Runx2 调控巨噬细胞的肝内浸润在NASH发病机制中的作用研究

Nonalcoholic steatohepatitis (NASH) is an intermediate part for fatty liver disease progressing to severe ones. Recently, more and more studies have demonstrated that the infiltration and activation of macrophages is crucial for the initiation of hepatic injury, but the mechanism is still unclear. Runx2 is a transcriptional factor related to metabolism syndrome, but the relationship between Runx2 and NASH is unknown. In our previous study, we have found that Runx2 directly promotes the expression of Monocyte chemoattractant protein-1 (MCP-1) and Receptor activator for nuclear Factor-κ B ligand (RANKL), and recruits macrophages in aorta. Additionally, the expression of Runx2 is increased in the liver tissue of high-fat-diet induced rat, which was established as NASH animal model. Therefore, we are going to utilize Runx2 hepatic specific knockout mice (Runx2f/f AlbCre +/-), and establish NASH model by feeding them with high-fat-diet. Then, the histopathologic changes, the infiltration of macrophages and the expression of Runx2/MCP-1/RANKL would be detected subsequently. Also, the mechanism of Runx2-induced MCP-1/RANKL expression and Runx2-mediated macrophages liver infiltration would be tested in vitro. That is due to elucidate the effect of hepatic recruitment of macrophages through Runx2-induced MCP-1/RANKL signaling pathway in NASH, and to find a new clinic therapeutic target for NASH patients.

非酒精性脂肪性肝炎（NASH）是脂肪肝向终末期肝病进展的中间环节。巨噬细胞的肝内浸润及活化是启动NASH肝损伤的关键步骤，但其具体机制尚不清楚。Runx2是一个与代谢综合征相关的核内转录因子，但目前尚无其与NASH的相关报道。我们前期研究发现Runx2不仅可以直接调控细胞因子MCP-1和RANKL的表达，还可以诱导巨噬细胞的血管内聚集，进一步研究发现Runx2在高脂饮食诱导的大鼠NASH模型中的表达显著增高。因此，本课题拟利用Runx2肝组织特异性基因敲除小鼠模型（Runx2f/f AlbCre+/-)，通过高脂饮食构建NASH动物模型，研究Runx2在NASH发病过程中肝脏的组织病理学变化、巨噬细胞的浸润及表型变化等，证实Runx2通过调控MCP-1/RANKL的表达募集巨噬细胞的肝内浸润在NASH发病过程中起着十分重要的作用，明确Runx2为NASH中一个重要的治疗靶点。

非酒精性脂肪肝病（NAFLD）是目前全球发病率最高的慢性肝病，巨噬细胞的肝内浸润及活化是启动NASH肝损伤的关键步骤。【目的】本项目主要研究Runx2通过上调MCP-1的表达募集巨噬细胞的肝内浸润在NAFLD发病过程中的重要作用。【结果】 我们发现：1）42%高脂诱导的小鼠脂肪肝模型，随着时间的延长，脂变越重，然而8M才进展为NASH，肝纤维于12M表现最明显；2）Runx2在4M高脂模型组出现上升，但主要表达于间质细胞中，进一步研究发现HSC的活化标志物α-SMA也于4M开始明显升高，免疫荧光共染色提示Runx2主要表达于α-SMA阳性的细胞中，提示Runx2在HSC中表达，并且与HSC的活化有关；3）巨噬细胞的标志蛋白F4/80随着高脂诱导的时间的推移其表达逐渐增多，4M、8M、12M均明显增多，12M增加更明显，另外，我们发现M1型巨噬细胞标志物逐渐上升，而M2型巨噬细胞标志物逐渐下降，M1/M2逐渐增大，提示巨噬细胞的肝内浸润主要表现为诱导炎症为主的M1型；4）于体外转染过表达或干扰Runx2于HSC细胞株中，我们发现过表达Runx2后MCP-1表达明显升高，而抑制Runx2可降低MCP-1的表达；5）Transwell研究发现，Runx2通过MCP-1诱导巨噬细胞的迁移。【结论】通过以上研究我们证实了Runx2主要表达于活化的HSC中，Runx2通过诱导MCP-1的表达募集巨噬细胞的肝内浸润，促发NASH的发生。