Munc13-1、Munc18-1对锰染毒SH-SY5Y细胞中多巴胺分泌障碍的影响  

Effects of Munc13-1 and Munc18-1 on dopamine secretion dysfunction in manganese-exposed SH-SY5Y cells

作　　者：李昌哲 于春 赵华 李军[2] 胡婷[2] LI Changzhe;YU Chun;ZHAO Hua;LI Jun;HU Ting(Toxicity Testing􀀁Center,Guizhou Medical University,Guiyang,Guizhou 550025,China;School of Public Health,Guizhou Medical University,Guiyang,Guizhou 550025,China)

机构地区：[1]贵州医科大学毒性检测中心,贵州贵阳550025 [2]贵州医科大学公共卫生与健康学院,贵州贵阳550025

出　　处：《环境与职业医学》2023年第3期268-272,共5页Journal of Environmental and Occupational Medicine

基　　金：国家自然科学基金项目:(81960578);贵州省教育厅自然科学研究项目[黔教合KY字(2021)146]。

摘　　要：[背景]慢性锰中毒诱发神经递质分泌障碍一直是其造成机体损伤的重要原因之一,但锰致神经递质分泌障碍的机制目前并不清楚。[目的]探究突触前膜胞内蛋白13-1(Munc13-1)与突触融合蛋白结合蛋白18-1(Munc18-1)对氯化锰(MnCl_(2))致人神经母细胞瘤细胞(SH-SY5Y细胞)多巴胺(DA)分泌障碍的影响。[方法]建立MnCl_(2)诱导SH-SY5Y细胞模型,根据MTT法测细胞存活率,实验分组为对照组和低、中、高浓度染锰组(0、100、200、400μmol·L^(−1)MnCl_(2)),处理24 h。用酶联免疫吸附试验试剂盒测定培养基中DA分泌量。通过实时荧光定量PCR检测细胞突触融合蛋白-1(Syntaxin-1)mRNA表达水平。提取细胞总蛋白,采用Western blotting检测Munc13-1、Munc18-1以及Syntaxin-1蛋白表达水平。并对MnCl_(2)染毒浓度和DA分泌量与Munc13-1、Munc18-1蛋白表达量之间做Pearson相关性分析。[结果]与对照组比较,随着锰染毒浓度的增加,细胞存活率逐渐下降,中、高浓度染锰组差异具有统计学意义(P<0.05);各锰染毒组细胞培养液中DA浓度随锰浓度增加呈下降趋势,与对照组和低浓度染锰组比,中、高浓度染锰组差异具有统计学意义(P<0.05);Syntaxin-1的表达量在mRNA和蛋白层次上,在各组间变化不明显,差异不具有统计学意义(P>0.05)。与对照组比较,Munc13-1的蛋白表达量随染锰浓度增加而依次下降,Munc18-1的蛋白表达量依次上升(P<0.05),其中与低浓度染锰组相比,高浓度染锰组Munc13-1和中、高浓度染锰组Munc18-1蛋白变化有统计学意义(P<0.05),与中浓度染锰组比,高浓度染锰组Munc18-1蛋白变化有统计学意义(P<0.05)。相关性分析显示,MnCl_(2)染毒浓度与Munc13-1蛋白表达呈负相关(r=-0.898,P<0.05),与Munc18-1蛋白表达呈正相关(r=0.678,P<0.05);DA浓度与Munc13-1蛋白表达呈正相关(r=0.932,P<0.05),与Munc18-1蛋白表达呈负相关(r=-0.817,P<0.05)。[结论]锰染毒SH-SY5Y细胞致DA的分泌抑制,与�[Background]Neurotransmitter secretion disorder induced by chronic manganese poisoning has always been one of the important causes of body injury,but the mechanism of neurotransmitter secretion disorder caused by manganese is not clear at present.[Objective]To investigate the effects of presynaptic membrane intracellular protein 13-1(Munc13-1)and synapse fusion protein binding protein 18-1(Munc18-1)on dopamine secretion dysfunction induced by manganese chloride(MnCl_(2))in human neuroblastoma(SH-SY5Y)cells.[Methods]A SH-SY5Y cell model induced by MnCl_(2) was established.Cell viability was measured by MTT assay.Four experimental groups were set up:control group and low-,medium-,and highdose manganese groups(0,100,200,and 400μmol·L^(−1) MnCl_(2)).They were treated with corresponding doses of MnCl_(2) for 24 h.The secretion of dopamine was measured by enzyme-linked immunosorbent assay.The mRNA expression of Syntaxin-1 was detected by real-time quantitaive PCR.Total cell proteins were extracted,and the protein expression levels of Munc13-1,Munc18-1,and Syntaxin-1 were detected by Western blotting.The correlations of MnCl_(2) exposure and dopamine secretion with the protein expressions of Munc13-1 and Munc18-1 were also analyzed by Pearson correlation.[Results]Compared with the control group,the cell viability rate decreased gradually with the increase of manganese exposure concentration,and the difference between the medium-and the high-dose manganese groups was statistically significant(P<0.05).The concentration of dopamine in cell culture medium of all manganese exposure groups decreased with the increase of manganese concentration,and compared with the control group and the low-dose manganese group,the medium-and the high-dose manganese groups were statistically significant(P<0.05).The expression of Syntaxin-1 at mRNA or protein level did not change significantly among groups(P>0.05).Compared with the control group,the protein expression of Munc13-1 decreased and that of Munc18-1 increased with the increase o

关 键 词：锰 SH-SY5Y细胞 多巴胺 突触前膜胞内蛋白13-1 突触融合蛋白结合蛋白18-1 

分 类 号：R114[医药卫生—卫生毒理学]