自噬在造影剂诱导的肾小管上皮细胞损伤中的作用

Role of autophagy in contrast media-induced renal tubular epithelial cells injury

目的 观察造影剂对肾小管上皮细胞自噬及凋亡的影响,探讨自噬在造影剂诱导的肾小管上皮细胞损伤中的作用.方法 体外培养大鼠肾小管上皮细胞（NRK-52E）,以不同浓度（25、50、100、200 gI/L）造影剂碘普罗胺处理NRK-52E细胞1h或以50 gI/L碘普罗胺刺激NRK-52E细胞不同时间（0.5、1、3、6、12、24 h）,吖啶橙染色观察细胞内自噬体形成,Western印迹检测自噬标志蛋白微管相关蛋白1轻链3（LC3）的表达,流式细胞术检测细胞凋亡率.雷帕霉素（Rap,1μg/L）或3-甲基腺嘌呤（3-MA,2 mmol/L）与碘普罗胺（50 gI/L）共处理肾小管上皮细胞后,Western印迹法检测各组LC3及自噬相关蛋白Beclin-1的表达,绿色荧光蛋白标记的LC3（GFP-LC3）转染后荧光显微镜观察GFP-LC3的表达,Hoechst-33342染色观察细胞凋亡.结果 （1）随碘普罗胺刺激浓度增加肾小管上皮细胞自噬体表达先上升后下降.（2）随碘普罗胺刺激浓度及时间的增加肾小管上皮细胞LC3-Ⅱ/LC3-Ⅰ的表达先上升后下降（P＜0.05）.（3）碘普罗胺刺激后,肾小管上皮细胞的凋亡率呈浓度及时间依赖性增加（P＜0.05）.（4）Rap与碘普罗胺共处理肾小管上皮细胞后,LC3-Ⅱ/LC3-Ⅰ、Beclin-1及GFP-LC3表达上调,细胞凋亡减少（P＜0.05）;3-MA与碘普罗胺共处理肾小管上皮细胞后,LC3-Ⅱ/LC3-Ⅰ、Beclin-1及GFP-LC3表达下调,细胞凋亡增加（P＜0.05）.结论 造影剂可诱导肾小管上皮细胞凋亡和自噬,小剂量造影剂刺激下肾小管上皮细胞自噬和凋亡同时发生,但高峰早于凋亡.在刺激早期一定程度增强自噬可抑制细胞凋亡,反之抑制自噬后细胞凋亡增加.

Objective To observe the effect of contrast media on autophagy and apoptosis of renal tubular epithelial cells,evaluate the role of autophagy in contrast media-induced renal tubular epithelial cells injury.Methods NRK-52E cells were exposed to iopromide at different concentration for 1 hour or at 50 gI/L for variable incubation time.Rapamycin （1 μg/L） and 3-methyadenine （2 mmol/L） were further introduced to investigate the role of autophagy in the process.The formation of autophagy was observed by acridine orange staining and Green fluorescent protein tagged LC3 （GFP-LC3）.The expression of autophagy protein LC3 and Beclin-1 was examined by Western blotting,and the apoptosis level was examined by flow cytometry and Hoechst 33342-staining.Results （1） Autophagy could be enhanced by contrast media in renal tubular epithelial cells.（2） The expression of LC3-Ⅱ/LC3-Ⅰ in renal tubular epithelial cells rose at first and then dropped with the increase of iopromide stimulation time and concentration （P ＜ 0.05）.（3） Iopromide promoted renal tubular epithelial cell apoptosis in dose-and time-dependent manner （P ＜ 0.05）.（4） Co-culture with rapamycin further increased LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and GFP-LC3 expression,but obviously prevented iopromide-induced apoptosis of renal tubular epithelial cells （P ＜ 0.05）.On the contrary,Coculture with 3-methyadenine reduced iopromide-induced LC3-Ⅱ/LC3-Ⅰ,Beclin-1 and GFP-LC3 overexpression,but aggravated the apoptosis induced by iopromide （P＜0.05）.Conclusions Contrast media can induce renal tubular epithelial cells apoptosis as well as autophagy.Enhancing autophagy appropriately has a protective effect on iopromide-induced renal tubular epithelial cells apoptosis,which conforms that autophagy plays an important role in antagonizing iopromide-induced renal tubular epithelial cells injury.