慢性间歇性低氧活化NLRP3炎症小体的促癌作用及机制研究

Obstructive sleep apnea (OSA) is a common disorder characterized by repetitive chronic intermittent hypoxia (CIH) ventilation during sleep. Recently, several studies have demonstrated that patients with OSA have a higher prevalence of cancer and cancer-related mortality. CIH caused oxidative stress and inflammatory reaction, induced downstream products, and promoted tumor metastasis. NLRP3 inflammasome is a large cytosolic multi-protein complex and results in the activation of inflammatory responses. It is found that the serum IL-1β and IL-18 in patients with OSA and cancer are higher than in health controls. Based on these studies, we hypothesis that CIH can promote cancer progression by activating NLRP3 inflammasome through ROS/TXNIP pathway. To investigate biological role of NLRP3 engaged in CIH-induced tumor progression，we are going to use NLRP3 mutant mice to analyze the effect of NLRP3 downregulation in a mouse model of CIH-induced tumor. To investigate the molecular pathways of NLRP3 inflammasome activation, this project is going to use biology techniques to analyze the effect of NLRP3 downregulation or overexpression on ROS signaling in vitro CIH-induced cell model. The study will provide a new therapeutic target to prevent the occurrence of tumors and effective interventions in the treatment of cancer.

OSA人群的肿瘤发病率死亡率高于正常人群，其中肺癌病死率最高，且与OSA的低氧程度及持续时间相关，但机制不明确。OSA主要病理特征为间歇低氧IH，缺氧复氧过程产生大量ROS，引起氧化应激和炎症反应，持续炎症反应可促进肿瘤进展。NLRP3炎症小体是启动和参与炎症反应的核心环节，前期研究发现，OSA合并肺腺癌者血清IL-1β和IL-18高于单纯肺腺癌及健康者，NLRP3在肺腺癌中高表达，活化后促进腺癌细胞增殖迁移。我们推测“间歇低氧过程可激活ROS/TXNIP通路活化NLRP3炎症小体，促进肺腺癌进展”。本项目拟整合小鼠IH模型和肺癌模型，利用NLRP3-/-小鼠研究NLRP3敲除后IH模型下小鼠肺癌的进展，研究mROS清除剂及siTXNIP阻断信号通路后IL-1β和IL-18的表达，明确NLRP3在IH促癌过程中的作用及分子机制，为研究靶向NLRP3药物预防治疗OSA合并肺腺癌提供理论基础。

流行病学研究发现，阻塞性睡眠呼吸暂停综合征（Obustructive Sleep Apnea Syndrom，OSAS）人群的肿瘤发病率和病死率高于正常人群，其中肺癌的病死率最高，且与OSA的低氧程度及持续时间相关，但目机制尚不明确。. 本项目从在体和离体两个方面研究了NLRP3炎症小体活化在OSA模式IH中促进癌症细胞的增殖和转移的作用。首先，OSAS患者血清中IL-1β、IL-18明显高于健康对照者，提示OSA模式的慢性间歇性低氧（intermittent hypoxia，IH）可以活化NLRP3炎症小体；OSAS合并肺癌的患者血清中IL-1β和IL-18明显高于单纯肺腺癌患者，提示NLRP3炎症小体活化可能参与了OSAS患者肿瘤的发展。其次，动物在体水平，通过原位肿瘤模型和皮下成瘤构建小鼠肺癌模型，通过病理、免疫组化、ELISA等观察了OSA模式IH对肿瘤生长、转移的影响。使用NLRP3过表达和敲低的肿瘤细胞株，通过活体肿瘤荧光成像技术研究小鼠肺腺癌细胞在体内瘤块形成的大小、胸腔内及全身转移的情况。结果表明OSA模式IH促进小鼠肿瘤生长及侵袭，该过程中NLRP3炎症小体活化发挥了作用。再次，细胞离体水平，通过免疫荧光、免疫印迹、EdU、Transwell、划痕实验等发现了IH可以促进正常支气管上皮发生EMT，使肿瘤细胞增殖、迁移能力增强。通过qPCR、免疫共沉淀等发现这一现象与NLPR3炎症小体的活化有关。然后，进一步深入机制研究，发现OSA模式IH通过TLR9/NF-κB信号通路激活NLRP3炎症小体，促使IL-1β和IL-18分泌到细胞外，参与炎症反应，促进肿瘤进展。. 综上所述，本项目明确了NLRP3炎症小体活化在OSA模式IH促癌过程中的功能及其作用机制，对研究开发靶向调控NLRP3炎症小体的药物，治疗OSAS合并肺腺癌者，提供一定的理论基础。