LILRB4在急性肺损伤中的功能和机制研究

Acute lung injury (ALI) is a severe respiratory disease with high mortality but no effective treatment currently. Excessive activation of inflammatory responses is a major cause of development of ALI. Previous studies have shown that LILRB4 plays an important role in many diseases such as atherosclerosis by regulating immune and inflammatory responses, but the function of LILRB4 in ALI has not been reported. In the preliminary study, we have found that LILRB4 mRNA and protein expression were significantly increased in LPS-induced lung tissues of mice, which mainly located in macrophages. Therefore, we hypothesize that LILRB4 may regulate the pathogenesis of ALI by modulating macrophage inflammatory response. In this study, we intend to determine the exact function of LILRB4 in the pathogenesis of ALI by establishing LPS-induced ALI model in LILRB4 knockout mice. Then we will isolate primary macrophages and evaluate the effect of LILRB4 on the inflammatory response in vitro. Finally, using RNA-Seq and interaction proteomics, we will elucidate the mechanism by which LILRB4 regulates the function of macrophage and ALI. This work will provide theory evidence for searching for the novel therapeutic targets of ALI.

急性肺损伤(acute lung injury，ALI)是一种具有高死亡率的呼吸重症疾病，目前并无有效治疗手段。炎症反应过度激活是导致急性肺损伤的主要因素。以往研究显示LILRB4通过调节免疫和炎症反应在动脉粥样硬化等多种疾病中发挥重要作用，但在急性肺损伤中的功能未见报道。本项目前期研究发现，在LPS诱导的急性肺损伤小鼠肺组织中，LILRB4的mRNA和蛋白表达均显著上升，且集中在巨噬细胞中。因此，我们推断LILRB4可能通过调节巨噬细胞炎症反应调控急性肺损伤。本项目拟通过应用LILRB4基因敲除小鼠建立LPS诱导的急性肺损伤模型，评价LILRB4在急性肺损伤中的作用。并通过体外原代巨噬细胞阐明LILRB4在调控巨噬细胞炎症反应的功能，最后再应用转录组学和互作蛋白组学联合分析寻找LILRB4调控急性肺损伤的分子机制，为寻找防治急性肺损伤的药物靶点提供重要的理论基础。

背景：急性肺损伤(acute lung injury，ALI)是一种具有较高发病率及病死率的呼吸重症疾病。天然免疫与炎症反应之间的失调是引起ALI的主要发病机制之一。寻找ALI过程中介导天然免疫和炎症反应的关键调控分子，对ALI疾病预防及临床治疗具有重要意义。 .主要研究内容、结果数据及其科学意义：为探究ALI的发病机制和潜在治疗靶点，结合我们前期研究的成果，在明确LILRB4在ALI小鼠肺组织中表达情况的实验基础上，利用LILRB4敲除小鼠（LILRB4 KO）构建LPS诱导的ALI损伤模型，借助病理和生化分析探究了其对ALI的影响。骨髓细胞移植实验用于鉴定LILRB4缺失加重ALI炎症反应的细胞来源。分离小鼠原代肺泡巨噬细胞和骨髓巨噬细胞，体外实验探究了LILRB4对巨噬细胞炎症反应的影响。利用分子生物学手段，阐明LILRB4调控急性肺损伤的分子机制及通路。本研究为临床上干预或治疗急性肺损伤提供了理论基础和实验依据。.成功构建稳定的体内外LPS诱导的ALI模型后，肺组织及原代肺泡巨噬细胞中LILRB4的表达显著上调并呈现时间依赖性。与WT组相比，LILRB4敲除小鼠肺组织损伤程度、肺通透性及炎症反应均显著恶化。在γ照射后对WT和LILRB4 KO小鼠进行骨髓移植。与WT-WT组相比，KO-WT组BALF中的总蛋白、IgM含量及总细胞数均显著升高；分离出原代肺泡巨噬细胞和原代骨髓源性巨噬细胞进行体外实验，与WT组相比，LILRB4 KO组中炎症因子的表达增加。这表明LILRB4对ALI中炎症反应的调控作用依赖于骨髓来源巨噬细胞。体内和体外实验表明，LILRB4缺乏导致的巨噬细胞依赖性炎症反应是由NF-κB信号通路的激活增加介导的。因此，LILRB4可能是ALI疾病预防和治疗的新型靶点。