基于海马神经元线粒体自噬探讨脾气虚心神不足的可能机制

Possible Mechanism Of Uneasiness Caused By Spleen Qi Deficiency Based On Hippocampal Neuron Mitophagy

目的:通过检测脾气虚大鼠行为学及其海马神经元线粒体超微结构、基本功能和自噬水平,探讨脾气虚所致心神不宁的可能机制。方法:16只雄性SD大鼠随机分为正常组和脾气虚组(模型组),每组8只;旷场实验检测大鼠行为学;透射电镜观察海马神经元线粒体超微结构;JC-1法检测海马神经元线粒体膜电位;ELISA法检测海马神经元线粒体ATP和反应性氧簇(ROS)水平;Western blot法检测海马线粒体PTEN诱导激酶1 (PINK1)、Parkin、微管相关蛋白轻链3(LC3)-I和II表达。结果:与正常组相比,模型组大鼠在旷场的中心活动时间明显延长,周边活动时间缩短,运动距离和直立次数显著减少;海马神经元部分线粒体嵴消失,自噬样结构减少;海马神经元线粒体的ATP水平和膜电位下降明显,PINK1、Parkin和LC3-II表达及LC3-II/I比值均显著下降。结论:脾气虚所致心神不足与海马神经元线粒体损伤及其自噬水平低下有关。

Objective: To study the possible mechanism of uneasiness caused by spleen Qi deficiency by testing behaviors and mitochondrial ultrastructure, basic function and mitophagy flux in hippocampal neurons. Methods: Sixteen male SD rats were randomly divided into the normal group and spleen Qi deficiency group(model group) with 8 rats in each group. The open-field test was used to observe behaviors and transmission electron microscope was chosen to observe hippocampal neuronal ultrastructures. JC-method was used to check the mitochondrial membrane potential. ELISA was applied to detect mitochondrial ATP and reactive oxygen species(ROS) levels. Western blot was employed to test expressions of PTEN-induced putative kinase 1(PINK1), Parkin, microtubule associated protein light chain(LC3)-I and II. Result: Compared with those in the normal group, rats in the model group spent more time in the center and less time in the periphery, covered shorter motion distances and reared less times. The hippocampal neuronal mitochondria exhibited decreased cristae and autophaged-like structures, lowered the ATP level and membrane potential, reduced expressions of PINK1, Parkin and LC3-II and the ratio of LC3-II/I. Conclusion: The uneasiness caused by spleen Qi deficiency might be caused by mitochondrial damage and mitophagy inhition in hippocampal neurons.