肝郁脾虚型抑郁症海马-杏仁核神经活性甾体变化及逍遥散的作用机理

Based on the close relationship of excitatory between neuroactive steroid and hippocampus-amygdala in depression and previous studies found that Xiaoyao Powder have an effect on balance regulation of neuronal plasticity of excitatory and inhibitory in hippocampus-amygdala with chronic stress depression rats.Therefore,this project proposed a hypothesis that Xiaoyao powder against depression by regulating the neuronal plasticity of excitatory and inhibitory in hippocampus-amygdala.Internationally recognized rats model with depression was established by chronic unpredicted mild stress (CUMS). Compared with Xiaoyao Powder composition of the serum, brain tissue and cerebrospinal fluid were using GC-Mesta to solve the main components scattered the blood brain barrier, the content of PREG, PROG, ALLO in peripheral blood, hippocampus and amygdale of neuroactive steroids was detected using ELISA and LC-MS;Neuroactive steroids enzyme P450scc, Srd5a2, 3 β-HSD and 3 α-HSD gene expression in the hippocampus- amygdale were measured by western blotting and RT-qPCR.Interpretation the mechanism of Xiaoyao powder has an effect on regulating neuroactive steroids of hippocampus-amygdale in liver stagnation and spleen deficiency. We hope to provide some scientific evidences for Xiaoyao powder against depression.

基于神经活性甾体与抑郁症海马-杏仁核兴奋性的密切关系，以及课题组前期研究发现逍遥散具有调节慢性应激抑郁症大鼠海马-杏仁核神经可塑性兴奋-抑制的平衡作用，本项目提出"逍遥散防治抑郁症可能通过调节海马-杏仁核神经活性甾体平衡"的假说。采用慢性轻度不可预期性应激建立国际公认的抑郁症大鼠模型；GC-MS方法对脑脊液、血清及脑组织匀浆液逍遥散成分进行比较，拟解决逍遥散透过血脑屏障的主要成分有哪些；分别采用ELISA、LC-MS方法检测外周血、海马-杏仁核神经活性甾体（PREG, PROG, ALLO）的含量，免疫印迹法（Western blotting）和RT-qPCR检测海马、杏仁核合成神经活性甾体限速酶（P450scc, 3β-HSD, Srd5a2, 3α-HSD）蛋白和基因表达，阐释逍遥散调节肝郁脾虚型抑郁症海马-杏仁核神经活性甾体的作用机理，为逍遥散防治抑郁症提供科学依据。

1.研究背景：.基于神经活性甾体与抑郁症海马-杏仁核兴奋性的变化有密切的关系，以神经甾体为切入点，提出“逍遥散可能通过调节海马-杏仁核神经活性甾体发挥对抑郁症的治疗作用”的假说。因此，通过探索海马-杏仁核神经甾体变化与逍遥散治疗抑郁症的关联机制，揭示中药复方逍遥散治疗抑郁症的靶点和机理。.2.主要研究内容：.采用慢性轻度不可预期性应激建立国际公认的抑郁症大鼠模型；采用LC-MS/MS法进行逍遥散的质量控制研究；分别采用ELISA、LC-MS 方法检测外周血、海马-杏仁核神经活性甾体（PREG, PROG, ALLO）的含量，免疫组化法和RT-qPCR 检测海马、杏仁核合成神经活性甾体限速酶（P450scc, 3β-HSD, Srd5a2, 3α-HSD）蛋白和基因表达。.3.重要研究结果：.（1）21天慢性轻度不可预知应激方法可制备应激性抑郁症模型：即动物接受CUMS造模后观察动物宏观表征、体重结合糖水消耗率、摄食潜伏期和旷场试验的行为学变化、中药逍遥散以方测病、氟西汀阳性对照，综合表明21天慢性轻度不可预知应激方法可以成功复制出应激性抑郁症大鼠模型。.（2）应激性抑郁大鼠模型大鼠海马和杏仁核中PREG、PROG和ALLO总体变化趋势一致，主要体现在PROG和ALLO含量减少和PREG含量增加。与该三种神经甾体相关的合成和代谢酶3α-HSD、3β-HSD、P450scc 和 5a-R在海马和杏仁核区的 mRNA表达总体变化趋势一致，主要体现在3α-HSD、3β-HSD、5a-R基因表达减少和P450scc基因表达增加。而逍遥散可以使海马和杏仁核PROG、ALLO含量增加，3α-HSD、3β-HSD、5a-R基因表达增加，而使PREG含量减少、P450scc基因表达减少，从而发挥一定的调节作用。.4.科学意义：.海马和杏仁核组织中PREG、PROG和ALLO的含量变化和及与其生成和代谢相关酶3α-HSD、3β-HSD、P450scc 和 5a-R mRNA的表达的变化是应激性抑郁症的内在生物学基础和中枢神经内分泌机制之一，同时初步揭示了逍遥散可能是通过对海马、杏仁核神经甾体的调节发挥对抑郁症的治疗作用，神经甾体可能是其作用靶点之一，丰富了逍遥散治疗抑郁症的科学内涵。