肝Tregs细胞抑制所致肝胰岛素抵抗介导高脂饮食致NAFLD的机制研究

Nonalcoholic fatty liver disease (NAFLD) is one of the important public health problems that endanger human health. The high-fat diet is the exact cause of NAFLD. Its pathological process can be divided into two stages: early reversible stage of simple fatty liver (SFL) and later stage of irreversible hepatitis (NASH). Therefore, it is very important to explore the regulatory factors of SFL, which is helpful to the early diagnosis and treatment of NAFLD. Regulatory T cells (Tregs) are important immunosuppressive cells in the body. Epidemiologic and experimental studies have confirmed that high fat diet-induced NAFLD is accompanied with inhibited Tregs. However, the current study only observed the role of Tregs in the hepatitis stage of NAFLD. There is no report on the role of Tregs in SFL. Our recent studies showed inhibited Tregs during the SFL stage. Insulin resistance (IR) is the initiating factor of NAFLD, while inhibited Tregs is closely related to IR in other organs. Thus we hypothesized that Tregs can launch NAFLD by inducing hepatic IR. This project were designed to confirm the hepatic Tregs inhibition occur in each stage of NAFLD development. And this inhibition occurs in the early stage (SFL stage) and then induces hepatocyte steatosis by inducing hepatic IR. Further, the mechanisms of high fat diet-induced hepatic Tregs inhibition and Tregs-induced hepatic IR are elucidated in cell and molecular level. Lastly, by injecting Tregs cells into the liver of mice, the important role of hepatic Tregs played in the early stage of NAFLD was confirmed. The results of this project will provide new directions and ideas for early diagnosis and treatment of NAFLD.

非酒精性脂肪肝（NAFLD）是危害人类健康的重要公共卫生问题，高脂饮食是其确切诱因。其分为早期可逆的单纯性脂肪肝（SFL）和后期不可逆的肝炎（NASH）阶段，因此探究SFL阶段的调控因素尤为重要。调节性T细胞（Tregs）是机体重要的免疫抑制细胞，已证实，高脂饮食致Tregs抑制参与了SFL向NASH转变的炎症过程，但在SFL阶段的研究尚是空白。我们近期发现SFL即伴随Tregs数目下降。胰岛素抵抗（IR）是NAFLD的启动因素，而Tregs抑制与其它脏器IR密切相关。我们推测，Tregs可诱导肝IR而启动NAFLD。本项目首先在整体水平证实肝Tregs抑制发生在SFL阶段甚至之前，并通过诱导肝IR导致NAFLD发生。进一步在细胞和分子水平阐明高脂饮食致Tregs抑制及Tregs致肝IR的机制。最后，通过回输Tregs观察其对NAFLD的改善作用，为NAFLD的早期诊治提供新的方向和思路。

非酒精性脂肪肝(NAFLD)是全球第一大慢性肝病且发病机制尚不明确。其分为早期可逆的单纯性脂肪肝(SFL)和后期不可逆的肝炎(NASH)阶段，因此探究SFL阶段的调控因素尤为重要。调节性T细胞(Tregs)是机体重要的免疫抑制细胞，已证实，高脂饮食致Tregs抑制参与了SFL向NASH转变的炎症过程，但在SFL阶段的研究尚是空白。本项目研究了肝Tregs 在NAFLD发生中的作用及其在SFL阶段减少的分子机制。我们观察到不仅在NASH阶段肝内Tregs降低，且在单纯性脂肪肝阶段甚至其形成之前即降低。通过高脂饲喂Tregs消除鼠，我们证实 IL-10 分泌减少介导了 Tregs 抑制所致肝内 IR。进一步研究发现肝内Tregs的减少与肝内巨噬细胞Notch1的异常激活密切相关，巨噬细胞Notch1敲除能明显逆转高脂饮食小鼠肝内Tregs细胞的减少，并阻止NAFLD进程。进而我们建立骨髓源性巨噬细胞（BMDM）与Naïve CD4+T细胞体外共培养模型，证实了Notch1在巨噬细胞调控Tregs的诱导分化中的调控作用。进一步的机制研究显示，通过使用外泌体抑制剂及外泌体摄取、共孵育实验证实了巨噬细胞Notch1通过外泌体途径调控Tregs。进而通过Exos-miRNA测序，筛选并验证了巨噬细胞Notch1激活通过调控外泌体miR-142a-3p来抑制Tregs。最后证实了扩增肝内Tregs可有效阻碍NAFLD进程。本项目首次提出肝脏Tregs细胞抑制发生在SFL阶段甚至其形成之前，推动 NAFLD 的发生。目前高脂饮食所致肝 Tregs细胞抑制尚无详细报道，更缺乏深入的机制研究。本项目首次阐明巨噬细胞通过外泌体miRNA调控Tregs分化的分子机制，将为NAFLD开展靶向治疗提供新思路，同时有助于人们更全面地认识肝内免疫微环境在NAFLD发生中的作用。