靶向α7烟碱型乙酰胆碱受体的胆碱能抗炎通路对缺血性脑卒中的免疫调节机制研究

Lymphocytic α7 nicotinic acetylcholine receptor (α7 nAChR) mediated peripheral cholinergic anti-inflammatory pathway is an important regulatory mechanism between the nervous system and the innate immune responses. Our previous study demonstrated that significant lymphocyte infiltration occurs in a mouse model of transient cerebral ischemia and subsequently NK cell mediated innate immunity exacerbated brain injury (results published in P.N.A.S，2014，IF 9.7). We also found a rapid lymphocyte-derived cholinergic over-activity in patients of acute ischemic stroke, which positively correlates with clinical severity. Thus, we hypothesize that miRNA regulates cholinergic anti-inflammatory pathway by targeting α7 nAChR and acts as a new mechanism of maintaining immunologic homeostasis after stroke. Based on the hypothesis, we will use α7 nAChR transgenic mice to establish the middle cerebral artery occlusion (MCAO) in mice. We intend to investigate α7 nAChR regulation of brain and peripheral immune functional changes after acute ischemic stroke. In addition, we will apply luciferase report assays to investigate a specific miRNA targets α7 nAChR in post-transcriptional regulation. Then we will examine the role of the miRNA in ischemic stroke and subsequent systemic immune responses by affecting α7 nAChR activity. In conclusion, our group will identify specific roles of cholinergic anti-inflammatory pathway in ischemic stroke which can be regulated by new miRNAs, providing more potential approaches to study new mechanisms of post-stroke immunity.

由淋巴细胞表面α7烟碱型乙酰胆碱受体（α7 nAChR）介导的胆碱能抗炎通路，是神经-固有免疫系统之间重要的炎性调节机制。课题组已发表的前期结果表明，急性缺血性脑卒中后脑内淋巴细胞浸润增多，以NK细胞为主的固有免疫系统会加重脑损伤（结果发表在P.N.A.S，2014，IF 9.7），而且淋巴细胞源性胆碱能抗炎通路过度活化，与患者病情严重程度呈正相关。据此，我们提出科学假说，靶向淋巴细胞α7 nAChR的胆碱能抗炎通路，是调控缺血性脑卒中免疫炎性反应的新机制。本课题拟利用α7 nAChR转基因小鼠，探讨淋巴细胞胆碱能抗炎通路对缺血性脑卒中脑内免疫损害及其它重要组织变化的影响；靶基因验证miRNA可能会介导α7 nAChR的转录后调控，并验证其通过影响α7 nAChR的功能调节卒中后系统性免疫反应。本研究将为阐明miRNA调控的胆碱能抗炎通路在缺血性脑卒中后脑损害和免疫抑制的具体机制奠定基础。

本课题将临床和基础研究结合，主要研究了胆碱能抗炎通路和卒中后肺炎之间的关系。为了研究缺血性脑卒中外周血胆碱能通路的功能上的变化，我们已经成功利用超高效液相色谱-质谱联用法（UPLC-MS/MS）精确测定了免疫细胞内的乙酰胆碱（ACh）含量。通过收集缺血性脑卒中急性期和缓解期以及合并肺炎的患者外周血，并利用影像学资料测定脑梗死体积。发现脑卒中严重程度外周免疫细胞内ACh含量相关，且与卒中后肺炎的发生有直接关系。脑卒中患者外周淋巴细胞胆碱能通路的过度活化与卒中后肺炎及不良预后相关。在基础研究中，我们利用小鼠脑缺血模型，探讨了卒中后免疫抑制和特殊类型免疫细胞之间的密切关系。我们首次发现人和小鼠骨髓来源的抑制性细胞（MDSC）具备合成和分泌ACh的能力，而且在卒中急性期小鼠肺组织内MDSC分泌ACh的能力增强。将小鼠MDSC和肺内II型肺泡上皮细胞共培养，我们发现MDSC可能通过自分泌或旁分泌ACh的形式，通过α7 nAChR介导的胆碱能通路降低II型肺泡上皮细胞防御细菌感染的能力。增强α7 nAChR的功能虽然在减轻了脑梗死体积，但增加了卒中后小鼠肺感染的机会。同时应用α7 nAChR激动剂和MDSC抑制剂可在一定程度上减轻肺内II型肺泡上皮细胞的炎症刺激作用，从而降低卒中后肺部细菌感染的可能。