肠道感觉神经HCN通道表达和功能变化的天然免疫机制与内脏痛敏的发生及转归

Functional gastrointestinal tract diseases(FGIDS) are considered as immune-neuro-endocrine disorders. Visceral hyperalgesia represents the most troublesome and characteristic symptom and sign of FGIDS but unfortunately still lacks effective treatments. The hyperpolarization-activated cyclic nucleotide-gated cation channels (HCN1-4) are known to regulate membrane excitability. Our previous work demonstrated that the spinal and vagal afferents of the small intestine differentialy express HCN2 and HCN3 subtypes and recent data further implicated up-regulated HCN2 expression and function in TNBS-induced intestinal hypersensitivity. In the present project, we propose to investigate the role of innate immunity in the regulation of the expression and function of HCN channels in vagal and spinal afferents and the development and maintanance of intestinal hypersensitivity following simulated bacterial/fungal infection and chemical injury. The time-dependent alterations in HCN2/3 expression/function and the mechanosensitivity of vagal and spinal afferents innervating the small intestine will be investigated following challenging the intestine with TNBS,zymosan and lipopolysacharrides.The effects of innate immunity receptor (TLR2,4,5) activation on HCN2/3 expression and function, direct (activation of neuronal TLRs) or indirect (via releasing cytokines and inflammatory mediators from immune cells),will then be studied through multiple approaches. And finally, we will explore the consequence of TLRs interference on the genesis and maintanance of intestinal hypersensitivity. These studies may reveal, at cellular and molecular levels, the common and the different features and the underlying innate immunity mechanisms of visceral hyperalgesia due to different pathogenic insults and provide novle clues for the diagnosis and treatment of FGIDs and visceral hyperalgesia.

功能性胃肠病（FGIDs）是神经-免疫-内分泌疾病，内脏痛敏是其重要体征，尚缺乏有效治疗。离子通道HCN1-4参与细胞膜兴奋性调节，我们的工作已证明肠道脊髓和迷走传入神经差异表达HCN2和HCN3通道，并提示HCN2表达和功能上调与TNBS诱导的肠道高敏有关。本项目拟采用文献常用的化学方法模拟细菌/真菌感染和化学损伤后肠易激模型，探讨天然免疫机制对肠道感觉神经HCN2/3表达和功能的调控作用：1）比较脂多糖（LPS）、酵母多糖和TNBS处理后，肠道传入神经HCN2/3表达和功能及机械敏感性的变化规律；2）分析天然免疫受体（TLR2,4,5）对肠道传入神经HCN表达和功能的直接或间接（通过细胞因子和炎性介质）调节作用；3）观察干预TLRs对肠道高敏发生和转归的影响。本项目的成果可能揭示不同致病因子引起的内脏痛敏在细胞分子水平的异同及其原理，从而为FGIDs和内脏痛敏的诊疗提供线索。

功能性胃肠病（FGIDs）与炎症性肠病（IBD）是神经-免疫-内分泌疾病，内脏痛敏与动力障碍是其重要体征，尚缺乏有效治疗。离子通道HCN1-4参与细胞膜兴奋性调节，我们的前期工作已证明肠道脊髓和迷走传入神经差异表达HCN2和HCN3通道，并提示HCN2表达和功能上调与TNBS诱导的肠道高敏有关。本项目计划采用化学方法模拟细菌/真菌感染和化学损伤后肠易激模型，探讨天然免疫机制对肠道感觉神经HCN2/3表达和功能的调控作用。在实验进程中，我们意外地发现肠道上皮的嗜铬细胞(EC)高表达HCN2，因此围绕HCN2对肠道内分泌和免疫炎症反应的调控作用，开展了一系列逐步深入的研究。.1）通过免疫荧光、western blot和qPCR等方法，细致地分析了HCN1-4在消化道分布的特征。证明小肠和结直肠EC细胞特异性表达对cAMP敏感的HCN2通道，而肠道粘膜固有层的免疫细胞可能表达HCN3与HCN4通道。.2）建立了肠道EC细胞急性分离与培养模型，通过膜片钳记录的方法证实EC细胞表达功能性HCN通道。.3）采用离体肠道模型，同步记录5-HT分泌与感觉神经活动，观察到cAMP能增加5-HT释放与感觉神经放电活动，而ZD7288(HCN阻断剂）可抑制5-HT分泌与感觉神经活动，从而证明HCN2能够驱动5-HT分泌。.4）在多种炎症性肠病小鼠模型（酵母多糖、TNBS和DSS模型），探讨了肠道初级传入神经元及肠道上皮中HCN2表达变化，发现DSS诱导的IBD小鼠结肠上皮EC细胞增生和HCN2表达升高。.5）聚焦DSS诱导的IBD小鼠模型，预防性和治疗性给药HCN通道阻断剂ivabradine, 发现能明显减轻肠道炎症和降低疾病活动指数，同时观察到DSS诱导的IBD小鼠血清5-HT和TNFa水平升高，而预防性和治疗性给药Ivabradine都能明显降低血清5-HT和TNFa水平。.6）在离体肠管模型，观察到DSS诱导的IBD小鼠结肠高动力与高敏感，离体或在体给药ivabradine都能显著抑制IBD模型小鼠的肠道高动力或高敏感状态。.7）对IBD患者血清的检测发现5-HT和TNFa水平与IBD指数存在明显相关性。.以上结果证明HCN2通道驱动5-HT分泌和肠道免疫炎症反应，在功能性和炎症性肠病的病理生理进程中发挥重要作用。并有力地提示HCN2是功能性和炎症性肠病的潜在治疗靶点。