胆碱能抗炎通路调节巨噬细胞M1/M2极化在CVB3诱导的急性病毒性心肌炎中的作用及机制研究

Macrophage M1/M2 polarization has been implicated in the pathogenesis of myocarditis after viral infection. Recently, we have found that excessive sympathetic activation aggravates the severity of viral myocarditis, and vagus nerve stimulation and activation of cholinergic antiinflammatory pathway (CAP) attenuate inflammation in viral myocarditis through α7 nicotinic acetylcholine receptor (α7-nAChR). Therefore, the results suggest that dysfunctional autonomic neural-immune circuit (sympathetic activation and vagal withdrawal) is associated with the development of viral myocarditis. However, the effects of CAP on macrophage polarization in viral myocarditis are unknown. Our previous studies have demonstrated that activation of CAP through α7-nAChR upregulates the mRNA expression of M2 macrophage polarization inducible cytokines and M2 macrophage-associated cytokines and downregulates the mRNA expression of M1 macrophage polarization inducible cytokines, and α7-nAChR is strongly expressed by macrophages. Hence, we postulate that activation of CAP could ameliorate the severity of viral myocarditis by regulating macrophage polarization through macrophage α7-nAChR. In the present study, we will investigate the effects of CAP on macrophage polarization and function in a coxsackievirus B3 murine myocarditis model. In addition, macrophages were isolated from murine cardiomyocytes and splenocytes and cultured in vitro. The agonist and antagonist of α7-nAChR will be administered, and the right cervical vagus nerve will be cut off in the infected mice. The proportion of M1 and M2 macrophages, levels of macrophages α7-nAChR, inflammatory cytokines, pJAK and pSTAT protein expression within the myocardium will be determined. The most novel insight of the study is that the nervous system may modulate the inflammatory response in viral myocaditis. The study will help elucidate the novel mechanism involved in the pathogenesis of viral myocarditis, and open new possibilities in the therapeutic management of viral myocarditis.

巨噬细胞M1/M2极化与病毒性心肌炎（VMC）的发生发展密切相关。我们前期研究发现，交感神经系统以及迷走神经相关的胆碱能抗炎通路（CAP）在VMC中分别介导促炎和抗炎作用，因此提出了自主神经-免疫调节系统失衡参与VMC发生发展的新观点。然而，CAP能否通过调节巨噬细胞极化来控制VMC炎症反应尚未见报道。申请者发现CAP兴奋后可刺激VMC小鼠M2型巨噬细胞极化诱导因子以及其相关炎症因子的表达，而且巨噬细胞膜大量表达α7nAChR，这些启示我们CAP很可能通过促进巨噬细胞向M2型极化来抑制VMC的炎症反应。本项目以VMC小鼠及巨噬细胞为研究对象，从兴奋/阻滞α7nAChR及切断颈部迷走神经着手，通过在体和离体两方面观察激活及阻断CAP对巨噬细胞极化的影响，解析CAP调控VMC小鼠巨噬细胞极化的机制。本项目最大特色是将VMC的机制研究深入到神经免疫学领域，从而为VMC的治疗提供一个崭新的思路。

巨噬细胞M1/M2 型极化与病毒性心肌炎（viral myocarditis，VMC） 的发生发展密切相关。我们在前期研究中发现，巨噬细胞膜表面有大量的α7-烟碱型乙酰胆碱受体 (α7-nAChR)表达，而我们前期研究发现， α7-nAChR 介导的胆碱能抗炎通路(CAP)在VMC中具有抗炎作用。这提示α7-nAChR介导的CAP很可能通过调节巨噬细胞M1/M2型极化来减轻VMC的炎症反应。然而，CAP调节巨噬细胞极化的具体机制尚不清楚。本研究旨在探讨α7-nAChR介导的CAP对VMC中巨噬细胞极化分型的作用及其潜在机制。在VMC模型中，我们利用α7-nAChR激动剂、抑制剂来激活或阻断CAP，利用氟达拉滨和AS1517499分别抑制STAT1和STAT6的磷酸化。我们发现激活CAP能够增加VMC中M2型巨噬细胞的数量以及IL-10、PPAR-γ和TGF-β的表达，促进STAT6磷酸化并降低STAT1的磷酸化水平。CAP的激活也能够改善VMC小鼠心脏功能、减轻心脏炎性细胞浸润、提高VMC小鼠存活率。阻断CAP则起到相反的作用。此外，抑制STAT1磷酸化能够增强CAP激活后的作用。而抑制STAT6磷酸化则逆转了CAP激活在VMC中的作用。我们的研究结果表明，α7-nAChR介导的CAP能够通过抑制JAK-STAT1和激活JAK-STAT6通路，诱导巨噬细胞向M2型分化，进而降低促炎细胞因子的表达，降低VMC炎症水平。本研究将VMC的机制研究深入神经免疫学领域，有助于阐明VMC发病机制，为VMC的治疗提供新的思路和理论依据。

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