靶向抑制黑质网状部GABA能神经元的DRP1改善肝性脑病小鼠运动功能

Targeted inhibition of GABAergic neuron’s DRP1 in substantia nigra pars reticulata improves motor function in mice with hepatic encephalopathy

目的:探讨黑质网状部(SNr)的GABA能神经元中线粒体分裂对急性肝性脑病(AHE)小鼠运动障碍的影响。方法:利用硫代乙酰胺(TAA)腹腔注射制备AHE小鼠模型,通过苏木精-伊红(HE)染色观察AHE小鼠肝小叶的变化,利用生化检测试剂盒检测AHE小鼠血清天冬氨酸转氨酶(AST)、丙氨酸转氨酶(ALT)和血氨的变化。接下来通过转棒疲劳实验、高架十字迷宫实验、旷场实验观察AHE小鼠运动功能。进一步利用透射电镜观察分析AHE小鼠SNr的线粒体面积、周长、圆率等形态学指标的变化,Western Blot观察AHE小鼠SNr的线粒体分裂融合相关分子的表达变化。接下来,利用重组腺相关病毒(AAV)靶向调控AHE小鼠SNr的线粒体动力相关蛋白1(DRP1)的表达,在荧光酶标仪上检测SNr的线粒体膜电位(MMP)、细胞的ATP和活性氧(ROS),并观察小鼠运动功能的变化。结果:较对照组,AHE小鼠运动功能明显降低,SNr的线粒体分裂明显增强,线粒体分裂相关蛋白表达显著升高;AHE小鼠SNr的MMP显著下降,细胞的ATP下降,ROS升高。靶向抑制AHE小鼠SNr的DRP1表达后,运动改善;进一步观察发现,AHE小鼠SNr的线粒体分裂被抑制后,MMP显著升高,细胞的ATP升高,ROS下降,证明线粒体功能明显改善。结论:靶向抑制AHE小鼠黑质网状部GABA能神经元的线粒体分裂,可以改善线粒体形态和功能,从而缓解其运动障碍。

Objective:To investigate the effect of mitochondrial division of GABAergic neurons in substantia nigra pars reticulata(SNr)on motor dysfunction in mice with acute hepatic encephalopathy(AHE).Methods:AHE mice model was established by intraperitoneal injection of thioacetamide(TAA).The changes of liver lobules in AHE mice were observed by hematoxylin-eosin(HE)staining.The changes of serum aspartate aminotransferase(AST),alanine aminotransferase(ALT)and blood ammonia in AHE mice were detected by biochemical detection kit.Then,the motor function of AHE mice was observed by rod fatigue test,elevated cross maze test and open field test.Furthermore,the changes of mitochondrial area,perimeter,roundness and other morphological indicators in SNr of AHE mice were observed and analyzed by transmission electron microscopy.The expression of mitochondrial division and fusion related molecules in SNr of AHE mice was observed by Western Blot.Then,the expression of mitochondrial dynamic related protein 1(DRP1)in SNr of AHE mice was targeted by recombinant AAV virus.The mitochondrial membrane potential(MMP),ATP and reactive oxygen species(ROS)in SNr were detected by fluorescence enzyme marker,and the changes of motor function of mice were observed.Results:Compared with the control group,the motor function of AHE mice was significantly decreased,the mitochondrial division of SNr was significantly enhanced,and the expression of mitochondrial division related proteins was significantly increased.The MMP in SNr of AHE mice was significantly decreased,the ATP of cells was decreased,and the ROS was increased.After targeted inhibition of DRP1 expression in SNr of AHE mice,the movement was improved;further observation found that after the mitochondrial division in SNr of AHE mice was inhibited,the MMP was significantly increased,the ATP of cells was increased,and the ROS was decreased,which demonstrated that the mitochondrial function was significantly improved.Conclusion:Targeted inhibition of mitochondrial division of GABAergic neurons in SNr of AHE mice can improve mitochondrial morphology and function,thus alleviating their movement disorders.