黄连解毒汤调控JNK/FoxO1信号通路介导的自噬对血管性痴呆大鼠突触可塑性的影响

Effects of Huanglian Jiedu Decoction on Synaptic Plasticity in Rats with Vascular Dementia Through JNK/FoxO1 Signaling Pathway-Mediated Autophagy

关键词:  黄连解毒汤  血管性痴呆  自噬  突触可塑性  JNK/FoxO1信号通路

Key words:  Huanglian Jiedu Decoction  vascular dementia  autophagy  synaptic plasticit  JNK/FoxO1 signaling pathway

摘要:

目的 探究黄连解毒汤对血管性痴呆(VD)大鼠神经元突触可塑性的影响及作用机制。方法 将60只雄性SD大鼠适应性喂养1周后,随机抽选10只作为假手术组,仅分离颈总动脉不结扎,剩余大鼠将采用颈总动脉分次结扎法制作VD大鼠模型。剔除死亡和造模不成功的大鼠后,将造模成功的大鼠随机分为模型组、黄连解毒汤低剂量组(1.5 g/kg)、 黄连解毒汤高剂量组(3.0 g/kg)、 黄连解毒汤高剂量＋JNK 激活剂(Anisomycin)组(3.0 g/kg＋5 mg/kg),每组10只。各组对应治疗4周后取材。Morris水迷宫实验检测各组逃避潜伏期、跨越平台次数；苏木素-伊红(HE)染色法观察海马区病理形态学变化；透射电镜观察大鼠海马区神经元和突触超微结构；蛋白免疫印迹法(WB)及实时荧光定量聚合酶链式反应(RT-qPCR)检测大鼠海马组织c-Jun氨基末端激酶(c-Jun N-terminal kinase,JNK)、叉头盒蛋白1(Forkhead box protein O1,FoxO1)、LC3蛋白的全称是微管相关蛋白1A/1B-轻链3(microtuble-associated protein light chain 3,LC3B)、泛素结合蛋白p62(ubiquitin-binding protein p62,p62)、肌球蛋白样BCL2结合蛋白(myosin-like BCL2 interacting protein,Beclin1)、自噬相关蛋白7(Autophagy related gene 7,Atg7)、生长相关蛋白43(growth-associated protein 43,GAP43)、突触素(synaptosin,SYP)、突触后致密蛋白95(postsynaptic dense protein95,PSD95)、N-甲基-D-天冬氨酸受体2B亚基(N-methyl-D-aspartate receptor (NMDAR) subunit 2B,NR2B)、脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)、蛋白及mRNA相对表达水平。结果 与模型组比较,黄连解毒汤干预后可明显提高模型大鼠的学习、空间记忆能力(P<0.05或P<0.01),减轻海马区的神经元和突触超微结构,自噬小体数量减少,下调JNK、FoxO1、LC3B、Beclin1、Atg7蛋白及mRNA的表达(P<0.05或P<0.01),上调p62、GAP43、SYP、PSD95、NR2B、BDNF蛋白及mRNA的表达(P<0.05或P<0.01)。然而,Anisomycin的使用可部分逆转黄连解毒汤对于突触可塑性的治疗效果。结论 黄连解毒汤能够通过调控JNK/FoxO1介导的细胞自噬,改善VD大鼠的突触可塑性。

Abstract:

ABSTRACT: OBJECTIVE To investigate the effect of Huanglian Jiedu Decoction on neuronalsynaptic plasticit in rats with vascular dementia（VD） and its mechanism. METHODS Sixty male SD rats underwent adaptive feeding for one week before the study. Ten rats were randomly assigned to the sham group,where the common carotid artery was isolated without ligation.The remaining rats were subjected to sequential ligation of the common carotid artery for the modeling of VD.The successfully modeled rats were randomly assigned into the following groups:model,the low-dose Huanglian Jiedu Decoction group(1.5 g/kg), the high-dose Huanglian Jiedu Decoction group(3.0 g/kg),the high-dose Huanglian Jiedu Decoction group(3.0 g/kg)+JNK activator (Anisomycin) group(5 mg/kg),with 10 rats in each group.After 4 weeks of treatment,samples were collected. The escape latency and cross-platform times in each group were detected by Morris water maze test. The pathomorphological changes in the hippocampus were observed by Hematoxylin-eosin(HE) staining. Transmission electron microscope was used to detect the ultrastructural observation of neurons and synapses in rat hippocampus. The protein and mRNA relative expression levels of c-Jun amino-terminal kinase (JNK),Forkhead box protein O1(FoxO1)、microtuble-associated protein light chain 3(LC3B)、ubiquitin-binding protein p62 (p62),myosin-like BCL2 interacting protein(Beclin1),Autophagy related gene 7(Atg7)、growth-associated protein 43 (GAP43), postsynaptic dense protein95 (PSD95), N-methyl-D-aspartate receptor (NMDAR) subunit 2B (NR2B) and brain-derived neurotrophic factor (BDNF) in hippocampus was detected by Western blotting(WB) and Real-time quantitative polymerase chain reaction(RT-qPCR). RESULTS Compared with the model group,the intervention of Huanglian Jiedu Decoction improved the learning and spatial memory abilities of model rats significantly(P<0.05 or P<0.01),alleviated pathological and morphological damage of hippocampal tissue, rthe ultrastructure of neurons and synapses in hippocampus was improved,decreased the number of autophagosomes,down-regulated the expression of JNK,FoxO1,LC3B,Beclin1,Atg7 proteins and mRNA (P<0.05 or P<0.01), Up-regulated the expression of p62,GAP43,SYP,PSD95,NR2B,BDNF protein and mRNA simultaneously (P<0.05 or P<0.01). However, the co-treatment of Anisomycin partially reversed the therapeutic effects of Huanglian Jiedu Decoction on synaptic plasticit.CONCLUSION Huanglian Jiedu Decoction can improve synaptic plasticity in VD rats via regulating JNK/FoxO1-mediated autophagy.