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氧化应激在尿白蛋白超负荷诱导肾小管上皮细胞自噬中的作用

Role of oxidative stress in urine albumin overload-induced autophagy in renal proximal tubular epithelial cells
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摘要 目的观察尿蛋白的主要成分白蛋白超负荷对体外培养的肾小管上皮细胞(HK-2)活性氧(ROS)水平及其对自噬激活的影响,以探讨白蛋白超负荷诱导肾小管上皮细胞自噬激活的可能机制。方法以体外培养的HK-2细胞为研究对象,分为正常对照组(NC)、白蛋白组(ALB)、白蛋白+N-乙酰-L-半胱氨酸(NAC)组、NAC组、白蛋白+氯喹(CQ)组和白蛋白+西罗莫司(RAP)组;采用免疫印迹检测自噬标记蛋白Becline-1与微管相关蛋白1轻链3(LC3)的表达,2′,7′-二氯荧光黄双乙酸盐(DCFH-DA)免疫荧光技术检测ROS水平。结果白蛋白超负荷诱导HK-2细胞自噬,表现为自噬标记蛋白LC3-Ⅱ与Becline-1表达显著增多,与正常对照组比较,差异具有统计学意义(P<0.05);白蛋白超负荷引起HK-2细胞氧化应激,与正常对照组ROS水平10.15±0.57比较,白蛋白组ROS水平22.47±0.79,表现为绿色荧光亮度增强,ROS生成增多(P<0.05);抗氧化剂NAC干预可以减少白蛋白超负荷处理诱导的HK-2细胞自噬增多(P<0.05);另外,与白蛋白组ROS水平22.62±0.92比较,CQ预处理组ROS水平为29.49±1.12,表明CQ处理可以进一步增加白蛋白超负荷所致的ROS释放,而RAP预处理组ROS水平为15.34+1.01,表明RAP可以减少白蛋白超负荷所致的ROS释放(P<0.05)。结论白蛋白超负荷诱导了HK-2细胞自噬激活,其机制可能与白蛋白超负荷引起的氧化应激有关。 Objective To monitor reactive oxygen species(ROS)levels in renal proximal tubular epithelial cells cultured in vitro under albumin overload and their effect on autophagy activation, and to investigate the underlying mechanisms. Methods HK-2 cells cultured in vitro were divided into a normal control group(NC), an albumin(ALB)group, an ALB+ N-acetyl-L-cysteine(NAC)group, an NAC group, an ALB+ chloroquine(CQ)group and an ALB+ rapamycin(RAP)group.The expression of molecular markers for autophagy, Beclin-1 and microtubule-associated protein 1 light chain 3(LC3), were detected by Western blotting.ROS levels were measured by a dichloro-dihydro-fluorescein diacetate(DCFH-DA)immunofluorescence method. Results Albumin overload-induced autophagy was activated in HK-2 cells as assessed by the significant upregulation of Beclin-1 and LC3-Ⅱ levels, compared with the control group(both P<0.05). Albumin overload triggered oxidative stress in HK-2 cells as revealed by the increased production of ROS and the enhancement of green fluorescence brightness, compared with the control group[(22.47±0.79)vs.(10.15±0.57), P<0.05]. The antioxidant NAC significantly inhibited albumin-induced autophagy(P<0.05). Moreover, the increase in ROS levels caused by albumin overload was promoted by chloroquine and blocked by rapamycin(both P<0.05). Conclusions The mechanisms for albumin overload-induced autophagy in HK-2 cells were related to oxidative stress.
作者 汪苗红 谭进 苗雨阳 张蔷 Wang Miaohong;Tan Jin;Miao Yuyang;Zhang Qiang(Department of Geriatrics, Tianjin Medical University General Hospital, Tianjin Geriatrics Institute, Tianjin 300052, China;Basic Medical college, Tianjin Medical University, Tianjin 300070, China)
出处 《中华老年医学杂志》 CAS CSCD 北大核心 2019年第3期312-316,共5页 Chinese Journal of Geriatrics
基金 国家级自然科学基金(81370183、81670086 ) 天津市自然科学基金(14JCYBJC27800,16JCQNJCU00R) 国家科技部国际科技合作与交流专项(ISTCP)(2015DFA50310)。
关键词 白蛋片尿 氧化性应激 自噬 Albuminuria Dxidative stress Autophagy
作者简介 通信作者:张蔷,Email:zhangqiangyulv@163.com。
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