大麻素受体介导中性粒细胞参与肝脏炎症及纤维化的机制研究

Our previous studies have shown that, between the liver and the bone marrow tissue, cannabinoid ligand is formed a gradient of high to low concentration in liver injury and drives bone marrow mononuclear macrophage (BMM) infiltration, as BMM expressed cannabinoid receptors (CB1, CB2). Neutrophils (PMN) are the terminally differentiated cells in bone marrow, also expressed cannabinoid receptors. As one kind of components of body's innate immunity, PMN also involved in non-pathogen-induced liver injury, inflammation and fibrosis. However, little is known about the cellular and molecular mechanisms how cannabinoid receptors control the infiltration and activation of PMN during liver injury, inflammation and fibrosis. In the project, we will prepare mouse models by CCl4 injection, bile duct ligation operation or feeding a methionine- and choline-deficient + high-fat diet (MCDHF), perform bone marrow transplantation to produce chimera mice of all bone marrow cells labelling with EGFP, or CB1-/-, CB2-/- and use CB1-/-, CB2-/- knockout mice, flow cytometry analysis, immunofluorescence, RNAi technology, to explore the functions and mechanisms of cannabinoid receptors on infiltration and activation of PMN in the liver injury, inflammation and fibrosis mice. Based on the above research works, we will select the appropriate time window, choose the molecules which play an important role in key cannabinoid receptors signaling pathway (e.g. enzyme or receptor agonist, blocker), intervene liver injury, inflammation and fibrosis in mice, and determine its outcome results. Accordingly, we will further confirm the roles of cannabinoid receptors and PMN during liver injury, inflammation and fibrosis in mice. The knowledge will help to represent novel therapeutic targets in liver injury, inflammation and fibrosis.

我们前期研究证明，在肝损伤时，大麻素配体在肝脏与骨髓之间形成由高到低的浓度梯度，驱动了表达大麻素受体(CB1，CB2)的骨髓单核巨噬细胞的募集；中性粒细胞(PMN)为骨髓来源的终末分化细胞，亦表达大麻素受体，其作为机体固有免疫组分，参与非病原体引发的肝损伤、炎症及纤维化之事实目前备受关注，但具体的细胞分子机制尚不清。本项目拟以四氯化碳、胆管结扎术、蛋氨酸胆碱缺失+高脂饮食等不同原因诱导的肝脏炎症及纤维化小鼠模型、小鼠及人PMN为研究对象，以大麻素受体为靶分子，应用骨髓细胞为EGFP+、CB1-/-或CB2-/-的嵌合体小鼠、流式细胞分析、高内涵分析、RNAi、免疫荧光等技术，探讨大麻素受体介导PMN参与肝脏炎症及纤维化的时相和分子机制；再用适宜的分子、选择合适的病程‘窗口’期，干预肝脏炎症及纤维化小鼠模型，确认大麻素受体的作用机理，为阐明肝脏炎症及纤维化发生、发展及转归提供理论和实验依据。

中性粒细胞(PMN)为骨髓来源的终末分化细胞，其作为机体固有免疫组分，参与非病原体引发的肝损伤、炎症及纤维化之事实备受关注，但具体的细胞分子机制尚不清。我们前期研究发现，在肝损伤时，大麻素配体在肝脏与骨髓之间形成由高到低的浓度梯度，驱动了表达大麻素受体(CBs)的骨髓单核巨噬细胞的募集；PMN亦表达CBs，但CBs是否参与PMN的浸润和活化，以及内在的分子机制尚不详。本项目以四氯化碳、胆管结扎术、蛋氨酸胆碱缺失+高脂饮食等不同原因诱导的肝脏炎症及纤维化小鼠模型、小鼠及人PMN为研究对象，以CBs为靶分子，应用流式细胞分析、高内涵分析、RNAi、免疫荧光等技术，探讨CBs介导PMN参与肝脏炎症及纤维化的分子机制，再用适宜的分子、选择合适的病程‘窗口’期，干预肝脏炎症及纤维化小鼠模型，确认CBs的作用机理，取得了一些重要发现：.1.明确了PMN的过度激活与肝脏炎症及纤维化进程相关，且通过不同病因导致肝损伤的小鼠模型的数据进一步证明了，PMN的参与是普遍现象，与病因无关；.2.阐明了CBs介导PMN迁移和活化的分子机制，具体为：CB1通过Gαi/o引起了中性粒细胞ROS的产生，并进一步激活p38 MAPK信号通路，最终介导了中性粒细胞的迁移和NETosis，参与了小鼠肝损伤；.3.阐明了另外一组与CBs高度同源的受体--磷酸鞘胺醇受体（S1PRs） 介导PMN迁移和活化的分子机制，具体为：S1P/S1PR1/2信号轴促进PMN的迁移，抑制其自发性凋亡，并诱导NETosis发生，这一过程依赖 Gαi/o/ERK，p38/ROS信号通路；.4.在小鼠肝损伤模型中，腹腔注射DNase I，清除中性粒细胞活化后产生的NETs，发现：血清ALT水平降低，肝组织中炎症因子和纤维化相关指标明显下调，肝组织炎症和纤维化区域均减小。在整体水平证明了PMN的过度激活与肝脏炎症及纤维化进程相关；.5.阻断或敲减CB1或S1PR2可以减少肝组织中性粒细胞的浸润和NETs的形成，进而缓解肝脏炎症和纤维化。.研究成果为肝脏炎症及纤维化提供了新的干预策略。