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    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

      摘要
    • 摘要:
      目的 探究黄连解毒汤(Huanglian Jiedu Decoction，HLJDD)对血管性痴呆(vascular dementia，VD)大鼠神经元突触可塑性的影响及作用机制。
      方法 随机选取10只大鼠作为假手术组；剩下大鼠以改良双侧颈总动脉阻断法制备VD大鼠模型，并随机分为模型组、HLJDD低剂量组(1.5 g·kg–1)、 HLJDD高剂量组(3.0 g·kg–1)、 HLJDD高剂量(3.0 g·kg–1)+JNK 激活剂(Anisomycin，5 mg·kg–1)组，每组10只。各组对应治疗4周后取材。Morris水迷宫试验检测各组逃避潜伏期、跨越平台次数；苏木素-伊红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；Western blotting及实时荧光定量聚合酶链式反应检测大鼠海马组织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)、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(synapsin，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，HLJDD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)，减轻海马组织病理损伤和形态结构异常，改善神经元与突触超微结构，减少自噬小体数量，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HLJDD对于突触可塑性的治疗效果。
      结论 HLJDD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:
      OBJECTIVE  To investigate the effect of Huanglian Jiedu Decoction(HLJDD) on neuronal synaptic plasticit in rats with vascular dementia(VD) and its mechanism.
      METHODS  Ten rats were randomly selected as the sham group. The remaining animals were used to establish the VD model via the modified method of cutting bilateral common carotid arteries method, and were randomly assigned to the model group, low-dose HLJDD group(1.5 g·kg–1), high-dose HLJDD group(3.0 g·kg–1), high-dose HLJDD(3.0 g·kg–1)+JNK activator(Anisomycin, 5 mg·kg–1) group, 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 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 N-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 and Real-time quantitative polymerase chain reaction(RT-qPCR).
      RESULTS  Compared with the model group, the intervention of HLJDD 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, ameliorated the ultrastructure of neurons and synapses in hippocampus, 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 HLJDD on synaptic plasticit.
      CONCLUSION  HLJDD can improve synaptic plasticity in VD rats via regulating JNK/FoxO1-mediated autophagy.

       

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