脑肠轴迷走传入通路调控海马小胶质细胞表型转化及黄连解毒汤干预研究

Regulation of microglia phenotype is presently and internationally an hot topic on the interference of neuroinflammation. Because the basic clinical applications of Huanglian Jiedu Tang (HLJDT) are antibacteria and therapeutic of cerebral ischemia & dementia. Meanwhile, our previous investigation indicated that HLJDT could regulate microglia phenotype-associated inflammatory cytokines in cerebral spinal fluid. Therefore, according to the cues from cholinergic anti-inflammatory and brain-gut axis theories, , we put forward a hypothesis that microbiota induce the dynamic variation of microglia phenotype and function in the hippocampus via brain-gut axis afferent vagus nerves pathway, which mediates neruoprotection of HLJDT through interfering neuroinflammation. Thus, the present investigation was designed to study the regulation of vagus nerves afferent of brain-gut axis on microglia phenotype and function in hippocampus, analyze microglia phenotype variation induced by gut microbiota in a vagus nerves afferent-dependent manner, explore the effection of HLJDT on a pathway comprised of gut microbiota, vagus nerves afferent of brain-gut axis and microglia phenotype. The final task will be realized by using primaryly the means of vagotomy, microbiota transplantation, intracerebral microinjection, and the technologies of fluorescence activated cell sortor (FACS), two photo laser scanning, neural electrophysiology and denatured gradient gel electrophoresis (DGGE). The ultimate aim of this study is to reveal the role of gut microbiota/ afferent vagus nerves /microglia pathway in the therapeutic effect of HLJDT on neuroinflammation which will provide a new idea on the treatment of various neurological diseases.

调控小胶质细胞表型干预中枢神经炎症是目前国际上研究的热点。基于黄连解毒汤抗菌、治疗脑缺血和痴呆的临床应用，借鉴迷走抗炎及脑肠轴理论，结合黄连解毒汤调节脑脊液中小胶质细胞表型相关炎症因子的研究基础。我们提出“肠道菌群通过脑肠轴迷走传入通路调控海马小胶质细胞表型和功能动态变化，黄连解毒汤通过该通路干预神经炎症实现神经保护”。本研究拟采用神经阻断、微生物移植、脑微量注射等手段，联合荧光激活细胞分类、双光子激光扫描、神经电生理和变性梯度凝胶电泳等技术，研究脑肠轴迷走传入神经对海马小胶质细胞表型和功能的调节，分析肠道菌群调控海马小胶质细胞表型及对脑肠轴迷走传入通路依赖性，观察黄连解毒汤对肠道菌群、脑肠轴迷走传入通路和海马小胶质细胞表型等环节的调控，并拆方研究君药黄连在全方对小胶质调节中的角色。揭示肠道菌群-迷走神经-小胶质细胞通路在黄连解毒汤干预中枢神经炎症中的作用。以期为中枢神经疾病治疗提供参。

通过调控肠道菌群如使用益生菌和药物可实现对多种中枢疾病的干预，如中风、痴呆、抑郁、焦虑、帕金森病和精神病等。迷走神经是联系肠道和脑组织的重要通路之一，一方面解剖学特征使得肠道的状态和肠道菌的代谢产物刺激迷走传入纤维，经孤束核投射到各个脑区；一方面刺激迷走神经可直接干预中枢功能，例如迷走神经刺激术治疗药物难治性癫痫。本项目的研究内容主要有：（1）黄连解毒汤及小檗碱对MCAO大鼠及APP/PS1小鼠肠道微生态多样性的影响；（2）小檗碱对迷走神经放电特征及海马小胶质细胞表型的影响；（3）迷走神经递质乙酰胆碱调控小胶质细胞表型分析；（4）代谢组学分析黄连解毒汤干预MCAO大鼠和APP/PS1小鼠的作用机制及潜在的生物标记物；（5）网络药理学分析黄连解毒汤干预阿尔茨海默病的机制及调控巨噬细胞代谢及表型的潜在机制。结果发现，（1）黄连解毒汤可显著调控Bacteroidales、Lachnospiraceae、Bacteroides、Akkermansia、Ruminocaccaceae和Coprostanoligenes水平，而小檗碱可显著调控Lachnospiraceae、Bacteroidales、Ruminocaccaceae和Coprostanoligenes水平；（2）小檗碱可兴奋膈下右迷走神经腹腔支，刺激延髓孤束核的c-fos和ChAT的高表达，增加海马CD163阳性小胶质细胞数量而降低CD86阳性小胶质细胞数量（3）迷走神经递质乙酰胆碱激活α7 nAChR阻断LPS诱导的小胶质细胞的M1型转化并促进M2型转化（4）代谢组学研究提示，黄连解毒汤使APP/PS1小鼠血浆14个代谢物回调，脑组织9个鞘脂代谢物回调；对于MCAO大鼠，黄连解毒汤显著调节炎症和代谢相关的19个代谢产物及10条代谢通路。（5）网络药理学分析提示，黄连解毒汤可通过清除/减少β淀粉样蛋白、抑制Tau蛋白过度磷酸化、抗炎和免疫活性等多途径对AD具有治疗作用，另外可通过调节糖酵解、鞘脂代谢和谷氨酰胺代谢的代谢产物和酶，调节免疫细胞的炎症反应。黄连解毒汤通过调节肠道菌种属和水平，激活迷走神经传入通路，调控海马区小胶质细胞表型，发挥干预中枢神经炎症的作用，而通过调节代谢进而影响小胶质细胞表型和功能是其干预中枢神经炎症的另一条通路。