BNIP3介导锰经线粒体凋亡途径诱导SH-SY5Y细胞凋亡的研究  

BNIP3-mediated manganese-induced apoptosis of SH-SY5Y cells involves in mitochondria-dependent apoptosis

作　　者：黄燕宁 王进[1] 文巧林 黄锦凤[1] HUANG Yan-ning;WANG Jin;WEN Qiao-lin;HUANG Jin-feng(Department of Neurology, The First Affiliated Hospital of Guangxi Medical University, Nanning Guangxi 530021 , China)

机构地区：[1]广西医科大学第一附属医院神经内科,广西南宁530021

出　　处：《毒理学杂志》2018年第2期112-117,共6页Journal of Toxicology

基　　金：国家自然科学基金(81460181)

摘　　要：目的探讨线粒体蛋白BNIP3在锰诱导线粒体依赖的SH-SY5Y细胞凋亡中的作用。方法取对数生长期的人神经母细胞瘤SH-SY5Y细胞,经MnCl_2处理24 h后应用透射电子显微镜直接观察MnCl_2作用下SH-SY5Y细胞的凋亡情况,使用流式细胞仪检测SH-SY5Y细胞线粒体膜电位(Δψm),通过蛋白免疫印记法(Western blot)检测BNIP3及凋亡相关蛋白Caspase-3的表达。并使用shRNA将SH-SY5Y细胞中的BNIP3沉默,观察BNIP3对MnCl_2诱导的SH-SY5Y细胞凋亡、线粒体膜电位丢失(Δψm)及凋亡相关蛋白Caspase-3表达的影响。结果 MnCl_2可使线粒体膜电位逐渐丢失(F=49.061,P<0.01)、并使线粒体蛋白BNIP3及凋亡相关蛋白Caspase-3的表达随着MnCl_2浓度的提高而上升(F=334.832,F=299.902,均P<0.01)、明显增加SH-SY5Y细胞的凋亡小体个数,上述差异均有统计学意义。沉默BNIP3(F=1.301,P=0.318,t=32.647,P<0.01)可以减少MnCl_2诱导线粒体膜电位的丢失(F=1.786,P=0.252;t=20.290,P<0.01),减少凋亡相关蛋白Caspase-3的表达(F=2.816,P=0.168;t=22.073,P<0.01)并使SH-SY5Y凋亡小体的个数减少,上述差异均有统计学意义。结论在MnCl_2通过线粒体凋亡途径诱导SH-SY5Y细胞凋亡的过程中BNIP3起到了重要作用,这种调控机制可能是锰产生神经毒性的作用机制之一。Objective To investigate the role of mitochondrial protein BNIP3 in manganese-induced mitochondria-dependent apoptosis of SH-SY5 Y cells. Methods SH-SY5 Y cells were treated with MnCl2 for 24 h. The apoptosis of SH-SY5 Y cells were observed by transmission electron microscopy(TEM）. The mitochondrial membrane potential(Δψm） were measured by flow cytometry. The expression of BNIP3 and apoptosis related protein Caspase-3 were detected by Western blot. And BNIP3 was silenced by shRNA to observe the effect of BNIP3 on the apoptosis,mitochondrial membrane potential(Δψm） depolarization and expression of Caspase-3 in SH-SY5 Y cells induced by MnCl2. Results After treated with MnCl2 for 24 h,the number of apoptotic bodies in SH-SY5 Y cells increased significantly. And the depolarization of mitochondrial membrane potential(Δψm）(F = 49. 061,P 〈0. 01）,expressions of BNIP3(F = 334. 832,P 〈0. 01） and Caspase-3(F =299. 902,P 〈0. 01） were in a dose-dependent manner. While silenced BNIP3(F = 1. 301,P = 0. 318; t = 32. 647,P 〈0. 01） suggest that BNIP3 expression is disadvantage to the mitochondrial membrane potential(Δψm） depolarization(F = 1. 786,P = 0. 252; t = 20. 290,P〈 0. 01）.Moreover,knockdown of BNIP3 protein reduced Caspace-3 expression significantly(F = 2. 816,P = 0. 168; t = 22. 073,P〈 0. 01） and decreased apoptotic body formation in SH-SY5 Y cells. Conclusion BNIP3 plays a key role in the process of mitochondria-dependent apoptosis in SH-SY5 Y cells induced by MnCl2. This regulatory mechanism may be one of the mechanisms of manganese-induced neurotoxicity.

关 键 词：锰 SH-SY5Y细胞 细胞凋亡 BNIP3 线粒体膜电位 

分 类 号：R114[医药卫生—卫生毒理学]