人类特异性乙酰胆碱受体在心肌缺血再灌注损伤中保护作用及机制研究

Inflammation plays a critical role in cardiac ischemia/reperfusion (I/R) injury. α7nAchR has been shown to mediate the anti-inflammatory response in I/R injury, leading to cardiac protection in animal models. The molecular mechanisms of α7nAchR-mediated anti-inflammatory response in I/R include vagus nerve stimulation (VNS)-dependent and -independent pathways. The ligands in the former are VNS released acetylcholine (Ach) and in the later are epithelia and immunocytes secreted mammalian Ly-6/urokinase plasminogen activator receptor-related protein-1/2 (SLURP1/2). Both ligands bind to, and activate α7nAchR receptors on leukocytes, which in turn inhibits NF-κB signaling pathway, leading to decreased neutrophil infiltration, markedly reduced production and release of TNFα, IL1β, HMGB1, IL6 and other proinflammatory cytokines from macrophages, and thus protects cardiomyocytes from I/R injury. However, the discovery of a human-specific, and partially duplicated α7nAchR, namely dupα7nAchR homologous to α7nAchR on the C-terminal 384 aminoacid residues that defines all functional domains of the ion-gated channel monomer except the 117-aminoacid extracellular portion of the N-terminus, challenged the protective role of α7nAchR in I/R injury in humans since (1) dup-α7nAchR encoded by CHRFAM7A is the predominant transcript relative to α7nAchR expressed in leukocytes, (2) LPS significantly up-regulated the expression of dup-α7nAchR, not α7nAchR, in gut primary epithelial cells and, (3) forced expression of CHRFAM7A in THP1 cells up-regulated the expression of α7nAchR, increased the binding affinity of α7nAchR to its ligands, changed the functions of the ion-channel, and modulated the expression of genes involved in cell adhesion and leukocyte trans-epithelial migration. These results suggest the potential regulatory roles of dup-α7nAchR in human leukocytes, dependent or independent of α7nAchR. We hypothesize that dup-α7nAchR contributed directly or indirectly a significant portion, if not more than α7nAchR, to the cardio-protective effect in cardiac I/R injury in humans. Here, we propose to investigate the tissue distribution and the mechanisms of anti-inflammation of dup-α7nAchR in vitro, and to analyze its role in cardiac I/R injury using the emerging ‘humanized’ mouse model.

炎症反应是造成心肌缺血再灌注(I/R)损伤的重要原因。动物研究发现,白细胞表达的乙酰胆碱受体(α7nAchR)在心肌I/R损伤中起重要的保护作用。其机理是：迷走神经释放的乙酰胆碱或上皮及免疫细胞分泌的SLURP作用于α7nAchR，通过NF-κB信号通路抑制巨噬细胞炎性介质的释放和中性粒细胞的渗出。近来发现,人白细胞存在人类特异的α7nAchR, 命名为 dup-α7nAchR。dup-α7nAchR (1) 和α7nAchR的碳端384个氨基酸残基完全相同;(2)在白细胞中的表达远高于α7nAchR;(3)在THP1细胞过度表达引起α7nAchR表达上调,并影响其受体的功能。我们推测, dup-α7nAchR在人类心肌I/R损伤中直接或间接地起抗炎保护作用。本研究将利用体外实验和人源性小鼠技术来阐明dup-α7nAchR的组织分布, 抗炎机理, 及对人源性小鼠心肌I/R损伤的保护作用。

a7-乙酰胆碱受体（a7-nAChR，简称a7）与人类认知及神经精神疾病的发生密切相关。研究表明：巨噬细胞表面的a7在‘胆碱抗炎通路’中起重要的作用。人类进化过程中出现了人特异性a7基因（CHRFAM7A，编码dup-a7-nAChR，简称dup-a7），该基因由FAM7和a7基因（CHRNA7）部分复制融合而成。CHRFAM7A转录产生一个1236bp开放阅读框架，编码411氨基酸残基（AA），其中N-端27AA（来自FAM7基因）取代了a7N-端146AA的胞外配体结合域，其余384AA与a7完全相同。由于dup-a7与a7可以同时转运到膜上，并形成杂合五聚体离子通道，dup-a7剂量依赖性地显性负调节a7结合a-Bungarotoxin（a-BTX）的能力及其通道电流。然而，dup-a7对a7在胆碱抗炎通路及中枢高级神经功能的调节作用及其机理尚未见报道。.共转染试验表明：dup-a7显著抑制a7结合a-BTX的能力及a7的通道电流；转染小鼠上皮细胞得到了类似的结果。提示人特异性CHRFAM7A基因可以在小鼠的遗传背景中发挥其功能。利用CHRFAM7A转基因小鼠（Tg），我们进行了如下研究：.dup-a7通过调节a7而促进炎症反应。到目前为止，我们提供了以下证据（1）Tg腹腔巨噬细胞结合αBTX的能力明显减弱，并呈现明显的炎性基因激活的基因表达谱；（2）在盲肠结扎穿孔术的小鼠模型中，Tg的存活率显著低，其外周血中炎性因子的水平及炎性细胞的数目显著上调；（3）dup-a-nAChR对炎症反应的调节作用涉及NFkB及MAPK信号传导通路；（4）腹膜炎患者CHRFAM7A的表达水平与IL6呈明显的正相关；（6）中国人群中0.54%（1/185）无此基因。.Tg全脑蛋白组学分析表明：CHRFAM7A通过抑制帕金森病、亨丁顿氏舞蹈病、氧化磷酸化、阿兹海默病等信号传导通路而调节a7在人类神经精神疾病的发生及发展中的作用。.上述结果说明：dup-α7通过显性负调控α7的功能而促进炎症反应、影响人类神经精神疾病的发生及发展。由于CHRFAM7A在人群中的差异分布（1%无该基因）及表达差异（含1-3个拷贝），该研究一方面为人类炎症及神经精神等疾病的个体差异提供了解释，另一方面也暗示了绝大多数靶向α7的先导药物在临床试验中的失败的原因：动物模型缺乏CHRFAM7A/dup-α7。