LXRβ调控胆固醇转运在铜绿假单胞菌致急性肺损伤肺部炎症中的作用及机制研究

Toll-like receptor 4(TLR4)- mediated inflammation response in alveolar macrophages caused by pseudomonas aeruginosa was involved in the development of acute lung injury. LXRβ regulated TLR4-mediated inflammatory response, and may be involved in the regulation of acute lung injury caused by pseudomonas aeruginosa. Previous studies have shown that, in the model of LPS induced acute lung injury, elevated LXR expression in macrophages lipid rafts reduced cholesterol content, disrupted lipid rafts and reduced TLR4-mediated inflammatory response. Since TLR4 translocation to lipid raft is a necessary condition for Pseudomonas aeruginosa initiating downstream inflammatory response, therefore, we hypothesized that LXRβ may reduce the level of cholesterol in lipid rafts by increasing cholesterol delivery, reduce translocation of TLR4 to lipid raft, and further reduce pulmonary inflammatory response caused by Pseudomonas aeruginosa. On the basis of previously study, the goal of this project is to confirm the role of LXRβ in the acute lung injury pulmonary inflammation caused by pseudomonas aeruginosa. Furthermore, with the experiment of cholesterol-efflux and cholesterol supplementation, the mechanisms for LXRβ regulatinginflammatory injury in alveolar macrophages caused by pseudomonas aeruginosa will be clarified. If successful, our results will contribute to elucidate the underlying mechanism for regulation of LXBβ on the acute lung injury caused by pseudomonas aeruginosa and provide a new target for treating acute lung injury.

TLR4介导的铜绿假单胞菌对肺泡巨噬细胞致炎作用参与急性肺损伤发生发展。LXRβ调控TLR4介导炎症反应，可能参与调控铜绿假单胞菌致急性肺损伤。既往研究表明在LPS所致急性肺损伤中，上调LXR的表达巨噬细胞脂筏中胆固醇减少，脂筏被破坏，TLR4介导的炎症反应减轻。由于TLR4易位至脂筏是铜绿假单胞菌启动下游炎症反应的必要条件，因此我们推测LXRβ可能通过增加胆固醇向胞外转运，降低脂筏中胆固醇水平，减少TLR4向脂筏易位从而减轻铜绿假单胞菌所致肺部炎症反应。在此基础上，本项目采用LXRβ敲除小鼠，探讨LXRβ在铜绿假单胞菌所致急性肺损伤小鼠肺部炎症中的作用；分离小鼠巨噬细胞脂筏，采用胆固醇外流、胆固醇补充等实验，明确LXRβ调控铜绿假单胞菌所致肺泡巨噬细胞炎症的作用机制。本研究可阐明LXRβ在铜绿假单胞菌所致ARDS中的作用及可能的机制，为治疗铜绿假单胞菌所致急性肺损伤提供新的作用靶点。

TLR4介导的铜绿假单胞菌对肺泡巨噬细胞致炎作用参与急性肺损伤发生发展。 LXRβ调控TLR4介导炎症反应，可能参与调控铜绿假单胞菌致急性肺损伤。既往研究表明上调LXR的表达巨噬细胞脂筏中胆固醇减少，TLR4介导的炎症反应减轻。因此推测LXRβ可能通过增加胆固醇向胞外转运，降低脂筏中胆固醇水平，减少TLR4向脂筏易位从而减轻铜绿假单胞菌所致急性肺损伤肺部炎症。本项目首先通过RT-PCR检测了各小鼠肺组织和RAW264.7细胞中LXRa表达情况，发现LXRa在LPS诱导的急性肺损伤小鼠肺组织及RAW264.7细胞中表达下调。给予LXR激动剂T0901317后LPS诱导的急性肺损伤小鼠肺组织肺部炎症减轻、小鼠肺组织中炎症因子TNFa以及RAW264.7细胞炎症因子TNFa和IL6表达降低。免疫荧光、Western Blot、电镜均提示LPS能够增加自噬在小鼠肺组织中的表达。进一步采用自噬抑制剂抑制自噬的表达，发现抑制自噬保护LPS诱导的急性肺损伤肺泡屏障功能，提示自噬过程可能参与调控LPS诱导急性肺损伤。电镜结果进一步证实给予LXR激动剂T0901317后LPS诱导的小鼠肺组织以及小鼠巨噬细胞系RAW264.7细胞中自噬小体减少。Western blot进一步证实发现给予LXR激动剂T0901317后LPS诱导的小鼠巨噬细胞RAW264.7细胞中LC3、P62以及NF-kB p65表达降低。本项目明确了1、激活LXR对LPS诱导的急性肺损伤小鼠起保护作用；2.自噬在LPS诱导的急性肺损伤肺组织中表达增加，抑制自噬保护LPS诱导的急性肺损伤肺泡屏障功能；3. LXR对急性肺损伤的保护作用可能是通过调节自噬过程和NF-KB介导的。这一结果可能为ARDS 的治疗提供一个新的靶点。