摘要
目的:通过观察雷帕霉素对PAN诱导的足细胞损伤及自噬相关蛋白表达的影响,探讨自噬在雷帕霉素保护PAN诱导的足细胞损伤中的作用及可能机制。方法:构建PAN诱导的足细胞损伤模型,将足细胞分成对照组(Control组),PAN组(加入50μg/ml PAN),雷帕霉素组(RAP组:分别加入100、200、300 ng/ml雷帕霉素),PAN+雷帕霉素组(PAN+RAP组:细胞在用含PAN的培养液培养前1 h,分别用100、200、300 ng/ml雷帕霉素进行预处理1 h)。采用Annexin V/PI双染法检测细胞凋亡,透射电镜观察自噬小体,Western blot检测LC3、p62、4EBP1、P70S6K、m TOR蛋白表达。结果:与对照组比较,PAN组足细胞凋亡增加,自噬体减少,LC3Ⅱ蛋白表达下调,p62上调,m TOR、4EBP1、P70S6K磷酸化水平上调;与PAN组比较,PAN+RAP组足细胞凋亡率下降,自噬体增加,LC3Ⅱ蛋白表达上调,p62下调,m TOR、4EBP1、P70S6K磷酸化水平下调。结论:PAN可以抑制足细胞自噬,促进足细胞凋亡;雷帕霉素可通过激活自噬改善PAN诱导的足细胞损伤,这种作用可能与雷帕霉素抑制m TOR/4EBP1、P70S6K信号通路有关。
Objective： To explore the protective effect of rapamycin and the role of autophagy on podocyte injury induced by puromycin amino nucleoside. Methods： The mice podocytes cell 5（ MPC5） were randomly divided into four groups： Control group,PAN group（ 50 μg/ml PAN）; rapamycin group（ RAP group,100 ng/ml,200 ng/ml,300 ng/ml rapamycin） and PAN ＋ rapamycin group（ PAN＋RAP group,treated with rapamycin before PAN）. The apoptosis of podocytes were detected by Annexin V/PI. Transmission electronic microscopy was used to observe the formation of autophagosome. The expression of autophagic markers,LC3,p62,and 4 EBP1,P70 S6 K,m TOR were detected by Western blot. Results： The podocyte apoptotic rate was significantly decreased when the podocytes were treated with rapamycin compared with PAN group. Further,more autophagosomes appeared in cytoplasm of podocytes in RAP and PAN＋RAP group compared with PAN group. And the expression of LC3Ⅱ was significantly increased in rapamycin treated podocytes,whereas the expression of p62 was decreased markedly. Phosphorylation of m TOR,4 EBP1 and P70 S6 K was decreased rapamycin treated groups. Conclusion： Podocyte autophagy was inhibited by PAN,and the apoptosis of podocytes was promoted. Rapamycin protect puromycin amino nucleoside induced podocyte injury by upregulating autophagy and it may be concerned with m TOR 4 EBP1 and P70 S6 K signaling pathway.
作者
詹慧芳
刘文娟
金娟
龚建光
赵黎
何强
ZHAN Hui-Fang;LIU Wen-Juan;JIN Juan;GONG Jian-Guang;ZHAO Li;HE Qiang(Zijingang Campus of ZhejiangUniversity Hospital,Hangzhou 310058,China)
出处
《中国免疫学杂志》
CAS
CSCD
北大核心
2018年第2期188-191,198,共5页
Chinese Journal of Immunology
基金
浙江医药卫生科技一般项目(2015KYA025)
浙江省中医药管理局项目(2016ZA023)资助
