哈巴苷对急性脑缺血小鼠神经细胞及caspase非依赖性凋亡通路的影响

摘要

目的 研究哈巴苷对急性脑缺血小鼠海马神经细胞、线粒体功能及caspase非依赖性细胞凋亡通路的影响.方法 采用大脑中动脉阻塞法(middle cerebral artery occlusion,MCAO)复制小鼠急性脑缺血模型.造模后,各组分别立即尾静脉注射哈巴苷(4、8、12 mg·kg-1)、依达拉奉(3.2 mg·kg-1),模型组及假手术组同法给予等量生理盐水(10 mL·kg-1).造模6 h后,采用流式细胞术检测MCAO小鼠海马神经细胞凋亡率及线粒体膜电位;透射电镜观察MCAO小鼠海马神经细胞线粒体内外膜的清晰度、线粒体嵴的完整性、线粒体基质电子密度的高低变化及线粒体肿胀情况;采用Western blot法检测MCAO小鼠脑组织线粒体中AIF及Endo G的蛋白表达;采用qPCR法检测MCAO小鼠海马组织中AIF及Endo G mRNA的表达.结果 造模6 h后,与假手术组相比,模型组小鼠脑组织海马神经细胞凋亡率明显增加(P<0.01);海马神经细胞线粒体膜电位明显降低(P<0.01);神经细胞线粒体超微结构受损严重,线粒体结构松散,可见明显肿胀;脑组织线粒体中AIF及Endo G蛋白表达明显降低,线粒体AIF及Endo G蛋白释放增加(P<0.01);海马组织AIF及Endo G mRNA表达明显增加(P<0.01);与模型组相比,哈巴苷各剂量组小鼠脑组织海马神经细胞凋亡率明显降低(P<0.05,P<0.01);海马神经细胞线粒体膜电位明显升高(P<0.01);神经细胞线粒体超微结构明显改善;哈巴苷8、12 mg·kg-1组小鼠脑组织线粒体中AIF及Endo G蛋白表达明显增加,线粒体AIF及Endo G蛋白释放减少(P<0.05,P<0.01),哈巴苷4 mg·kg-1组小鼠脑组织线粒体中Endo G蛋白表达明显增加,线粒体Endo G蛋白释放明显减少(P<0.05);哈巴苷8、12 mg·kg-1组小鼠海马组织AIF及Endo G mRNA表达明显降低(P<0.05,P<0.01).结论 哈巴苷对急性脑缺血有保护作用,其机制可能与保护大脑神经细胞及线粒体生理活性,抑制caspase非依赖性细胞凋亡通路相关.%Aim To study the effects of harpagide on hippocampal neurons, mitochondrial function and caspase-independent apoptosis pathway after cerebral ischemia in mice. Methods The middle cerebral ar-tery occlusion (MCAO ) was employed to establish MCAO model. After that,the mice were given harp-agide (4,8,12 mg·kg - 1 )and edaravone (3. 2 mg ·kg - 1 )by tail vein injection after MCAO immediate-ly,and the model and control mice were given equal a-mounts of saline by the same way. After MCAO for 6 h,the apoptosis rate of hippocampal neuron and the mitochondrial membrane potential (MMP)of MCAO mice were detected by flow cytometry. We observed the clarity of inner and outer membrane of the hipp-ocampal neuronal mitochondrial,the integrity of the mitochondrial cristae,the changes of matrix electron density of mitochondria,and mitochondria swelling by transmission electron microscopy. Western blot was employed to determine the expression of apoptosis in-duced factor (AIF)and endonuclease G (Endo G)in mitochondrion and pro-caspase-3 in endochylema. qPCR was employed to determine the expression of AIF and Endo G. Results Compared with control group, the apoptosis rate of hippocampal neuron of MCAO mice significantly increased(P < 0. 01),the MMP of hippocampal neuron significantly decreased and the mi-tochondrial ultrastructure of cerebral ischemic area was severely damaged, loosely arranged and obviously swollen;the expression of AIF and Endo G in mito-chondria of cerebral tissue of MCAO mice significantly decreased,and the releases of AIF and Endo G signifi-cantly increased(P < 0. 01);the expressions of AIF, Endo G mRNA were evidently up-regulated (P <0. 01). Compared with model group,each dose of harpagide could significantly decrease the apoptosis rate of hippocampal neurons of mice brain (P < 0 . 05 ,P < 0. 01);MMP markedly increased in hippocampal nerve cells(P < 0. 01);the ultrastructure of neuronal mitochondria was obviously improved. Compared with model group,harpagide (8,12 mg·kg - 1 )could sig-nificantly increase the expression of AIF and Endo G protein in mitochondria of mouse brain,and the release of mitochondrial AIF and Endo G protein decreased(P< 0. 05,P < 0. 01);harpagide (4 mg·kg - 1 )could significantly increase the expression of Endo G protein in mitochondria of mouse brain,and the release of En-do G protein markedly decreased (P < 0. 05);harp-agide (8,12 mg·kg - 1 )could significantly decrease the expression of AIF and Endo G mRNA in hippocam-pus of mice(P < 0. 05,P < 0. 01). Conclusion The protective effect of harpagide on MCAO may be related to the protective effects on cerebral nerve cells,the ac-tivity of the mitochondria and the inhibition of caspase-independent apoptotic signaling pathways.