肠道CCR9/CCL25信号通路对NAFLD中肠道免疫屏障功能的影响及机制研究

Nonalcoholic fatty liver disease (NAFLD), the common cause induced increment of live aminotransferasesbelongs, belongs to metabolic stress liver injury. The gut immune barrier and NAFLD interact each other. Studies showed that there had gut immune barrier dysfunction in the process of NAFLD; but its mechanism remains unclear. Dysfunction of T cell intestinal homing is the essential cause of gut immune disorder; but its mechanism in NAFLD has not been systematic studied. We focused on the axis of gut dendritic cell (DC), CCR9/CCL25 signaling pathway and T cell homing to investigate the effect of CCR9/CCL25 pathway on T cell intestinal mucosal homing in NAFLD through detecting the intestinal expression of CCL25 , proportion of CD4+T cell, CCR9+CD4+T cell in gut associated lymphoid tissue (GALT) and the migratory ability of these cells under different conditions. Then, GALT DC subsets regulating the CCR9/CCL25 signaling pathway were measured and sorted. Finally, we observed the DC subsets up-regulating CCR9 expression on T cells effects on gut barrier and liver functions in vivo. Our study aims to reveal the molecular mechanism of gut immune barrier dysfunction in NAFLD and provide the theoretical basis from the perspective of gut immune to treat NAFLD.

非酒精性脂肪性肝病（NAFLD）属代谢应激性肝损伤，是导致肝酶升高的常见原因。肠免疫屏障与NAFLD关系密切，NAFLD中伴肠免疫屏障障碍，但发病机制不明确。T细胞归巢障碍是肠道免疫失调的基础，但在NAFLD中缺乏系统研究。我们在前期证实肠道MHCⅡ+CD103+树突状细胞（DC）参与NAFLD肠免疫屏障障碍形成的基础上，着眼于肠道DC→CCR9/CCL25通路→T细胞归巢轴，分析肠道CCL25表达、肠黏膜相关淋巴组织（GALT）中CD4+T细胞、CCR9+CD4+T细胞数量及其在不同条件下的迁移能力，探讨NAFLD中肠道CCR9/CCL25通路对T细胞归巢的影响，并筛选出GALT中调控CCR9/CCL25通路的DC亚群，最后在体内观察上调CCR9表达的DC对NAFLD肠道屏障功能及肝脏的干预效果。本课题旨在揭示NAFLD中肠道免疫屏障障碍发生的机制，为从肠道免疫角度治疗该病提供理论依据。

非酒精性脂肪性肝病（NAFLD）属代谢应激性肝损伤，是导致肝酶升高的常见原因。肠道屏障障碍与NAFLD关系密切，两者相互作用，但具体机制尚未阐明。为了证实NAFLD中存在肠道屏障障碍，我们通过高脂饮食（HFD）及蛋氨酸胆碱缺乏饮食（MCD）分别建立大鼠及小鼠单纯性脂肪性肝病（NAFL）、脂肪性肝炎（NASH）模型，通过组织病理、肝脏生化、电镜观察小肠超微结构、小肠内容物细菌培养、流式细胞检测肠系膜淋巴结（MLN）树突状细胞（DCs）、调节性T细胞（T-regs），明确NAFLD存在肠道屏障障碍，且与肝脏炎症相关。然后，为进一步阐述肠道屏障对NAFLD影响，我们采用Morris法+HFD建立肠屏障障碍伴NAFLD模型，通过病理、生化、ELISA、实时PCR等方法发现NAFLD中肠道屏障障碍导致的肠源性LPS是加重肝脏炎症的重要因素之一。在NAFLD存在肠道DCs、T-regs数量改变的基础上，为进一步探讨其分子机制，我们通过免疫组化、免疫荧光、蛋白印迹、实时PCR等手段，阐述了肠黏膜及MLN中的趋化因子CCL25及其受体CCR9的表达异常导致的免疫细胞“归巢”失调参与肠道屏障障碍形成。然后，为了明确肠道屏障障碍对于肝细胞损伤的机制，我们在阐明肠源性LPS在导致KC功能障碍基础上，通过研究PPARγ、SOCS3的表达及其信号通路在肝细胞脂肪变中的作用，揭示了肝细胞PPARγ通过抑制JAK2/STAT3通路抑制SOCS3从而缓解肝细胞脂肪变性。综上，本研究发现：（1）NAFLD与肠道屏障功能障碍相互影响；（2）肠道CCL25/CCR9介导的免疫细胞归巢失调是NAFLD引起肠道屏障碍的分子基础；（3）NAFLD发展过程中，肠源性LPS导致肝脏KC功能障碍是加重肝脏损伤关键因素之一；（4）肝细胞PPARγ能够通过抑制JAK2/STAT3信号通路抑制SOCS3从而缓解肝细胞脂肪变性。