南方医科大学学报

南方医科大学学报 ›› 2021, Vol. 41 ›› Issue (9): 1409-1414.doi: 10.12122/j.issn.1673-4254.2021.09.17

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褪黑素可改善PBDE-47所致PC12细胞的异常自噬与凋亡

肖博雅，董理鑫，高 慧，杨凯朝，王亚飞，李晓凝，邱海霞，王爱国，张 舜   

  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；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
  • 出版日期:2021-09-20 发布日期:2021-09-30

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

XIAO Boya, DONG Lixin, GAO Hui, YANG Kaichao, WANG Yafei, LI Xiaoning, QIU Haixia, WANG Aiguo , ZHANG Shun   

  1. 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
  • Online:2021-09-20 Published:2021-09-30

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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、200 μmol/L）预处理 PC12 细胞 2 h 后，并联合浓度为 20 μmol/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25 μmol/L MT用于后续实验。实验分组为：溶剂对照组（0.5‰ DMSO）、20 μmol/L PBDE-47处理组、20 μmol/L PBDE-47+25 μmol/L MT处理组、25 μmol/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+25 μmol/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。

关键词: 褪黑素；PBDE-47；神经毒性；细胞自噬；细胞凋亡

Abstract: 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 200 μmol/L) of melatonin for 2 h before exposure to 20 μmol/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), 20 μmol/L PBDE-47, 25 μmol/L melatonin, or both PBDE-47 and melatonin. Immuno-fluorescence 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 25 μmol/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

Key words: melatonin; 2,2',4,4'-tetrabromodiphenylether; neurotoxicity; autophagy; apoptosis