褪黑素可改善PBDE-47所致PC12细胞的异常自噬与凋亡  

Effects of melatonin on PBDE-47-induced abnormal autophagy and apoptosis in PC12 cells

作　　者：肖博雅 董理鑫 高慧 杨凯朝 王亚飞 李晓凝 邱海霞 王爱国[1] 张舜[1] XIAO Boya;DONG Lixin;GAO Hui;YANG Kaichao;WANG Yafei;LI Xiaoning;QIU Haixia;WANG Aiguo;ZHANG Shun(Department of Occupational and Environmental Health,Ministry of Education Key Laboratory of Environment and Health,School of Public Health,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China;Department of Clinical Nutrition,Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology,Wuhan 430030,China)

机构地区：[1]华中科技大学同济医学院公共卫生学院劳动卫生与环境卫生学系,环境与健康教育部重点实验室,湖北武汉430030 [2]华中科技大学同济医学院附属同济医院临床营养科,湖北武汉430030

出　　处：《南方医科大学学报》2021年第9期1409-1414,共6页Journal of Southern Medical University

基　　金：中国博士后科学基金(2016T90694,2015M570643)。

摘　　要：目的探讨褪黑素(MT)对2,2',4,4'-四溴联苯醚(PBDE-47)所致大鼠肾上腺髓质嗜铬细胞瘤PC12细胞异常自噬与凋亡的影响。方法通过一系列浓度梯度MT(12.5、25、50、100、200μmol/L)预处理PC12细胞2 h后,并联合浓度为20μmol/L的PBDE-47染毒细胞24 h,采用细胞计数试剂盒(CCK8)检测细胞存活率,筛选25μmol/L MT用于后续实验。实验分组为:溶剂对照组(0.5‰DMSO)、20μmol/L PBDE-47处理组、20μmol/L PBDE-47+25μmol/L MT处理组、25μmol/L MT处理组。采用免疫荧光技术检测自噬体标志蛋白微管相关蛋白1轻链3(LC3)阳性着色情况;采用Western blot技术检测自噬关键蛋白自噬相关蛋白7(ATG7)、自噬选择性底物p62、LC3-Ⅱ水平,以及凋亡相关蛋白活化型含半胱氨酸的天冬氨酸-3(active caspase-3)和剪切型多(ADP-核糖)聚合酶(cleaved PARP)水平。结果CCK8结果表明,PBDE-47处理组细胞活力相比对照组有所下降(P=0.001);与PBDE-47处理组相比,PBDE-47+25μmol/L MT处理组细胞存活率上升且在80%以上,差异均有统计学意义(P=0.023)。与对照组相比,PBDE-47处理后PC12细胞LC3蛋白阳性着色增强;此外,PBDE-47处理组PC12细胞ATG7蛋白水平下降,p62、LC3-Ⅱ、active caspase-3、cleaved PARP蛋白水平上升,差异均有统计学意义(P均<0.001)。与PBDE-47处理组相比,PBDE-47+MT处理组LC3蛋白阳性着色减弱;此外,PBDE-47+MT处理组ATG7蛋白水平上升(P=0.034),p62蛋白水平下降(P=0.048),LC3-Ⅱ蛋白水平下降(P=0.018),active caspase-3蛋白水平下降(P<0.001),cleaved PARP蛋白水平下降(P=0.032)。结论PBDE-47可通过诱导PC12细胞自噬损伤引起自噬体蓄积,并能促进细胞凋亡,从而降低细胞存活;褪黑素可改善PBDE-47所致PC12细胞异常自噬与凋亡,进而提高细胞存活。Objective To explore the effect of melatonin(MT)on 2,2',4,4'-tetrabromodiphenylether(PBDE-47)-induced abnormal autophagy and apoptosis in rat adrenal medullary pheochromocytoma PC12 cells.Methods PC12 cells were pretreated with a concentration gradient(12.5,25,50,100,and 200μmol/L)of melatonin for 2 h before exposure to 20μmol/L PBDE-47 for 24 h to determine the optimal concentration of melatonin for cell treatment.In subsequent experiments,PC12 cells were treated with 0.5‰DMSO(control group),20μmol/L PBDE-47,25μmol/L melatonin,or both PBDE-47 and melatonin.Immunofluorescence assay was used to detect the positive staining of microtubule associated protein 1 light chain 3(LC3;a marker protein of autophagy);Western blotting was performed to determine the expression levels of the key autophagic proteins including autophagy-related protein 7(ATG7),LC3-Ⅱ and autophagy substrate p62,and the key apoptotic proteins including active cysteine-containing aspartate specific protease-3(active caspase-3)and cleaved poly(ADP ribose)polymerase(cleaved PARP).Results PBDE-47 treatment significantly reduced the viability of PC12 cells(P=0.001),but pretreatment with 25μmol/L melatonin maintained a cell viability over 80%following exposure to PBDE-47(P=0.023).PBDE-47-treated PC12 cells showed obviously enhanced immunofluorescent staining of LC3 protein,a significantly decreased expression of ATG7 and increased expression levels of p62,LC3-Ⅱ,active caspase-3 and cleaved PARP(P<0.001).The cells treated with both PBDE-47 and melatonin showed obviously reduced staining of LC3 protein with a signficantly increased expression level of ATG7(P=0.034)and decreased expressions of p62(P=0.048),LC3-Ⅱ(P=0.018),active caspase-3(P<0.001)and cleaved PARP(P=0.032).Conclusion PBDE-47 exposure impairs autophagy to cause autophagosome accumulation and promote apoptosis of PC12 cells.Melatonin can improve PBDE-47-induced abnormal autophagy and apoptosis and thus promote the survival of PC12 cells.

关 键 词：褪黑素 PBDE-47 神经毒性 细胞自噬 细胞凋亡 

分 类 号：R285[医药卫生—中药学]