中性粒细胞来源的颗粒蛋白Proteinase 3 通过调节肝脏-肠道菌群轴介导非酒精性脂肪肝炎的研究

Nonalcoholic steatohepatitis (NASH) is one of the most common chronic liver diseases closely associated with obesity-induced alteration in gut microbiota and increased infiltration of neutrophils in the liver. However, the molecular mechanisms underlying NASH development still remains largely unclear. Proteinase 3 (PR3) is a serine protease originally characterized in neutrophil azurophilic granule. In our pilot experiments, we found that PR3 contents in both liver and intestine were markedly increased in association with the enhanced neutrophil infiltration in mouse model of NASH. Therefore, we hypothesize that PR3 modulates obesity-related alteration of gut microbiota, thereby mediating the initiation and development of NASH through regulating of liver-gut axis. To test our hypothesis, we propose several strategies to comprehensively investigate the role of PR3 in NASH development and NASH-associated alteration of gut microbiota, including the comparison of diet-induced NASH between wild type and PR3-genetically modified mice, investigation of NASH development in PR3 chimera mice generated by bone marrow transplantation, exploration of the effect of PR3 pharmacological inhibitor on diet-evoked NASH in mice, and co-culture of primary mouse neutrophils from PR3 knockout mice with primary rat Kupffer cells in ex vivo condition, as well as the in vitro stimulation of primary mouse neutrophils with recombinant mouse PR3 protein. It is expected that the findings in current proposal will provide experimental evidence for the therapeutic strategy targeting PR3, neutrophils, and gut microbiota.

非酒精性脂肪肝炎（NASH）是一种与肥胖及肠道菌群失调紧密相关、并伴随中性粒细胞浸润的慢性肝病，但目前介导NASH肝脏炎症的分子机制尚未阐明。Proteinase 3（PR3）是一种从中性粒细胞中鉴定出的丝氨酸蛋白酶。我们在前期研究中发现，PR3在小鼠NASH模型的肝脏及肠道中丰度显著升高，并与中性粒细胞的浸润密切相关。因此，我们假设PR3通过加速肠道菌群失调而介导由肝脏-肠道菌群轴参与的肝脏炎症的激活及进展。为验证该研究假说，本课题将采用多种特异的研究方案，包括以PR3基因敲除小鼠诱导NASH模型、以骨髓移植形成PR3骨髓来源细胞嵌合体小鼠、以药物性抑制剂阻断PR3通路观察其对NASH发展的效应、离体条件下PR3敲除原代小鼠中性粒细胞与Kupffer 细胞共培养以及基于重组小鼠PR3蛋白的的功能学实验，深入研究PR3是否通过干预肠道菌群及中性粒细胞功能而介导肝脏炎症，并揭示分子机制。

非酒精性脂肪肝炎（NASH）是一种与肥胖、2型糖尿病密切相关的肝脏疾病。随着肥胖及2型糖尿病发病率的显著增加，NASH已经成为全世界范围内最常见的慢性肝病之一，其有效治疗是临床难题。导致NASH治疗难题的主要原因在于对其病理机制尚未完全阐明。中性粒细胞浸润增强是NASH的关键组织病理学特征，Proteinase 3（PR3）是一种在中性粒细胞中具有高丰度表达的丝氨酸蛋白酶。本课题聚焦于揭示中性粒细胞来源的PR3调控中性粒细胞炎性反应、介导NASH及其相关肝纤维化的功能和机理。.主要研究内容包括：①.通过两种NASH诱导方案，分析了PR3敲除小鼠的NASH表型，揭示了PR3是介导NASH及其相关肝纤维化的关键分子；鉴定出了PR3上游调节因子miR-223；②.通过骨髓移植方案产生PR3上游调节因子miR-223的骨髓细胞嵌合小鼠，发现调控PR3的miR-223在调控小鼠实验性NASH中发挥重要功能，其作用与调控骨髓来源细胞功能密切相关。③.通过 AAV介导的PR3抑制剂Elafin过表达阻断PR3信号，发现Elafin可显著保护小鼠实验性NASH及其诱发的肝纤维化，支持PR3是研发NASH及其相关肝纤维化的重要靶点分子。④.通过转录组学发现Flos2是miR-223调控PR3的关键转录因子。在ApoE-/-/PR3-/-敲除小鼠诱导的NASH模型中检测了外周血中性粒细胞丰度及其肝脏浸润表型；通过小鼠原代中性粒细胞和骨髓细胞分离，分析并鉴定出了调控PR3的上游分子miR-223，揭示了PR3介导中性粒细胞和肝脏巨噬细胞（包括Kupffer细胞）相互作用的功能。.本课题的科学意义在于：第一，本课题揭示了中性粒细胞来源的PR3介导的中性天然免疫反应是调控肝脏炎症、促进NASH进展的关键分子机制，发现PR3是介导NASH的关键分子，并鉴定出调控中性粒细胞PR3表达的上游分子miR-223。因此，本课题研究结果对于阐明NASH的关键机制具有重要科学意义。第二，针对目前NASH药物研发中关键靶点分子不清的难点问题，本课题揭示出干预PR3信号可显著影响小鼠实验性NASH的进展，说明调控PR3是干预NASH的潜在治疗策略，支持PR3及其信号通路的关键分子是针对NASH进行药物研发的潜在靶点。因此，本课题研究结果对于NASH的新型药物研发具有重要的转化应用前景。