炎症在脂肪性肝病发生发展过程中的关键作用与分子机理

The key factor in the occurrence of non-alcoholic fatty liver disease (NAFLD) is uncontrolled inflammatory response which characterized by the monocyte /macrophages M1 and M2 subtype imbalance, or polarization disorder. Our previous study found that mesenchymal stem cells (MSC) with immuno-modulatory activity, 15d-PGJ2 with function of suppressing inflammation, and S1P with pro-inflammatory function, participated in liver inflammation by different ways. However, little is known about the cellular and molecular mechanisms that MSC, 15d-PGJ2, and S1P control the polarization disorder of monocyte/macrophages M1 and M2 subtype during NAFLD. In the project, we will prepare a NAFLD mouse model by feeding a methionine- and choline-deficient + high-fat diet (MCDHF), perform bone marrow transplantation to produce chimera mice of all bone marrow cells or MSCs lebelling with EGFP, and use the receptors (S1PR2 or S1PR3) gene knockout mice, flow cytometry analysis, cell co-culture, immunofluorescence, RNAi technology, to explore the functions and mechanisms of 15d-PGJ2 or S1P on infiltration and polarization of monocyte/macrophages (including Kupffer cells and bone marrow-derived monocyte/macrophages) and MSCs, and the interaction between each other in the liver of NAFLD mice. Based on the above research works, we will select the appropriate polarized monocyte/macrophages or MSCs (for cell therapy), choose the molecules which play an important role in key 15d-PGJ2 or S1P signaling pathway (e.g. enzyme or receptor agonist , blocker), intervene NAFLD in mice, and determine its outcome results. Accordingly, we will further confirm the roles of monocyte/macrophages, MSCs, 15d-PGJ2 or S1P during NAFLD in mice. The knowledge will help to represent novel therapeutic targets in NAFLD.

脂肪性肝病发生的关键因素是以单核巨噬细胞M1和M2亚型比例失调、极化紊乱为特征的炎症反应失控，而其细胞分子机制尚不清。我们前期研究发现，具有免疫调节活性的间充质干细胞、具有抑炎功能的15d-PGJ2、具有促炎功能的S1P以不同方式参与肝脏炎症反应，但具体到脂肪性肝病时调节单核巨噬细胞极化的机制尚不详。本项目拟以蛋氨酸胆碱缺失+高脂饮食制成的小鼠脂肪性肝病模型、小鼠单核巨噬细胞（肝脏或骨髓来源）、间充质干细胞为研究对象，以15d-PGJ2或S1P为靶分子，应用骨髓细胞为EGFP+的嵌合体小鼠、S1P受体2/3 基因敲除小鼠、流式细胞分析、RNAi等技术，探讨15d-PGJ2或S1P对小鼠单核巨噬细胞或间充质干细胞向脂肪性肝病肝脏浸润、极化及相互之间作用的机制；再用适宜的细胞或分子干预脂肪性肝病小鼠，确认15d-PGJ2或S1P的作用机理，为阐明脂肪性肝病发生发展及转归机制提供理论和实验依据。

脂肪性肝病发生的关键因素是以单核巨噬细胞M1和M2亚型比例失调、极化紊乱为特征的炎症反应失控，而其细胞分子机制尚不清。我们前期研究发现，具有免疫调节活性的间充质干细胞、具有抑炎功能的15d-PGJ2、具有促炎功能的S1P以不同方式参与肝脏炎症反应，但具体到脂肪性肝病时调节单核巨噬细胞极化的机制尚不详。本项目以蛋氨酸胆碱缺失+高脂（MCDHF）饮食制成的小鼠脂肪性肝病模型、小鼠单核巨噬细胞（肝脏或骨髓来源）、间充质干细胞为研究对象，以15d-PGJ2或S1P为靶分子，应用骨髓细胞为EGFP+的嵌合体小鼠、流式细胞分析、RNAi 等技术，深入研究了15d-PGJ2、S1P对小鼠单核巨噬细胞或间充质干细胞向脂肪性肝病肝脏浸润、极化及相互之间作用的机制，取得了一些原创性重要发现：.1.阐明了S1P募集单核巨噬细胞并诱导其向M1亚型极化的机制：S1P/S1PR2/3 诱导 BMM 向 M1 型巨噬细胞极化依赖于 G(α)i/o/PI3K/JNK 信号通路；.2.阐明了15d-PGJ2抑制单核巨噬细胞向M1亚型极化的机制：15d-PGJ2 / PPARγ 轴抑制 M1 BMMs 的极化依赖于上调 miR-27b-3p、miR-181a-1-3p 以及 miR-326-5p 的表达；.3.间充质干细胞归巢由S1PR3介导，而RNA结合蛋白HuR与miR30e通过协同方式调节S1PR3的表达，从而参与间充质干细胞向受损肝脏归巢；.4.间充质干细胞通过向周细胞方向分化，促进脂肪性肝病小鼠血管新生，从而参与肝纤维化，该过程的分子机制是：PDGF受体（PDGFR）是关键靶分子，执行间充质干细胞向周细胞方向分化的任务，而PDGFR的表达受长链非编码RNA（MEG3）和小RNA分子（miR26b-5p）共同调控；.5.阐明了与S1PRs 高度同源的受体CB1 介导 BMM 向 M1 型巨噬细胞极化分别依赖于 RhoA/NF-κBp65 和 ERK1/2 信号通路。.研究成果为脂肪性肝病提供了新的治疗策略。