雷帕霉素通过下调微小RNA-27a介导的雷帕霉素靶分子信号通路调控糖尿病肾脏疾病大鼠足细胞自噬的影响研究

Rapamycin regulates podocyte autophagy in rats with diabetic kidney disease by down-regulating miR-27a mediated mTOR signaling pathway

目的探讨微小RNA-27a(miR-27a)介导的雷帕霉素靶分子(mTOR)信号通路调控DKD大鼠足细胞自噬的作用。方法将30只SD大鼠随机分为正常对照组(NC,n=10)、糖尿病模型组(DKD,n=10)和雷帕霉素治疗组(Rap,n=10)。检测各组肾功能相关指标和细胞凋亡率,HE染色及免疫组化评估肾组织形态学变化。Western blot法检测各组肾组织中自噬相关指标Beclin1、微管相关蛋白1A/1B-轻链3(LC3)及mTOC蛋白表达,qRT-PCR检测足细胞特异性蛋白miR-27a、Podocalyxin、Nephrin、LC3-Ⅰ、LC3-Ⅱ、Bax、Beclinl mRNA表达量。结果与NC组比较,DKD组第6、12周FPG、24 hUAlb、BUN、血肌酐(Scr)、肾脏指数(KI)表达升高(P<0.05)。与NC组比较,DKD组细胞凋亡、mTOC蛋白、miR-27a、mTOR、Bax mRNA表达升高(P<0.05),Beclinl、LC3-Ⅰ、LC3-Ⅱ蛋白、LC3-Ⅰ、LC3-Ⅱ、Beclin1、Podocalyxin、Nephrin mRNA表达降低(P<0.05)。与DKD组比较,Rap组Beclinl、LC3-Ⅰ、LC3-Ⅱ蛋白、LC3-Ⅰ、LC3-Ⅱ、Beclinl、Podocalyxin、Nephrin mRNA表达升高(P<0.05),细胞凋亡、mTOC蛋白、miR-27a、mTOR、Bax mRNA表达降低(P<0.05)。结论DKD大鼠肾组织miR-27a过表达可能介导mTOR自噬信号通路损伤足细胞,雷帕霉素能下调miR-27a表达,抑制mTOR/Beclinl/LC3信号通路,保护肾小球足细胞。

Objective To investigate the role of microRNA27a(miR-27A)mediated mTOR signaling pathway in regulating podocyte autophagy in rapamycin diabetic nephropathy rats.Methods Thirty SD rats were randomly divided into normal control group(NC,n=10),model group(DKD,n=10)and rapamycin group(Rap,n=10).HE staining and immunohistochemistry were used to evaluate the changes of renal histomorphology.Cell apoptosis rate was detected.Beclinl,microtubule associated protein 1A/1b-light chain 3(LC3)and mTOC protein expressions in renal tissues of each group were detected by Western blot.mRNA expression levels of Podocalyxin,miR-27a,Nephrin,LC3-I,LC3-II,Bax and Beclinl were detected by qRT-PCR.Results Compared with NC group,FPG,24 hUAlb,BUN,serum creatinine(Scr)and renal index(KI)increased at the 6th and 12th week in DKD group(P<0.05).Compared with NC group,the apoptosis,the expression of mTOC protein,miR-27a,mTOR and Bax mRNA increased(P<0.05),while the expressions of Beclinl,LC3-I,LC3-II protein,LC3-I,LC3-II,Beclinl,Podocalyxin and Nephrin mRNA decreased in DKD group(P<0.05).Compared with DKD group,the mRNA expressions of Beclinl,LC3-I,LC3-II protein,LC3-II,LC-3II,Beclinl,Podocalyxin and Nephrin mRNA increased(P<0.05),while the mRNA expressions of apoptosis,mTOC protein,miR-27a,mTOR and Bax decreased in Rap group(P<0.05).CConclusion The overexpression of miR-27amay mediate the damage of mTOR autophagy signaling pathway to podocytes in renal tissue of DKD rats,and rapamycin can down-regulate the expression of miR-27a and inhibit mTOR/Beclinl/LC3 signaling pathway to protect glomerular podocytes.