基于线粒体自噬介导的凋亡探讨左归降糖解郁方对糖尿病并发抑郁证海马突触微环境损伤的保护作用

Protective effect of Zuogui Jiangtang Jieyu Formula against damage of hippocampal synaptic microenvironment induced by mitophagy-mediated apoptosis in diabetes-related depression

目的:基于线粒体自噬介导的凋亡探讨左归降糖解郁方(ZGF)对糖尿病并发抑郁症(DD)海马突触微环境损伤的保护作用。方法:建立DD大鼠动物模型及模拟DD环境的体外细胞模型,实验设正常组、模型组、GluR2阻断剂(CNQX)组和ZGF组。采用免疫荧光法检测GluR2、Parkin、Cyt-C、SYN及PSD-95蛋白表达;mRFP-GFP-LC3自噬双标法检测海马神经元自噬流;JC-1法检测线粒体膜电位;MitoSOX法检测线粒体活性氧(ROS);Tunel染色法检测海马神经元凋亡;ELISA法检测Glu、5-HT、DA、ATP水平;旷场试验和强迫游泳试验评估大鼠抑郁样行为;透射电子显微镜观察大鼠海马神经元自噬小体;Western blotting检测大鼠海马组织中GluR2、Parkin、Cyt-C蛋白表达。结果:ZGF能有效抑制体外海马神经元线粒体自噬流、线粒体ROS释放及海马神经元凋亡(P<0.01),缓解线粒体膜电位下降、单胺神经递质失衡、突触前膜SYN及突触后膜PSD-95蛋白表达下调(P<0.05,P<0.01),同时改善DD大鼠抑郁样行为、线粒体自噬过度激活、单胺神经递质分泌不足,并显著调控GluR2-Parkin-Cyt-C信号通路相关蛋白表达(P<0.05,P<0.01)。结论:ZGF对线粒体自噬介导的海马神经元凋亡致DD海马突触微环境损伤具有明显的保护作用,这可能是其防治DD的重要机制。

Objective:To investigate the protective effect of Zuogui Jiangtang Jieyu Formulation(ZGF)against damage of hippocampal synaptic microenvironment induced by mitophagy-mediated apoptosis in diabetes-related depression(DD).Methods:The rats model of DD and the in vitro cell model simulating DD conditions were both established.The cultured cells or rats were randomly divided into normal group,model group,GluR2 blocker(CNQX)group,and ZGF group.The protein expression of GluR2,Parkin,Cyt-C,SYN and PSD-95 were detected by immunofluorescent staining.Autophagy flux was tested by autophagy double-standard adenovirus(mRFP-GFP-LC3)assay.Mitochondrial membrane potential was detected by JC-1 assay.Mitochondrial reactive oxygen species(ROS)was analyzed by MitoSOX fluorescent probe.Hippocampal neurons apoptosis was detected by Tunel staining.The levels of Glu,5-HT and DA were measured by enzyme-linked immunosorbent assay.Depressive-like behaviors of rats was assessed by open field test and forced swim test.Transmission electron microscopy was used to evaluate mitochondrial impairment and autophagosome formation.Western blotting assay was used to detect the protein expression of GluR2-ParkinCyt-c pathway.Results:ZGF can significantly restrain autophagic flux increasement,mitochondrial ROS release,hippocampal neuron apoptosis(P<0.01),and ameliorate mitochondrial membrane potential reduction,monoamine neurotransmitter deficiency,presynaptic SYN and postsynaptic PSD-95 protein expression downregulation(P<0.05,P<0.01).Interestingly,ZGF can also alleviate mitochondrial autophagy overstimulation,the neurotransmitter deficiency and depressive-like behaviors,and GluR2-Parkin-Cyt-c pathway abnormity in DD rats(P<0.05,P<0.01).Conclution:ZGF has remarkable protective effects against damage of hippocampal synaptic microenvironment induced by mitophagy-mediated apoptosis in DD,which may be the important mechanism of ZGF for the prevention and treatment of diabetes-related depression.