下丘脑室旁核TLR4介导的AVP能神经元敏感化在急性胃黏膜损伤中的双向调节作用及其机制

AVP Neurons of the hypothalamus paraventricular nucleus play an important role in the gastric ischemia-reperfusion injury. Our preliminary experimental results confirmed that the strength of the Greater Splanchnic Nerve (GSN) activity is closely related to the degree of gastric ischemia-reperfusion injury, and in the region of the hypothalamus, giving the TLR4 or the NF-κB blockers can reduce GSN tensity, decrease the gastric ischemia-reperfusion injury. This reminder us that AVP neurons sensitization and the connection between its and the GSN are worth to study . Data have showed that microglia activation is the main source of inflammatory mediators in central nervous system and the key of neurons sensitization. Therefore, this project put the microglia TLR4 mediating dependency MyD88 and independence MyD88 signaling pathway as the breakthrough point in paraventricular nucleus, and the NF-κB as a center of research and application of gene knockout, nucleus accumbens trace injection, RT-PCR, Western Blot, Confocal, Electrophysiology, Immunofluorescence, such as experimental method, in gastric mucosa damage index, the gastric mucosal blood flow and GSN activity changes to observe the finish and the analysis method, explore the TLR4 mediating the NF-κB activated microglial cells can neurons on the AVP, renin - angiotensin system, the influence of inflammatory cytokines and reactive oxygen species, provide a new perspective for acute gastric mucosal ischemia-reperfusion injury.

下丘脑室旁核AVP能神经元在胃缺血-再灌注损伤调控方面发挥重要作用。我们的前期工作证实，内脏大神经的活动强弱与胃缺血-再灌注损伤程度密切相关；而在下丘脑区域，给予TLR4或NF-κB阻断剂可使内脏大神经紧张度降低，减轻胃缺血-再灌注损伤。这提示我们TLR4和NF-κB与AVP能神经元敏感化以及其和内脏大神经之间的联系值得关注。而TLR4在下丘脑室旁核内主要表达于小胶质细胞。因此，本项目拟以室旁核小胶质细胞TLR4介导的MyD88依赖性和MyD88非依赖性信号通路为切入点，以NF-κB为研究重心，应用基因敲除、核团微量注射、RT-PCR、免疫印迹、共聚焦、电生理、微透析、免疫荧光等方法，以胃黏膜损伤指数、胃黏膜血流量和内脏大神经活动变化为观察终点和分析手段，探讨小胶质细胞TLR4介导NF-κB激活对炎症因子、AVP能神经元、肾素-血管紧张素系统等的影响，为胃黏膜缺血-再灌注损伤提供新的视角。

临床的实践经验告诉我们，下丘脑做为生物体神经调节和体液调节两大调节系统的结合点，在应激（如：休克、高渗、脱水）状态下，势必对内脏活动产生影响。下丘脑区神经元和胶质细胞的相互影响，对机体的调节至关重要。为此我们观察了GI-R 损伤程度与下丘脑区小胶质细胞的激活之间的相关性以及小胶质细胞激活的机制，重点观察了GABA（一种神经系统内广泛存在的抑制性神经递质）通路对胃粘膜缺血再灌注损伤的影响，结果显示微量注射GABAB受体激动剂(巴氯芬)减轻了胃粘膜缺血再灌注损伤。而GABAB受体拮抗剂逆转了这一保护作用方面。相关成果已发表在Frontiers in Physiology杂志上。另外，不同程度的 GI-R 损伤模型大鼠下丘脑室旁核给予 TLR4 与 NF-κB 抑制剂，并用NF-κB基因敲除技术来佐证上述的关系，进一步侧脑室给予肾素-血管紧张素系统（RAS）的抑制剂（Aliskiren），观察胃粘膜血流量、外周内脏大神经活动规律、小胶质细胞激活标记蛋白CD11b的表达、LHA区神经元凋亡情况、Western-Blot、PCR/RT-PCR等方法观察了GABAB 受体、NOX2/NOX4、胃组织内p-AMPK表达、胃粘膜组织内的炎症因子（IL-1β,NOX2,NOX4）等动态表达量的变化。上述结果还在完善和整理中，预计今年能完成全部实验、撰写、投稿。.自噬做为细胞对外界应激的一种反应，我们在研究中意外发现了重度GI-R 大鼠 PVN 区小胶质细胞内有自噬小体的过度沉积，我们推测可能是由小胶质细胞的溶酶体-自噬通路不通导致其过度激活，进一步影响周边神经元的兴奋性，最后加强了胃肠活动的内脏大神经紧张性，其理论基础有待进一步研究。