摘要
目的:探讨左归降糖解郁方通过调控谷氨酸受体2(GluR2)/帕金蛋白(Parkin)信号改善线粒体自噬介导的糖尿病并发抑郁症(DD)大鼠海马突触微环境损伤的作用机制.方法:将80只雄性SD大鼠适应性饲养5 d后,随机抽取10只作为正常组大鼠,其余70只通过2周高脂饲料喂养+链脲佐菌素(STZ)注射+28 d慢性温和不可预测性应激(CUMS)加孤养复制DD大鼠模型,实验设正常组、模型组、GluR2阻断剂组(脑内注射,CNQX,5 μg·kg-1)、GluR2激动剂组(脑内注射,C1-HIBO,10 µg·kg-1)、二甲双胍+氟西汀组(灌胃,二甲双胍0.18 g·kg-1+氟西汀1.8 mg·kg-1)、左归降糖解郁方高、低剂量组(灌胃,20.52、10.26 g·kg-1).除正常组和模型组外,GluR2阻断剂组、激动剂组分别从应激造模开始每周 1次连续海马齿状回内微量注射CNQX、C1-HIBO,而左归降糖解郁方高、低剂量、二甲双胍+氟西汀组在应激造模的同时连续灌胃给药28 d.通过旷场、强迫游泳实验评估大鼠抑郁样行为;采用生化分析检测血清胰岛素、海马组织中三磷酸腺苷(ATP)水平;酶联免疫吸附测定法(ELISA)检测海马组织中5-羟色胺(5-HT)、多巴胺(DA)水平;透射电镜观察海马神经元自噬小体;高尔基染色观察大鼠海马神经元树突、树突棘形态结构;蛋白免疫印迹法(Western blot)检测海马组织中GluR2、Parkin蛋白表达水平;免疫荧光检测海马齿状回中GluR2、Parkin、突触相关前膜蛋白3(RIMS3)、突触后致密蛋白95(PSD95)蛋白表达水平.结果:与正常组比较,模型组大鼠旷场总活动路程减少,强迫游泳不动时间增加(P＜0.01);血清胰岛素、海马组织中ATP、5-HT和DA明显下降(P＜0.01);海马神经元自噬小体明显增加,海马神经元树突、树突棘及突触结构明显损伤;海马组织中GluR2、RIMS3、PSD95表达降低,而Parkin表达明显增高(P＜0.05,P＜0.01).与模型组比较,GluR2阻断剂和激动剂分别加重和减轻上述异常改变(P＜0.05,P＜0.01);左归降糖解郁方高、低剂量组大鼠上述抑郁样行为均有不同程度改善,血清胰岛素、海马组织中ATP、5-HT和DA含量均明显升高(P＜0.05,P＜0.01),海马神经元自噬小体数量减少、树突、树突棘及突触结构情况明显缓解,同时海马组织中GluR2、RIMS3、PSD95蛋白表达上调、Parkin蛋白表达明显下调(P＜0.05,P＜0.01).结论:左归降糖解郁方能改善线粒体自噬介导的DD大鼠海马突触微环境损伤,其作用机制可能与调控GluR2/Parkin信号通路有关.
Abstract
Objective:To reveal the mechanism of Zuogui Jiangtang Jieyu prescription against damage to hippocampal synaptic microenvironment via suppressing glutamate receptor 2(GluR2)/Parkin signal-mediated mitophagy in rats with diabetes-related depression(DD).Methods:Eighty male SD rats underwent adaptive feeding for 5 days before the study.Ten rats were randomly assigned to the normal group.The model of DD rats was established with the rest by 2-week high-fat diet+streptozotocin(STZ)tail intravenous injection+28 days of chronic unpredictable mild stress(CUMS)combined with isolation.The rats were randomly divided into a normal group,a model group,a GluR2 blocker group(5 μg·kg-1),a GluR2 agonist group(10 μg·kg-1),a metformin+fluoxetine group(0.18 g·kg-1 metformin+1.8 mg·kg-1 fluoxetine),and high-and low-dose Zuogui Jiangtang Jieyu prescription groups(20.52 and 10.26 g·kg-1,respectively).The rats in the GluR2 blocker group and the GluR2 agonist group were continuously injected with CNQX and C1-HIBO in the dentate gyrus of the hippocampus once a week starting from stress modeling,respectively,while the metformin+fluoxetine group and the high-and low-dose Zuogui Jiangtang Jieyu prescription groups were continuously given intragastric administration for 28 d at the same time of stress modeling.Depression-like behavior was evaluated by open field and forced swimming experiments.The levels of serum insulin and adenosine triphosphate(ATP)in hippocampus were detected by biochemical analysis.The levels of 5-hydroxytryptamine(5-HT)and dopamine(DA)in hippocampus were detected by enzyme-linked immunosorbent assay(ELISA).The autophagosomes of hippocampal neurons were observed by transmission electron microscopy.The morphology and structure of dendrites and spines of hippocampal neurons were evaluated by Golgi staining.Western blot detected the expression levels of GluR2 and Parkin proteins in hippocampus.The expression levels of GluR2,Parkin,regulating synaptic membrane exocytosis protein 3(RIMS3),and postsynaptic density protein 95(PSD95)in the dentate gyrus of the hippocampus were detected by immunofluorescence.Results:Compared with the normal group,the model group exhibited reduced total activity distance in the open field and increased immobility time in forced swimming(P＜0.01),lowered levels of serum insulin and ATP,5-HT,and DA in hippocampus(P＜0.01),increased autophagosomes of hippocampal neurons,significantly damaged morphology and structure of dendrites and spines of hippocampal neurons,decreased expression levels of GluR2,RIMS3,and PSD95 in hippocampus,and an increased Parkin expression level(P＜0.05,P＜0.01).Compared with the model group,the GluR2 blocker group and the GluR2 agonist group showed aggravation and alleviation of the above abnormal changes,respectively(P＜0.05,P＜0.01).The above depression-like behavior was significantly improved in the high-and low-dose Zuogui Jiangtang Jieyu prescription groups to different degrees.Specifically,the two groups saw elevated levels of serum insulin and ATP,5-HT,and DA in hippocampus(P＜0.05,P＜0.01),restrained increase in autophagosomes and damage to morphology and structure of dendrites and spines of hippocampal neurons,up-regulated protein expression levels of GluR2,RIMS3,and PSD95,and down-regulated Parkin expression level(P＜0.05,P＜0.01).Conclusion:Zuogui Jiangtong Jieyu prescription can ameliorate the mitophagy-mediated damage to hippocampal synaptic microenvironment in DD rats,the mechanism of which might be related to the regulation of GluR2/Parkin signaling pathway.
国家科技期刊平台
NSTL
维普
万方数据