急性脑缺血诱导的Sirt3蛋白表达下调通过破坏线粒体功能导致神经元损伤

Acute cerebral ischemia-induced down-regulation of Sirt3 protein expression contributes to neuronal injury via damaging mitochondrial function

本文旨在观察急性脑缺血对神经元沉默信息调节因子2相关酶类3(silent mating type information regulator 2 homolog 3,Sirt3)蛋白表达水平的影响,并阐明Sirt3在急性脑缺血中的病理意义。建立小鼠大脑中动脉栓塞(middlecerebralartery occlusion,MCAO)和Wistar大鼠原代培养海马神经元氧糖剥夺(oxygen glucose deprivation,OGD)模型,用Western blot检测Sirt3蛋白表达水平,用携带Sirt3的慢病毒感染海马神经元后,用CCK8试剂盒检测细胞存活率,用免疫荧光染色法检测海马神经元线粒体功能,用透射电子显微镜检测线粒体自噬情况。结果显示,与常氧组相比,OGD1 h/复氧2 h(R2 h)和OGD1 h/R12 h组海马神经元Sirt3蛋白表达水平均显著下调;与对侧正常脑组织相比,MCAO1 h/再灌注24 h(R24 h)和MCAO1 h/R72 h组小鼠大脑损伤侧半影区Sirt3蛋白表达水平均显著下调,而假手术组两侧Sirt3蛋白表达水平之间无显著差异。OGD1 h/R12 h处理损伤海马神经元线粒体功能,激活线粒体自噬,降低细胞存活率,而Sirt3过表达可减轻OGD1 h/R12 h对海马神经元的上述损伤作用。以上结果提示,急性脑缺血后Sirt3蛋白表达下调可通过破坏线粒体功能稳态影响神经元存活,纠正Sirt3蛋白可减轻急性脑缺血造成的损伤,这可为防治缺血性脑卒中提供新思路。

This study was aimed to determine the effect of acute cerebral ischemia on the protein expression level of silent mating type information regulator 2 homolog 3(Sirt3)in the neurons and clarify the pathological role of Sirt3 in acute cerebral ischemia.The mice with middle cerebral artery occlusion(MCAO)and primary cultured rat hippocampal neurons with oxygen glucose deprivation(OGD)were used as acute cerebral ischemia models in vivo and in vitro,respectively.Sirt3 overexpression was induced in rat hippocampal neurons by lentivirus transfection.Western blot was utilized to measure the changes in Sirt3 protein expression level.CCK8 assay was used to detect cell viability.Immunofluorescent staining was used to detect mitochondrial function.Transmission electron microscope was used to detect mitochondrial autophagy.The results showed that,compared with the normoxia group,hippocampal neurons from OGD1 h/reoxygenation 2 h(R2 h)and OGD1 h/R12 h groups exhibited down-regulated Sirt3 protein expression levels.Compared with contralateral normal brain tissue,the ipsilateral penumbra region from MCAO1 h/reperfusion 24 h(R24 h)and MCAO1 h/R72 h groups exhibited down-regulated Sirt3 protein expression levels,while there was no significant difference between the Sirt3 protein levels on both sides of sham group.OGD1 h/R12 h treatment damaged mitochondrial function,activated mitochondrial autophagy and reduced cell viability in hippocampal neurons,whereas Sirt3 over-expression attenuated the above damage effects of OGD1 h/R12 h treatment.These results suggest that acute cerebral ischemia results in a decrease in Sirt3 protein level.Sirt3 overexpression can alleviate acute cerebral ischemia-induced neural injuries by improving the mitochondrial function.The current study sheds light on a novel strategy against neural injuries caused by acute cerebral ischemia.