姜黄素抑制Cu2+诱导的转APP695基因SH-SY5Y细胞氧化损伤和凋亡作用
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ts of Curcumin on Cu2+-Induced Oxidative Damage and Cell Apoptosis in Transgentic APP695 SH-SY5Y Cells
ZHENG Bowen, JIANG Zhaofeng, HUANG Hanchang* (Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing
100191, China)
Abstract: Objective: This study was focused on the protective effect of curcumin on cellular oxidative damage and cell apoptosis induced by Cu2＋ in human neuroblastoma SH-SY5Y cells transfected by human amyloid-β precursor protein (APP) (SH-SY5Y-APP 695). Methods: SH-SY5Y-APP 695 cells were treated with 50 μmol / L Cu 2＋ at 37 ℃ for 24 h with or without curcumin pre-protection. Cell viability was detected by cell counting kit-8 (CCK-8). Extracellular lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by commercial assay kits. The enzymatic activities of caspase-3, caspase-8 and caspase-9 were assessed. Phosphorylation levels of NF-E2-related factor 2 (Nrf2) at Ser-40 (pSer40-Nrf2) and HO-1 were detected by Western blot analysis. Results: Compared with the control group, Cu 2＋ administration led to decreased cell viability and mitochondrial membrane potential, increased levels of LDH, intracellular ROS and caspase-3, caspase-8 and caspase-9 activities, and elevated levels of pSer40-Nrf2 and HO-1. Conversely, curcumin increased cell viability and mitochondrial membrane potential, decreased the levels of LDH and intracellular ROS, significantly mitigated caspase-9 and caspase-3 activities, and reduced the expression levels pSer40-Nrf2 and HO-1. Conclusion: Curcumin can attenuate Cu 2＋-induced cellular oxidative damage and cell apoptosis. Key words: Alzheimer’s disease; Cu2+; oxidative damage; cell apoptosis; curcumin; Nrf2-antioxidant response element pathway DOI:10.7506/spkx1002-6630-201703027 中图分类号：Q26 文献标志码：A 文章编号：1002-6630（2017）03-0164-06 收稿日期：2016-03-18 基金项目：国家自然科学基金面上项目（31471587）；北京市属高等学校高层次人才引进与培养计划项目（CIT&TCD201504034） 作者简介：郑博闻（1990—），男，硕士研究生，研究方向为食品生物活性物质功能评价。E-mail：tz19901111@ *通信作者：黄汉昌（1975—），男，副教授，博士，研究方向为食品神经分子营养学。E-mail：hanchang@
164 2017, Vol.38, No.03
食品科学
※营养卫生
姜黄素抑制Cu2＋诱导的转APP695基因SH-SY5Y 细胞氧化损伤和凋亡作用
郑博闻，姜招峰，黄汉昌*
（北京联合大学 生物活性物质与功能食品北京市重点实验室，北京 100191） 摘 要： 目的：研究 Cu 2＋诱导的转 β- 淀粉样前体蛋白（ amyloid-β precursor protein ， APP ）基因 SH-SY5Y （ SHSY5Y-APP 695 ）细胞氧化损伤、凋亡及姜黄素的抑制作用。方法：在姜黄素预保护和无姜黄素预保护条件下， 50 μmol / L Cu 2＋处理细胞 24 h ，测定细胞存活率、胞外乳酸脱氢酶（ lactate dehydrogenase ， LDH ）水平、胞内 活性氧（ reactive oxygen ， ROS ）水平、线粒体膜电位、半胱氨酸天冬氨酸蛋白酶（ caspase ） -3 、 caspase-8 和 caspase-9 活力，蛋白免疫印迹法测定核转录 NF-E2 相关因子 2 （ NF-E2-related factor 2 ， Nrf2 ） Ser40 位点磷酸化 （pSer40-Nrf2）水平以及血红素加氧酶（heme oxygenase，HO）-1蛋白表达水平。结果：与空白对照相比，Cu2＋ 损伤组细胞存活率降低，胞内ROS水平和胞外LDH活性升高，线粒体膜电位下降，caspase-9和caspase-3活性明显升 高，pSer40-Nrf2和HO-1含量增加，而姜黄素保护组细胞存活率升高，胞内ROS水平和LDH释放水平降低，线粒体 膜电位恢复升高，caspase-9和caspase-3活性明显降低，pSer40-Nrf2和HO-1表达量减少。结论：姜黄素在一定程度 上减弱了Cu ＋诱导的氧化损伤和细胞凋亡作用。 关键词：阿尔茨海默病；Cu2＋；氧化损伤；细胞凋亡；姜黄素；Nrf2/ARE信号通路
ZHENG Bowen, JIANG Zhaofeng, HUANG Hanchang* (Beijing Key Laboratory of Bioactive Substances and Functional Foods, Beijing Union University, Beijing
100191, China)
Abstract: Objective: This study was focused on the protective effect of curcumin on cellular oxidative damage and cell apoptosis induced by Cu2＋ in human neuroblastoma SH-SY5Y cells transfected by human amyloid-β precursor protein (APP) (SH-SY5Y-APP 695). Methods: SH-SY5Y-APP 695 cells were treated with 50 μmol / L Cu 2＋ at 37 ℃ for 24 h with or without curcumin pre-protection. Cell viability was detected by cell counting kit-8 (CCK-8). Extracellular lactate dehydrogenase (LDH), intracellular reactive oxygen species (ROS) and mitochondrial membrane potential were determined by commercial assay kits. The enzymatic activities of caspase-3, caspase-8 and caspase-9 were assessed. Phosphorylation levels of NF-E2-related factor 2 (Nrf2) at Ser-40 (pSer40-Nrf2) and HO-1 were detected by Western blot analysis. Results: Compared with the control group, Cu 2＋ administration led to decreased cell viability and mitochondrial membrane potential, increased levels of LDH, intracellular ROS and caspase-3, caspase-8 and caspase-9 activities, and elevated levels of pSer40-Nrf2 and HO-1. Conversely, curcumin increased cell viability and mitochondrial membrane potential, decreased the levels of LDH and intracellular ROS, significantly mitigated caspase-9 and caspase-3 activities, and reduced the expression levels pSer40-Nrf2 and HO-1. Conclusion: Curcumin can attenuate Cu 2＋-induced cellular oxidative damage and cell apoptosis. Key words: Alzheimer’s disease; Cu2+; oxidative damage; cell apoptosis; curcumin; Nrf2-antioxidant response element pathway DOI:10.7506/spkx1002-6630-201703027 中图分类号：Q26 文献标志码：A 文章编号：1002-6630（2017）03-0164-06 收稿日期：2016-03-18 基金项目：国家自然科学基金面上项目（31471587）；北京市属高等学校高层次人才引进与培养计划项目（CIT&TCD201504034） 作者简介：郑博闻（1990—），男，硕士研究生，研究方向为食品生物活性物质功能评价。E-mail：tz19901111@ *通信作者：黄汉昌（1975—），男，副教授，博士，研究方向为食品神经分子营养学。E-mail：hanchang@
164 2017, Vol.38, No.03
食品科学
※营养卫生
姜黄素抑制Cu2＋诱导的转APP695基因SH-SY5Y 细胞氧化损伤和凋亡作用
郑博闻，姜招峰，黄汉昌*
（北京联合大学 生物活性物质与功能食品北京市重点实验室，北京 100191） 摘 要： 目的：研究 Cu 2＋诱导的转 β- 淀粉样前体蛋白（ amyloid-β precursor protein ， APP ）基因 SH-SY5Y （ SHSY5Y-APP 695 ）细胞氧化损伤、凋亡及姜黄素的抑制作用。方法：在姜黄素预保护和无姜黄素预保护条件下， 50 μmol / L Cu 2＋处理细胞 24 h ，测定细胞存活率、胞外乳酸脱氢酶（ lactate dehydrogenase ， LDH ）水平、胞内 活性氧（ reactive oxygen ， ROS ）水平、线粒体膜电位、半胱氨酸天冬氨酸蛋白酶（ caspase ） -3 、 caspase-8 和 caspase-9 活力，蛋白免疫印迹法测定核转录 NF-E2 相关因子 2 （ NF-E2-related factor 2 ， Nrf2 ） Ser40 位点磷酸化 （pSer40-Nrf2）水平以及血红素加氧酶（heme oxygenase，HO）-1蛋白表达水平。结果：与空白对照相比，Cu2＋ 损伤组细胞存活率降低，胞内ROS水平和胞外LDH活性升高，线粒体膜电位下降，caspase-9和caspase-3活性明显升 高，pSer40-Nrf2和HO-1含量增加，而姜黄素保护组细胞存活率升高，胞内ROS水平和LDH释放水平降低，线粒体 膜电位恢复升高，caspase-9和caspase-3活性明显降低，pSer40-Nrf2和HO-1表达量减少。结论：姜黄素在一定程度 上减弱了Cu ＋诱导的氧化损伤和细胞凋亡作用。 关键词：阿尔茨海默病；Cu2＋；氧化损伤；细胞凋亡；姜黄素；Nrf2/ARE信号通路