基于肠黏膜免疫LPDC-Th17活化致敏外周神经研究肝郁脾虚夹湿证PI-IBS的病理机制

Post infectious irritable bowel syndrome (PI-IBS) is a common type of IBS disease which is short of effective therapy because of still unknown of pathogenesis. The pathological mechanisms of PI-IBS contain not only the syndrome of liver depression and spleen deficiency which is the key pathological mechanisms of IBS but also exogenous pathogens which turn out to be dampness verified by our clinical trial. As the characteristic pathological mechanisms of PI-IBS, the abnormal of intestinal immunity and visceral hypersensitivity are interrelated with the syndrome of liver depression and spleen deficiency and the syndrome of dampness. Lamina propria dendritic cells (LPDC) play an important regulatory role in post- infectious immune response. Recent researches found that inflammation mediated by the activation of LPDC-Th17 is associated with peripheral nerve sensitization of PI-IBS, in which serotonin, calcitonin generelated peptide (CGRP) and capsaicin receptor (TRPV1) are involved. Therefore, we propose a hypothese: the principal pathogenesis of PI-IBS is liver depression and spleen deficiency combined with dampness, of which biological foundation is the visceral hypersensitivity induced by sensitization of peripheral nerves which is sensitized by the activation of intestinal immune response. The inflammation induced by the activation of LPDC-Th17 not only upregulate the expression of TRPV1, but also activate the pathway of CGRP-5HT-5HTR3 to sensitize the peripheral nerves. This project intends to establish a disease animal model of PI-IBS presenting the characters of disease and syndrome to define the relationship between inflammation and peripheral nerve sensitization through observing the dynamic changes of intestinal immune and peripheral nerve ensitization. We aim to expound the pathologic mechanism of PI-IBS with syndrome of liver depression and spleen deficiency with dampness from perspective of immunology and neuroendocrinology, and it is a kind of supplement and innovation of research of IBS using method of disease integrating with syndrome.

感染后肠易激综合征（PI-IBS）是IBS的常见疾病类型，因发病机制不明而缺乏有效的治疗，其在肝郁脾虚核心病机的基础上，兼具正虚邪恋、邪正交争的特点，我们发现湿证与其密切相关。而作为PI-IBS的病理特征，内脏高敏感及肠道免疫活化与肝郁脾虚及湿证密切相关。固有层树突状细胞（LPDC）对感染后免疫应答有重要调节作用，新近发现，LPDC诱导Th17分化介导的炎症与PI-IBS神经敏化相关；5-HT、降钙素基因相关肽（CGRP）及辣椒素受体（TRPV1）参与了炎症后的神经敏化。因此假设：肝郁脾虚夹湿是PI-IBS的核心病机，LPDC-Th17介导的炎症通过TRPV1和CGRP-5HT-5HTR3通路致敏外周神经是其关键病理机制。本研究拟建立PI-IBS病证结合动物模型，通过动态观察肠道免疫及神经内分泌的变化明确两者的关系，从免疫-神经内分泌互作角度阐明其病机，是对病证结合研究IBS的补充与创新。

本课题假说为：肝郁脾虚夹湿是PI-IBS的核心病机，肠道黏膜免疫活化致敏外周神经从而导致内脏高敏感是其关键病理机制；其中LPDC-Th17免疫活化介导的肠道特异性炎症状态诱导肠神经纤维TRPV1受体的过表达和激活CGRP-5HT-5HTR3通路导致DRG去极化致敏外周神经是这一机制的关键环节。研究通过两个部分内容对假说进行验证，首先，通过理论探讨明确了PI-IBS的核心病机为肝郁脾虚夹湿证，并运用旋毛虫灌胃联合慢性应激及人工气候高湿的复合因素构建了PI-IBS肝郁脾虚夹湿证病证结合大鼠模型，在造模的第一、二、四、六、八周分别通过一般情况、粪便含水率、腹壁撤退试验、糖水消耗实验、旷场实验、肠组织病理学观察等指标对模型进行评价，结果表明在第六至八周肠道炎症消退后大鼠能持续形成粪便含水率升高、腹壁撤退反射敏感性增高、肠组织5-HT含量升高及AQP4表达下降的状态，具有疾病与证候的特点。第二部分在病证结合模型的基础上加用Th17细胞分化抑制剂HF，并在不同时间点通过免疫磁珠分选法分离肠组织LPDC并观察其内吞活力和细胞因子分泌特点，同时观察Th17细胞特异性细胞因子IL-17的表达变化以明确模型进展过程中两者间的关系，结果表明LPDC呈现趋化功能为主时IL-17的表达出现增高，而HF对LPCD的功能无影响，但在下游抑制了IL-17的表达。另一方面，通过免疫组化及荧光等方法动态检测肠组织及背根神经节中神经递质及其受体的表达情况，结果表明在PI-IBS病证模型的进展过程中，肠组织中神经递质5-HT和CGRP的表达出现了持续的升高趋势，随着后期的成模5-HTR3的表达逐渐升高，且背根神经节中5-HTR3和TRPV1的表达也明显升高，而HF的干预均能在一定程度逆转这类神经递质及其受体的表达，提示其抑制的Th17细胞及其分泌的特异性炎症因子可能干预了5-HT、CGRP的分泌及其相关受体5-HTR3和TRPV1的表达从而参与了外周神经敏化的过程。.综合上述结果，本研究构建了PI-IBS肝郁脾虚夹湿证病证结合大鼠模型及评价方法，同时观察了病证形成过程中LPDC-Th17的免疫活化状态及神经内分泌变化情况，并初步明确了Th17细胞及其介导的免疫状态与外周神经敏化的关系。将为进一步阐释PI-IBS的发病理论及其证候基础研究提供借鉴，也为中医药防治PI-IBS有效方药的开发奠定基础。

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