喹乙醇致肾脏毒性的内质网应激相关凋亡途径研究  

Research on olaquindox induced endoplasmic reticulum stress related apoptosis on nephrotoxicity

作　　者：李忠生[1] 于常艳[1] 陈宵[1] 张斌[1] 曹鹏[1] 李斌[1] 肖经纬[1] 

机构地区：[1]中国疾病预防控制中心职业卫生与中毒控制所毒理室,中国疾病预防控制中心化学污染物与健康重点实验室,北京100050

出　　处：《卫生研究》2015年第3期444-450,共7页Journal of Hygiene Research

基　　金：环保公益性行业科研专项(No.201109038)

摘　　要：目的通过喹乙醇染毒人源肾小管上皮细胞(HK-2细胞)并检测活性氧(ROS)和凋亡相关蛋白的表达,探讨喹乙醇肾脏毒性产生过程及其可能的内质网应激相关凋亡机制。方法分别以不同浓度(1、2、3、4、5、6、7和8μmol/ml)喹乙醇染毒HK-2细胞24 h,噻唑蓝(MTT)比色法检测细胞增殖率以确定剂量-效应关系;Hoechst-33258荧光染色检测各组细胞凋亡形态,流式细胞仪检测各组细胞凋亡率和细胞内活性氧含量;免疫印迹法(Western blot)检测内质网应激相关凋亡蛋白葡萄糖调节蛋白78(GRP78)、葡萄糖调节蛋白94(GRP94)和凋亡促进因子CCAAT增强子结合蛋白同源蛋白(CHOP)的表达。结果根据MTT毒性实验结果 ,喹乙醇对HK-2细胞凋亡效应的适宜剂量确定为1、2、3和4μmol/ml。在喹乙醇不同剂量观察组中,喹乙醇染毒2μmol/ml以上剂量组,细胞凋亡率、内质网凋亡相关蛋白GRP79、GRP94和CHOP表达增加,喹乙醇各染毒剂量均可见活性氧水平增加(P<0.05);在喹乙醇不同染毒时间观察组中,喹乙醇染毒12和24 h组,细胞凋亡率、内质网凋亡相关蛋白GRP79、GRP94表达增加,喹乙醇染毒6、12和24 h组,活性氧水平、内质网凋亡相关蛋白CHOP表达增加(P<0.05)。结论喹乙醇可致肾小管上皮细胞发生细胞凋亡从而造成肾脏毒性,凋亡的发生可能与内质网应激相关凋亡途径相关。Objective Renal tubular epithelial cell were exposed to olaquindox and detected the ROS and apoptosis related proteins, to investigate the renal tubular epithelial cell apoptosis through endoplasmic reticulum stress mediated pathway induced by olaquindox. Methods MTT assay （1, 2, 3, 4, 5, 6, 7and 8 μmol/ml olaquindox exposure） was used to detect the effects of olaquindox on renal tubular epithelial cell proliferation to determine test concentrations. Hoechst-33258 was used to detect morphological changes on apoptotic cells in each group. Flow cytometry method was applied to detect the apoptosis rate and intracellular reactive oxygen, and western blot assay was performed to detect the levels of endoplasmic reticulum stress-related apoptosis proteins, GRP78, GRP94 and CHOP. Results According to results of the MTT test, 1, 2, 3 and 4 μmol/ml olaquindox concentrations were determined for apoptosis analysis. With the increase of olaquindox concentration, apoptosis rate and levels of endoplasmic reticulum stress related apoptosis pathway protein GRP78, GRP94 and CHOP increased, levels of ROS were increased in every groups （ P 〈 0.05 ） in 2μmol/ml olaquindox groups and above. With the prolongation of olaquindox exposure, apoptosis rate and levels of endoplasmic reticulum stress related apoptosis pathway protein GRP78 and GRP94 increased in 12 and 24 h olaquindox exposure groups , whereas in groups of olaquindox exposed for 6, 12 and 24 h, levels of ROS and endoplasmic reticulum stress related apoptosis pathway protein CHOP increased （ P 〈 0. 05 ）. Conclusion Olaquindox can induce renal tubular epithelial cells to apoptosis and cause the renal toxicity, and the endoplasmic reticulum stress-related apoptosis maybe the associated toxicity pathway.

关 键 词：喹乙醇 内质网应激 细胞凋亡 人源肾小管上皮细胞 

分 类 号：Q593[生物学—生物化学] Q26