线粒体钙单向转运体在匹鲁卡品致痫大鼠r海马神经损伤中的作用

摘要

目的 研究线粒体钙单向转运体(mitochondrial calcium uniporter,MCU)在氯化锂-匹鲁卡品(pilo-carpine,PILO)癫痫大鼠模型神经损伤中的作用.方法 将84只雄性SD大鼠随机分成空白对照(CON)组、PILO组(2 h、8 h、24 h和72 h 4个亚组)、Ru360组和精胺(Spermine)组.观察各组大鼠行为学改变,并荧光染色法测定海马组织线粒体Ca2+浓度,TUNEL染色检测海马CA3区神经元凋亡,并采用Western Blot法检测凋亡相关蛋白Bax、Bcl-2、Cyt C和caspase-3表达变化.结果 (1)与CON组比较,PILO组海马神经元线粒体Ca2+浓度明显升高,凋亡明显增加(P<0.05);与CON组比较,PILO组海马Bax表达水平、线粒体Cyt C释放及caspase-3激活明显增多,而Bcl-2表达水平明显减少(P<0.05).(2)与PILO组比较,Ru360组海马神经元线粒体Ca2+浓度明显降低,凋亡明显减少(P<0.05);与PILO组比较,Ru360组海马Bax表达水平、线粒体Cyt C释放及caspase-3激活明显减少,而Bcl-2表达水平明显增多(P<0.05).(3)与PILO组比较,Spermine组海马神经元线粒体Ca2+浓度明显升高,凋亡明显增加(P<0.05);与PILO组比较,Spermine组海马Bax表达水平、线粒体Cyt C释放及caspase-3激活明显增多,而Bcl-2表达水平明显减少(P<0.05).结论 MCU在PILO癫痫大鼠海马神经损伤中发挥重要作用,抑制MCU可发挥对海马神经元凋亡的保护作用,其机制可能是通过抑制线粒体介导的细胞凋亡途径.%Objective To investigate the effect of mitochondrial calcium uniporter ( MCU) on hippocampal neuronal damage in pilocarpine-induced seizures .Methods 84 male SD rats were randomly divided into 4 groups:control group ,PI-LO group ( divided into 4 subgroups:2 h,8 h,24 h and 72 h) ,Ru360 group and Spermine group .The behavioral changes of each group were observed .Fluorescent staining was used to detect the free calcium concentration of hippocampus mitochon -dria and hippocampal neuronal apoptosis was detected by TUNEL staining .The expressions of Bax , Bcl-2, Cyt C and caspase-3 were detected by Western blotting .Results (1) Compared with control group ,the mitochondrial calcium con-centration and apoptotic neurons in PILO group increased (P<0.05).Compared with control group,expression of Bax, caspase-3 activation and Cyt C release in PILO group increased ,while expression of Bcl-2 decreased (P<0.05).(2)Com-pared with PILO group,Ru360 reduced the mitochondrial calcium concentration and apoptotic neurons (P<0.05).Com-pared with PILO group ,Ru360 reduced expression of Bax ,caspase-3 activation and Cyt C release ,while increased expression of Bcl-2 (P<0.05).(3)Compared with PILO group,Spermine increased the mitochondrial calcium concentration and ap-optotic neurons (P<0.05).Compared with PILO group,Spermine increased expression of Bax ,caspase-3 activation and Cyt C release,while reduced expression of Bcl-2 (P<0.05).Conclusion MCU plays an important role in hippocampal neuronal damage in pilocarpine-induced seizures .Inhibition of MCU can significantly attenuate hippocampal neuronal apop -tosis,and the underlying mechanism maybe through mitochondria-mediated apoptosis pathway .