自噬减轻造影剂诱导足细胞氧化应激损伤  

Autophagy alleviate podocytes injury induced by contrast media via oxidative stress

作　　者：汪雄攀 杨定平[1] 杨定位[1] 贾汝汉[1] 丁国华[1] 朱吉莉[1] 简永红 

机构地区：[1]武汉大学人民医院肾内科,430060

出　　处：《中华肾脏病杂志》2014年第9期684-688,共5页Chinese Journal of Nephrology

基　　金：国家自然科学基金（81370841;81270762;81100519）

摘　　要：目的 研究自噬在造影剂诱导足细胞氧化应激损伤中的作用.方法 培养的小鼠永生化足细胞共分为6组：（1）正常对照（Con）组;（2）造影剂（CM）组;（3）雷帕霉素（Rap）组;（4）造影剂加雷帕霉素（CM＋Rap）组;（5）3-甲基腺嘌呤（3-MA）组;（6）造影剂加3-甲基腺嘌呤（CM＋3-MA）组,予以相应的刺激2h.观察自噬增强剂雷帕霉素与自噬抑制剂3-甲基腺嘌呤对造影剂诱导的足细胞氧化应激及细胞活性的影响.Western印迹检测各组足细胞内自噬相关蛋白LC3-Ⅱ、Beclin-1及氧化应激相关蛋白Catalase、MnSOD的表达,丹酰戊二胺（MDC）染色检测足细胞内自噬体变化,采用氯甲基-2＇,7＇-二氯二氢荧光素二乙酯（CM-H2DCFDA）荧光染色检测足细胞内活性氧（ROS）水平,CCK8试剂盒检测细胞活性.结果 （1）造影剂抑制了足细胞内氧化应激相关蛋白Catalase、MnSOD的表达,促进了细胞内ROS的生成,导致足细胞活性下降（均P＜ 0.05）;（2）3-MA共处理足细胞后,细胞内自噬相关蛋白LC3-Ⅱ、Beclin-1的表达及自噬体生成均减少,氧化应激相关蛋白Catalase、MnSOD的表达进一步减少,ROS生成进一步增加,导致足细胞活性下降更加显著（均P＜ 0.05）;（3）雷帕霉素共处理足细胞后,自噬相关蛋白LC3-Ⅱ、Beclin-1的表达及自噬体生成增加,氧化应激相关蛋白Catalase、MnSOD的表达增强,ROS生成减少,促进了足细胞活性恢复（均P＜0.05）.结论 自噬在造影剂诱导的足细胞氧化应激损伤起保护性作用,一定程度自噬上调可以减轻其氧化应激水平从而抑制足细胞损伤.Objective To evaluate the effects of autophagy on oxidative stress induced by contrast media in podocytes.Methods The differentiated mouse podocytes were exposed to contrast media （Iopromide,50 mg/L）、rapamycin （Rap,autophagy enhancer,1 ng/L）,3-methyladenine （3-MA,autophagy inhibitor,2 mmol/L） for 2 hours.The expression of autophagy protein LC3-Ⅱ and Beclin-1 as well as oxidative stress-related proteins Catalase,MnSOD were detected by Western blot.The formations of autophagy were observed by MDC staining,and the levels of reactive oxygen species （ROS） by CM-H2DCFDA staining.Cell activity was evaluated by CCK8 assay.Results Both the levels of oxidative stress and autophagy in podocytes increased when stimulated by contrast media,the expression of LC3-Ⅱ and Beclin-1 were enhanced,Catalase and MnSOD were inhibited （all P ＜ 0.05）.Rapamycin increased the expression of Catalase,MnSOD and cell activity of podocytes,reduced the generation of ROS （all P ＜ 0.05）,but in Rap group,cell activity showed no significant difference （P ＞ 0.05）.3-MA decreased the expression of Catalase 、MnSOD and inhibited the cell activity of podocyte,increased the generation of ROS （all P ＜ 0.05）.Conclusion Autophagy protects podocyte from contrast media by the means of reducing oxidative stress.

关 键 词：自噬 造影剂 足细胞 氧化应激 

分 类 号：R587.2[医药卫生—内分泌]