NLRP3炎症小体活化加重TLR4介导的急性肺损伤及其机制研究

Critical incidence feature of acute lung injury (ALI) is characterized by excessive and uncontrolled inflammatory response，but the mechanisms of uncontrolled inflammation are not yet fully elucidated.Recent literatures and our previous work has shown that complex interactions between Toll like receptor 4 (TLR4) signal pathway and NOD-like recptor（NLR) signal pathway cause inflammation development of out-of-control direction in ALI.Recent studies also suggest that TLR4-mediated NLRP3 inflammasome activation induced inflammatory response,but it is still unclear whether NLRP3 inflammasome activation will encourage the TLR4 and NLR pathways dialogue,thereby exacerbate pulmonary inflammation and injury.In the present study,we firstly research the impact of the regulation of NLRP3 expression on TLR4 signal pathway and alveolar macrophage inflammatory response by using over-expression and RNA interference technique. Secondly,we use LPS-induced mouse model of ALI to study the different function of NLRP3 inflammasome at different time point and under different intervention,as well as clarify the relationship between NLRP3 inflammasome activation and lung injury.By blocking TLR4 signal pathway,we further observe the impact of TLR4 pathway changing on NLRP3 inflammasome activation in vitro and in vivo.Taken toghther,we will explore the mechanism of NLRP3 inflammasome activation involved in the TLR4 pathway mediated ALI from multiple dimensions.Results of this study may provide some experimental data and a basis for looking for ways to control the ALI inflammation runaway.

急性肺损伤(ALI)的关键发病特征是失控的炎症反应，但炎症失控的机制尚未完全阐明。近年文献报道和我们的前期工作表明ALI中Toll样受体4(TLR4)通路与NOD样受体(NLR)通路间存在复杂的相互作用，导致炎症向失控的方向发展。又有研究提示TLR4介导的NLRP3炎症小体活化参与了炎症反应，但目前尚未明确NLRP3炎症小体活化是否会促使TLR4和NLR通路对话，加重肺内炎症和损伤。因此本课题首先采用过表达和RNAi技术，研究NLRP3对肺泡巨噬细胞TLR4通路和炎症反应的调控作用；然后通过LPS诱导小鼠ALI模型，研究不同时间节点和不同干预措施下NLRP3炎症小体活化与肺损伤的相关性；进一步通过阻断TLR4通路，用体内外研究方法观察TLR4通路改变对炎症小体活化的影响。从多个层面探讨NLRP3炎症小体活化参与TLR4途径介导ALI炎症失控的机制，为寻找控制ALI炎症失控的方法提供依据。

肺泡巨噬细胞（AMs）是识别PAMPs和启动肺部内源性免疫防御的主要效应细胞。NLRP3炎症小体活化促使TLR4与NLR两条通路对话，加重肺内炎症反应，促使炎症向失控方向发展。在前期研究基础上，本研究通过体外培养大鼠肺泡巨噬细胞NR8383和小鼠巨噬细胞RAW264.7 ，经1μg/ml LPS刺激细胞6h，采用q RT-PCR、Western blot、流式细胞术、免疫荧光和激光共聚焦等方法检测NLRP3、caspase-1、IL-1β、TLR4、MD-2、MyD88和NF-κB P65 mRNA和/或蛋白的表达；进一步分别采用MD-2 shRNA、MD-2 pCDH、NLRP3 shRNA、NLRP3 pCDH、caspase-1抑制剂AP-IEC-1和NF-κB抑制剂SN50处理NR8383细胞，观察其对炎症小体活化和细胞炎症的调节作用。结果显示，LPS刺激NR8383和RAW264.7细胞后，NLRP3、caspase-1、IL-1β、TLR4、MD-2、MyD88和NF-κB P65的表达均明显上调，并增加细胞焦亡。MD-2、NLRP3基因敲除抑制LPS诱导的NR8383细胞炎症反应，减少NLRP3和MyD88/NF-κB信号通路活化。相反，MD-2、NLRP3基因过表达加重LPS诱导的NR8383细胞炎症反应。SN50抑制NLRP3、caspase-1表达和炎症小体活化，AP-IEC-1抑制NLRP3炎症小体活化和减少IL-1β合成。动物实验部分，本研究采用C57BL/6小鼠经气道内滴入5mg/kg LPS复制ALI模型，检测肺泡灌洗液中炎症细胞计数和IL-1β浓度、肺组织病理形态学改变、TLR4/NF-κB以及NLRP3炎症小体信号通路相关分子的表达。结果显示：模型组BALF中炎症细胞数明显增多，IL-1β浓度增高，肺组织病理损伤加重，MD-2、TLR4、NLRP3、caspase-1p10 mRNA和蛋白表达均上调。上述结果表明在LPS诱导的AMs炎症及ALI中，NLRP3炎症小体活化加重TLR4途径介导的AMs炎症和肺损伤，抑制MD-2-TLR4/NF-κB通路可减轻NLRP3炎症小体活化及肺损伤。该研究初步明确NLRP3炎症小体活化是TLR4和NLR通路对话的关键环节，为进一步阐明ALI炎症失控的机制和调控方法提供依据，为治疗ALI提供新方法。