基于NMDAR-NO-cGMP通路研究海马参与胃俞募配穴调节胃运动的分子机制

The preliminary results of the project team show that Back-Shu and Front-Mu points stimulation have synergistic effect on regulating gastric motility. The acupuncture information of Back-Shu and Front-Mu Points was gathered in the spinal cord Dorsal vagal complex(DVC), and then participated in the regulation of gastric motility by regulating the interaction between the central neurotransmitter glutamate (Glu) and its ionic receptor NMDA. The hippocampus, as the visceral integration center of the marginal system, also plays an important role in regulating gastric motility.NMDAR is widely distributed in the hippocampus and is activated by Glu to activate its downstream signaling pathway, resulting in a large number of NO release, thereby inhibiting gastric motility, and NO is also regulated by cGMP. Therefore, in this paper, the relationship between the regulation of gastric motility by Back-Shu and Front-Mu points stimulation and the hippocampus as the starting point. We detected the expression of NMDAR, NOS and guanylate cyclase (GC) in the hippocampus by immunofluorescence and Western blotting technique around the NMDAR-NO-cGMP pathway. The neuronal activity in the hippocampus was detected by recording extracellular neuron discharge. The changes of Glu content in hippocampal dialysate were analyzed by the technique of in vivo microdialysis. At the same time, using the method of destroying the central nucleus and injecting NMDAR, NOS, GC blocking agent into the hippocampus, to explore the central action target and molecular pathway of the synergistic effect between the Back-Shu and Front-Mu points stimulation and gastric motility. Explaining the molecular biological mechanism of regulating gastric motility by Back-Shu and Front-Mu points stimulation, and providing the biological basis for the clinical application.

前期研究结果表明胃俞募配穴调节胃运动具有协同效应，且胃俞、募穴针刺信息在延髓迷走孤束复合体（DVC）汇聚后，通过调控中枢递质谷氨酸（Glu）与其离子型受体NMDA的相互作用，参与对胃运动的调节。海马作为边缘系统的内脏整合中枢，在调节胃运动方面也起着重要作用。NMDAR广泛分布于海马，被Glu活化后激活其下游信号通路，导致NO大量释放抑制胃运动，而NO的作用会被cGMP所调节。因此，本课题以胃俞募配穴调整胃运动与海马的关系为切入点，围绕NMDAR-NO-cGMP通路，应用免疫荧光、蛋白印迹技术检测海马组织中NMDAR、NOS、GC表达；细胞外记录神经元放电方法,记录海马中神经元电活动，在体微透析动态分析海马透析液中Glu含量；同时借助中枢核团损毁和海马微量注射阻断剂技术，探讨胃俞募配穴协同效应的中枢靶点和分子通路，阐明胃俞募配穴调节胃运动的分子生物学机制，为俞募配穴的临床应用提供生物学依据。

本课题组一直从事胃俞募配穴调节胃运动的中枢机制研究，前期研究结果已表明胃俞募穴针刺信息在DVC、PVN汇聚，通过迷走神经参与对胃运动的调整作用。边缘系统海马有“内脏脑”之称，在调节胃运动方面也起着重要作用。因此本课题进一步探讨边缘系统海马是否参与胃俞募配穴对胃运动的调节作用，结果发现胃俞募穴针刺信息在边缘系统海马汇聚，通过调控海马谷氨酸及其受体信号通路NMDAR-NO-cGMP，达到调节胃运动的作用。同时运用脑片膜片钳技术记录海马谷氨酸神经元动作电位发放，化学遗传技术抑制海马谷氨酸神经元活性观察胃运动的变化等，结果进一步显示海马谷氨酸神经元参与电针对胃运动的调节作用，实现从特定神经元水平揭示针刺调节胃运动的中枢机制。近年来随着病毒示踪、光遗传、钙信号成像技术等高效应用，可从大脑内部神经环路层面，精准调控脑区特定神经元的活性，构建直观清晰的神经环路，从而明确某一神经环路在针刺效应中的作用，以实现从中微尺度更精准的揭示腧穴配伍的中枢整合机制，这将是我们下一步的研究目标。在本项目实施过程中，已围绕神经环路开展部分工作，这对基于神经环路研究腧穴配伍的中枢整合机制研究奠定了重要的实验基础。