PAR4在肥大细胞和TRPV1介导的内脏高敏感的作用和机制研究

Accumulating evidence has demonstrated that protease-activated receptor-4 (PAR4) as a modulator of visceral nociception has a role as inhibitor of visceral pain and hypersensitivity. Because PAR4 operates via G-protein-coupled receptors, it seems feasible to look for its downstream effectors among the pain-transducing signal in primary afferent. Recent studies suggest that mast cells and TRPV1 exert a key role in the regulation of irritable bowel syndrome （IBS） visceral sensitivity. It is also found that the down-regulation of PAR4 in mast cells followed by the increase in the activity of mast cells in IBS patients. Therefore, mast cells and TRPV1 appear to be the potential targets for expressing the analgesic action of PAR4. PAR4 may directly act on mast cells and TRPV1 mediating visceral pain signal transduction pathways. However, Whether PAR4 could directly influence the mast cell activation and the function of TRPV1 in primary sensory neurons to inhibit the visceral pain and hypersensitivity remains to be clarified. The present subject is designed to investigate whether the effect of PAR4 on visceral hypersensitivity is mediated by mast cells and TRPV1 by using morphological，electrophysiological and molecular metheds. The aims of this study are to examine: (i) Using IBS rats as a visceral hypersensitive model, we investigate the expression of PAR4 in gastrointestinal sensory nerve endings, sensory neurons of dorsal root ganglion (DRG) and mast cells in gastrointestinal tract; the effect of PAR4 on mast cells activation and expression of TRPV1,the effect of PAR4 on visceral pain and hypersensitivity mediated by mast cell and TRPV1; (ii) Using, as a model, the cultured mast cells of the rat, we study the effect of PAR4 and its molecular mechanism on mast cell activation, the effect of PAR4 on visceral hypersensitivity mediated by mast cells pathways; (ⅲ) Using cultured dorsal root ganglionic neurons of the rat as a model, we investigate the effect of PAR4 on TRPV1 expression and functional properties of the regulatory role of visceral pain, to explore the molecular and physiological mechanism and intracellular signal transduction pathways of PAR4 on visceral hypersensitivity mediated by TRPV1 receptors.

研究发现蛋白酶活化受体4(PAR4)具有抑制内脏痛和内脏高敏感作用，内脏高敏感的发生与肥大细胞和TRPV1受体活化以及PAR4表达下调密切相关，推测肥大细胞和TRPV1可能是PAR4参与内脏镇痛的下游效应靶点，PAR4可能通过作用于肥大细胞和TRPV1介导内脏痛觉信号的转导。然而其内脏镇痛作用是否直接与肥大细胞活化以及是否直接影响TRPV1功能还不清楚。本课题拟应用形态学、电生理和分子生物学等方法①以内脏高敏感大鼠为研究模型，观察PAR4在胃肠道感觉末梢及感觉神经元（DRG）的表达变化，PAR4活化对肠肥大细胞和TRPV1表达的作用；PAR4活化对胃肠道内脏高敏感诱发的痛觉信号转导的影响；②以培养的肥大细胞为研究模型，观察PAR4对肥大细胞活化的作用及分子机制；③以感觉神经元为研究模型，观察PAR4对TRPV1表达和功能特性的调节作用，探讨PAR4参与内脏痛信号转导和调控的生理和分子机制。

本项目以离体培养的大鼠肥大细胞（MCs）、脊神经(DRG)感觉神经元、结合内脏高敏感性大鼠模型为研究对象，应用逆行追踪、免疫荧光组织化学、Western blotting、实时定量PCR、细胞因子蛋白芯片、流式细胞技术和电生理等方法，研究了蛋白酶活化受体4(PAR4)在肥大细胞和瞬时受体电位香草酸亚型1(TRPV1)介导的内脏高敏感发生中的作用和分子机制。首次证实了PAR4通过肥大细胞MAPK细胞内信号转导机制调节炎症性细胞因子的表达参与内脏高敏感性疼痛的调控。主要结果如下：（1）形态学研究显示PAR4受体在DRG感觉神经元和肠道黏膜MC有广泛的表达，并且PAR4在MCs与TRPV1, IL-1β，iNOS 和P2X7存在广泛的共存；（2）内脏高敏感性疼痛的发生与肠黏膜MCs的浸润、上调MCs表达IL-1β，iNOS 和P2X7有关；（3）在内脏高敏感大鼠，结肠内注射PAR4活化肽（PAR4-AP）活化PAR4可明显降低结肠扩张诱发的内脏高敏感性疼痛；其机制可能是通过MAPK细胞内信号途径下调MC表达IL-1β，iNOS 和P2X7基因和蛋白水平；（4）细胞因子蛋白芯片技术显示PAR4-AP可明显抑制促炎症因子表达，如NGF, IL-1α, IL-1β, IL-1R6, IL-2, IL-4, IL-6, TIMP-1和TNF-α，以及增加抑炎症因子（如IFN-γ和MMP-8)表达；（5）内脏高敏感性疼痛可增加DRG感觉神经元表达TRPV1，PAR4活化可通过PKA/cAMP信号调节DRG内脏感觉神经元TRPV1功能、细胞内分布和表达。