摘要
目的:探讨线粒体钙离子单向转运体(MCU)在无镁致痫海马神经元凋亡中的作用及可能的机制。方法:将海马神经元分为空白对照组、无镁诱导组、Ru360(MCU特异性抑制剂)组、Spermine(MCU特异性激动剂)组、Mito-Q(线粒体特异性抗氧化剂)组。空白对照组用正常的含镁细胞外液处理3 h。无镁诱导组用无镁细胞外液处理3 h。Ru360组、Spermine组和Mito-Q组分别用5μmol/L的Ru360、10μmol/L的Spermine、500 nmol/L的Mito-Q处理1 h,然后用无镁细胞外液处理3 h,再维持培养24 h。取空白对照组、无镁诱导组、Ru360组、Spermine组神经元,分别采用MTT法、TUNEL染色法、钙离子荧光探针Rhod-2染色法、线粒体荧光探针MitoSOX染色法和Western blot法检测存活率、凋亡率、线粒体钙离子浓度、线粒体ROS生成水平及内质网应激相关蛋白GRP78、CHOP和Caspase-12的表达。取空白对照组、无镁诱导组、Mito-Q组神经元,检测线粒体ROS生成水平及GRP78、CHOP和Caspase-12的表达。结果:与空白对照组比较,无镁诱导组神经元存活率下降,凋亡率增加,线粒体钙离子浓度及ROS生成水平升高,GRP78、CHOP和Caspase-12表达增加(P<0.05)。与无镁诱导组比较,Ru360组神经元存活率增加,凋亡率降低,线粒体钙离子浓度、ROS生成水平和GRP78、CHOP、Caspase-12蛋白表达降低(P<0.05);而Spermine组各指标变化与Ru360组相反(P<0.05)。与无镁诱导组相比,Mito-Q组线粒体ROS生成水平和GRP78、CHOP和Caspase-12表达降低(P<0.05)。结论:MCU可能通过调节氧化应激介导的内质网应激反应,从而在无镁致痫海马神经元凋亡中发挥重要作用。
Aim:To investigate the effects and mechanism of mitochondrial calcium uniporter(MCU)on hippocampal neuronal apoptosis induced by Mg^2+-free extracellular fluid.Methods:Primary cultured hippocampal neurons were randomly allocated into the blank control group,Mg^2+-free group,Ru360(MCU-targeted inhibitor)group,Spermine(MCU-targeted agonist)group and Mito-Q(mitochondria-targeted antioxidant)group.The blank control group was incubated with normal extracellular fluid for 3 hours.The Mg^2+-free group was treated with Mg^2+-free extracellular fluid for 3 hours.TheRu360 group,Spermine group,and Mito-Q group were respectively incubated with 5μmol/L Ru360,10μmol/L Spermine,500 nmol/L Mito-Q for 1 hours,respectively,and then incubated with Mg^2+-free solution for 3 hours.After 24 hours of maintenance culture,MTT assay,TUNEL assay,calcium fluorescence probe Rhod-2,mitochondrial fluorescence probe MitoSOX and Western blot were respectively used to detect cell viability,cell apoptosis,mitochondrial calcium concentration,the production of mitochondrial ROS and the expressions of endoplasmic reticulum stress-related proteins(GRP78,CHOP and Caspase-12).Results:Compared with the blank control group,the hippocampal neuronal viability of the Mg^2+-free group decreased,the apoptosis rate increased,the mitochondrial calcium concentration and mitochondrial ROS level increased,and the expressions of GRP78,CHOP and Caspase-12 increased(P<0.05).Compared with the Mg^2+-free group,the hippocampal neuronal viability of the Ru360 group increased,the apoptosis rate decreased,the mitochondrial calcium concentration and mitochondrial ROS level decreased,and the expression of GRP78,CHOP and Caspase-12 decreased;however,the changes of the indexes mentioned above of the Spermine group was opposed to Ru360 group(P<0.05).Compared with the Mg^2+-free group,the mitochondrial ROS level and the expressions of GRP78,CHOP and Caspase-12 of the Mito-Q group decreased(P<0.05).Conclusion:MCU may play a crucial role in Mg^2+-free solution-induced epileptic h
作者
谢南昌
李英娇
王翠
连亚军
孟祥荷
李钰娟
杜丽媛
XIE Nanchang;LI Yingjiao;WANG Cui;LIAN Yajun;MENG Xianghe;LI Yujuan;DU Liyuan(Department of Neurology,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052;Department of Clinical Laboratory,the First Affiliated Hospital,Zhengzhou University,Zhengzhou 450052)
出处
《郑州大学学报（医学版）》
CAS
北大核心
2020年第4期458-463,共6页
Journal of Zhengzhou University(Medical Sciences)
基金
国家自然科学基金项目(81571260,81701272)。
关键词
线粒体钙单向转运体
癫痫
内质网应激
氧化应激
mitochondrial calcium uniporter
epilepsy
endoplasmic reticulum stress
oxidative stress
