大黄素诱导自噬改善顺铂导致肾小管细胞损伤机制探索

目的：观察大黄素对顺铂所致的肾小管上皮细胞（NRK-52E）损伤的影响，探讨其可能分子调节机制。方法：观察大黄素对顺铂所致NRK-52E细胞形态学改变的影响；采用Western Blot方法检测顺铂单独处理和加入大黄素共同处理细胞后，凋亡相关蛋白Caspase-3和cleaved Caspase-3的表达情况；用大黄素干预细胞不同的时间点，观察微管相关蛋白1轻链3（LC3）II/I的表达和pmRFP-LC3质粒转染细胞后荧光颗粒的变化情况，同时观察雷帕霉素处理细胞不同时间点后LC3-II/I的表达情况及其对顺铂环境下细胞形态学改变的影响，并观察大黄素对自噬上游通路AMPK的活化和mTOR信号的影响。结果：顺铂可以诱导NRK-52E细胞出现形态学改变，大黄素能够改善顺铂导致的变化。另外，大黄素干预可以下调顺铂导致的cleaved Caspase-3蛋白表达的增多；大黄素处理细胞不同时间点LC3-II/LC3-I的比值明显上升，pmRFP-LC3转染观察到大黄素处理细胞后自噬颗粒明显增多。同时雷帕霉素处理细胞后LC3-II/LC3-I的比值明显上升，其与顺铂共同干预能明显改善顺铂诱导的细胞凋亡；大黄素干预时间的延长，p-mTOR蛋白表达明显下调，p-AMPK的表达明显上调。结论：大黄素可以改善顺铂诱导的NRK-52E细胞凋亡，其作用机制可能是通过调节AMPK/mTOR信号通路诱导自噬来发挥肾保护作用。

Mechanism Exploration on Emodin Ameliorates Cisplatin-induced Renal Tubular Cell Injury through Activation of Autophagy

This study was aimed to observe the effect of emodin on cisplatin-induced renal tubular epithelial cells (NRK- 52E) injury, in order to explore its possible molecular mechanisms. Firstly, effects of emodin on cisplatin- induced morphological changes in NRK-52E cells were observed. Secondly, the apoptosis-related protein expression of Caspase- 3 and cleaved Caspase-3 were detected after the treatment of cisplatin alone or cisplatin together with emodin by western blot. Then, the expression of microtubule-associated protein 1 light chain 3 (LC3) II/I was detected after the treatment of emodin or rapamycin at different time points by western blot. Changes of pmRFP- LC3 fluorescent particles were observed by fluorescence microscopy. And effects of rapamycin on cellular morphological changes were observed in the environment of cisplatin. Finally, effects of emodin on the activation of AMPK and mTOR signal pathway were further observed, which is considered as the upstream of autophagy signaling pathway. The results showed that cisplatin can induce morphological changes in NRK- 52E cell, which was obviously ameliorated by the intervention of emodin. Additionally, the increased protein expression of cleaved Caspase-3 induced by cisplatin was obviously reduced after the intervention of emodin. The LC3-II/LC3-I ratio was significantly increased after the treatment of emodin or rapamycin at different time points. Rapamycin can significantly ameliorate NRK- 52E cell apoptosis induced by cisplatin. Simultaneously, the number of pmRFP- LC3 fluorescent particles increased after the treatment of emodin. As the extension of time by intervention of emodin, the protein expression of p-mTOR was significantly reduced. In contrast, the protein expression of p- AMPK was significantly increased. It was concluded that emodin can ameliorate cisplatininduced apoptosis in NRK- 52E cells. Its potential mechanism may be attributed to the activation of autophagy by regulating AMPK/mTOR signaling pathway. And thus, it played a role in renal protective effects.