining rats were injected with streptozotocin(STZ)55 mg·kg^(−1) into tail vein to induce DN rat model.The model rats were randomly divided into model group,low dose SA group,high dose SA group and benazepril group,with 10 rats in each group.Rats in low-dose SA group and high-dose SA group were given 40 mg·kg^(−1),80 mg·kg^(−1) intragastric administration of SA,respectively.The benazepril group were given Benazepril Hydrochloride Tablets 10 mg·kg^(−1) intragastric administration,while the model group and normal group were given normal saline intragastric administration,for 21 consecutive days.The 24-hour urinary albumin(Alb)content was detected by ELISA.The levels of fasting blood glucose(FBG),serum creatinine(Scr)and blood urea nitrogen(BUN)were detected by biochemical analyzer.Staining were used to observe the renal histological changes.Transmission electron microscopy(TEM)was used to observe the formation of autophagosome in kidney tissue.Western blotting was used to detect the relative protein expression levels of microtubule-associated protein 1 light chain 3(LC3),Bcl-2 interacting protein 1(Beclin-1),E-cadherin(E-cad),vimentin(Vim),silencing information regulator 1(Sirt1)and nephropathy protein nephrin in renal tissues.Results The DN rat model was successfully constructed.Compared with normal group,the 24-hour Alb content,FBG,Scr and BUN levels in other 4 groups were increased,the pathological damage of glomerulus and renal tubules occurred,the collagen fiber and glycogen deposition were obvious,the mesangial and basement membrane of glomerulus were thickened,the number of autophagosomes in renal tissue was decreased,and the LC3Ⅱ/LC3Ⅰratio was increased.The relative expression levels of Beclin-1,E-cad,nephrin and Sirt1 proteins were decreased,while the relative expression level of Vim protein was increased(P<0.05).Compared with model group,the 24-hour Alb content,FBG,Scr and BUN levels were decreased in 3 administration groups,and the pathological damage of glomerular and renal tubules was alleviated to varying degrees,the collagen fiber and glycogen deposition were decreased,the thickening of mesangial and basement membrane of glomerular was improved,the number of autophagosomes was increased,and the LC3Ⅱ/LC3Ⅰratio was increased.The relative expression levels of Beclin-1,E-cad,nephrin and Sirt1 proteins were increased,while the relative expression level of Vim protein was decreased(P<0.05).The effect on DN in benazepril group and low dose SA group was gradually increased(P<0.05).Conclusion SA could improve renal dysfunction in DN rats,and its mechanism may be related to the activation of Sirt pathway to promote podocyte autophagy and alleviate EMT.