CGRP介导肥大细胞活化参与内脏高敏感疼痛调控的作用和机制研究

The activation of mast cell (MCs) plays important roles in the regulation of visceral hyperalgesia. Studies have shown that calcitonin gene-related peptide (CGRP) released from visceral afferents in the gut of IBS patients may affect the activation of MCs, the synthesis and release of MCs mediators that is involved in the regulation of visceral hyperalgesia. CGRP receptor component protein (RCP) that mediated cAMP/PKA and RIG-I signaling pathways is involved in a variety of pathophysiological processes. However, the expression of CGRP receptor in MCs, and its effects and mechanisms on the activation of MCs, the synthesis and release of MCs mediators are not clear. We hypothesize that the activation of CGRP receptors by CGRP released from visceral afferents in the gut of IBS patients may regulate the visceral hyperalgesia through cAMP/PKA and RIG-I signaling pathways in MCs. The applicant has demonstrated that the visceral hyperalgesia is associated with release of mediators from MCs activation and the changes of expression profiles of cytokines in MCs. The activation of MCs expressed inflammatory mediators and cytokines is involved in the regulation of visceral hypersensitivity has also been published (Neurogastroenterol Motil. 2016 and GUT 2017). Preliminary experimental results suggested that the CGRP receptors are extensively expressed in mucosal MCs (MMCs) of gut in IBS patients and visceral hyperalgesia animal model. In the present subject, using mouse visceral hyperalgesia model, intestine and intestinal mucosa tissue and cultured MCs in vitro, as models, we investigate the expression of CGRP receptors on MCs, the effects of CGRP on activation and infiltration of MCs in gastrointestinal tract, the synthesis and release of MCs mediators, nerve-MCs interactions, and cAMP/PKA as well as RIG-I signal pathways, then, to explore the molecular mechanism of CGRP-mediated MCs activation in the regulation of visceral hyperalgesia.

肥大细胞(MCs)活化在内脏高敏感性疼痛发生中起着重要作用。有证据显示肠道感觉神经末梢释放CGRP可诱导肥大细胞(MCs)活化和介质释放参与内脏高敏感信号的调节。CGRP通过受体组分蛋白(RCP)介导cAMP/PKA和RIG-I信号通路参与各种病理生理过程。而其受体在MCs表达，以及对MCs活化、介质合成与释放的作用和机制未明。申请人前期研究发现MCs活化、介质合成与释放、细胞因子表达谱的改变等参与内脏高敏感信号的调控。预实验结果提示黏膜型MCs(MMCs)广泛的表达CGRP受体。本课题拟应用内脏高敏感动物模型、离体肠黏膜组织和培养MCs为研究模型，研究CGRP对肠MCs活化和浸润，介质合成与释放，神经-MCs相互作用及其cAMP/PKA和RIG-I信号途径的调节作用，进而探讨CGRP活化MCs参与内脏高敏感性疼痛发生的调控作用和分子机制。

肥大细胞(MCs)活化在内脏高敏感性疼痛发生中起着重要作用。降钙素基因相关肽(CGRP)可能参与肠道粘膜MCs的募集，这可能与肠易激综合征(IBS)的发生有关，但CGRP在MC激活中的作用还不清楚。本项目以离体培养的小鼠MCs结合慢性避水应激(WAS)诱导内脏高敏感小鼠为模型，综合免疫荧光组织化学、Western blotting、高通量基因测序(RNA-seq)、 基因敲除、细胞实时增殖分析(RTCA)和电生理等方法，研究了CGRP介导MCs活化参与内脏高敏感疼痛的调控作用和分子机制。首次证实了CGRP通过肥大细胞核转录因子Nr4a2/Nr4a3调节炎症性细胞因子表达参与内脏高敏感性疼痛的调控机制。主要结果：(1) 慢性应激可诱导结直肠黏膜CGRP释放并介导MCs活化、浸润和释放炎症因子参与内增高敏感性疼痛调控。(2) RNA-seq结果显示CGRP可活化MCs并诱导其基因表达谱的改变，包括28个基因上调和一个基因下调；生物信息学分析显示高表达基因与促肾上腺皮质激素释放激素(CRH)介导的基因转录、MCs活化、脱颗粒和炎症因子的释放有关，这些作用与应激诱导的MCs激活参与IBS调控有关。(3) CGRP刺激MCs可上调关键基因Nr4a2，Nr4a3, Crem, Gpr35, FosB和Sphlk1表达， RTCA证明了CGRP可促进MCs分裂增殖，Nr4a3敲除可阻断CGRP对MC增殖的促进作用；WAS小鼠可见结肠粘膜CGRP阳性纤维周围大量MCs表达关键基因Nr4a2/Nr4a3，但没有见到 Crem, Gpr35, FosB, Sphlk1阳性MCs。(4) 慢性应激和CGRP刺激可通过PKA/PKC、NFAT1和维甲酸诱导基因I(RIG-I)信号上调MCs表达关键基因Nr4a2/Nr4a3介导炎症因子合成与释放。(5) CGRP还可通过Nr4a2/Nr4a3 活化Src家族激酶(Src family kinase, SFKs) Fyn进而引起MCs活化、脱颗粒和介质释放。我们的结果表明，CGRP及其受体是MCs激活和内脏高敏感性疼痛的关键调节因子。Nr4a2/Nr4a3作为MCs功能新发现的调节受体，可能对应激诱导的内脏高敏感性疼痛具有重要调节作用，他们可能是治疗IBS药物开发的新靶点。