肠胶质细胞NGF/TrkA通路在胃电刺激缓解胃痛觉过敏中的作用及机制研究

Visceral pain is a common symptom in clinic and there is no effective treatment for it. Never growth factor (NGF) is a key transmitter involved in inflammatory pain generation and transmission, through activation of the high-affinity NGF receptor TrkA. The role of enteric glial cell (EGC) in visceral pain and the relationship between EGC and NGF/TrkA signaling pathway are still unclear. Our previous study demonstrated GES ameliorated visceral pain induced by acetic acid and NGF in a rodent model of gastric hyperagesia. There is no reports about the ameliorating effects of GES on gastric hyperalgesia via NGF/TrkA signaling pathway of EGC. In this study, the effect of GES on phenotypic changes of EGC and electrophysiological feature of stomach-specific doral root ganglia (DRG) neurous will be investigated in gastric hyperalgesia rats, using neuronal tracting, double immunofluorescence histochemistry， electronmicroscope and electrophysisology technique. The role of NGF/TrkA signaling pathway in EGC activated and in the effect of GES on EGC will be studied by molecular biology technique. This study will provide insights into the positive autocrine feedback loop of EGC and clarify NGF mechanism of GES on ameliorating visceral pain. The results will provide important insights and novel method for treatment of visceral hyperalgesia.

内脏痛是临床上常见的症状，其治疗尚缺乏有效的方法。NGF激活TrkA受体在痛觉过敏中发挥作用。肠胶质细胞（EGC）在内脏痛中的作用及与NGF/TrkA信号通路的关系尚不明确。我们前期研究发现胃电刺激（GES）对乙酸及NGF诱导的胃痛觉过敏大鼠具有改善作用，其具体机制不明。GES能否通过作用于EGC的激活及NGF/TrkA通路发挥减轻胃痛觉过敏的作用未有文献报道。本课题拟采用乙酸诱发胃痛觉过敏大鼠模型，通过神经示踪、免疫双标、电镜、膜片钳等技术，观察GES对胃痛觉过敏大鼠胃组织中EGC表型和胃特异DRG神经元电生理特征的影响，并从NGF/TrkA信号通路入手，借助分子生物学手段探讨该信号通路在EGC活化及GES调节EGC功能中的作用，旨在确定EGC-NGF“自分泌模式的正反馈循环”及GES对其的调节作用，阐明GES缓解胃痛觉的作用机制，为内脏痛的治疗提供新思路和新方法。

内脏痛是功能胃肠病主要的发病机制，与腹痛、腹胀等症状有关，目前临床上对其尚缺乏有效的治疗方法。NGF激活其受体Trk A介导痛觉过敏的发生，而肠胶质细胞（EGC）在内脏痛中的作用及其与NGF/Trk A信号通路的关系尚不明确。胃电刺激（GES）已成为临床治疗功能性胃肠疾病的新方法，但GES能否通过调节NGF/TrkA通路，影响EGC功能，在内脏痛中发挥作用尚未有文献报道。本课题采用乙酸诱导的胃痛觉过敏大鼠模型和体外EGC培养技术，探讨EGC、NGF/Trk A信号通路在GES缓解内脏高敏中的作用。研究发现：1.FD患者胃黏膜GFAP（EGC特异性标志物）、NGF和Trk A的表达水平明显高于健康对照组。GFAP与Trk A存在共表达，表明Trk A定位于EGC。GFAP、NGF和Trk A表达的变化与上腹痛、餐后饱胀感和早饱症状呈正相关。提示胃黏膜GFAP、NGF和Trk A的表达增加可能参与了FD的病理生理和症状感知变化。2.胃痛觉过敏大鼠在不同胃气囊扩张压力作用下斜方肌肌电图（EMG）频率和幅度变化高于对照组；适宜参数的GES可降低EMG的变化，下调组织中GFAP、NGF、Trk A的表达，降低疼痛介质SP、CGRP的释放；提示GES缓解内脏高敏感可能与抑制胃痛觉过敏大鼠EGC活化和调控NGF/Trk A信号通路有关。3.体外实验表明，EGC激活后，GFAP和EGC所分泌的神经介质NGF表达水平增高，活化的EGC可能会通过ERK1和ERK2信号通路进一步发挥其下游效应。4.电针缓解大鼠内脏高敏感可能与抑制EGC活化，调控EGC、DRG中AQP1的表达有关；电针对AQP1的调控可能由NF-κB信号通路介导。5.电针刺激可通过调控结肠肥大细胞，抑制其增殖活化，降低肠道TLR4表达，下调外周血浆炎性因子水平而改善内脏高敏感。