RNS诱导肺泡巨噬细胞焦亡促进体外循环术后急性肺损伤启动因子HMGB1分泌的机制研究

Acute lung injury (ALI) and its more severe form, the acute respiratory distress syndrome (ARDS), are common complications of cardiopulmonary bypass (CPB) operation. Applicant found that high mobility group box-1 protein (HMGB1) generated during ischemia-reperfusion injury(IRI) could induce ALI. However, the mechanism contributed to HMGB1 secretion during CPB remains unknown. Other studies indicated that macrophage pyroptosis might induce HMGB1 secretion and then aggravate inflammation. Increased synthesis of ROS and NO during IRI may react with each other and result in formation of reactive nitrogen species (RNS). Applicant found that RNS may induce macrophage pyroptosis. Thus, we hypothesized that IRI during CPB induced formation of RNS. Then, RNS actived macrophage pyroptosis that might induce HMGB1 secretion and ALI. At present study, we want to establish pulmonary macrophage IRI model induced by oxygen-glucose deprivation (OGD) and animal ALI model after CPB operation to investigate RNS synthesis, activation of macrophage pyroptosis, HMGB1 accumulation and inflammatory reaction in lung tissue during CPB. RNA interference and other strategies will also be used to interfere RNS formation and macrophage pyroptosis to alleviate inflammatory reaction in lung tissue during the process of ALI after CPB operation. The present study focuses on the activation of macrophage pyroptosis by RNS to investigate the mechanism of ALI after CPB operation, which can provide theoretical foundation for formulating perioperative lung protective strategy for CPB operation and can lay foundation for exploring gene therapy approach for ALI after CPB operation.

急性肺损伤（ALI）是体外循环（CPB）术后的严重并发症。我们已发现CPB能引起HMGB1分泌进而诱发ALI，但机制未明。巨噬细胞焦亡能引起HMGB1分泌，并相互促进放大炎性反应。CPB术后缺血再灌注损伤（IRI）可引起活性氧（ROS）及一氧化氮（NO）释放，两者极易相互作用形成氧化能力更强的活性氮（RNS）。我们发现RNS可能诱导肺泡巨噬细胞焦亡，由此推测：CPB术后因IRI产生的ROS与NO能生成RNS并诱导巨噬细胞焦亡，致HMGB1释放和ALI。本课题利用大鼠CPB模型以及肺泡巨噬细胞氧糖剥夺模型，深入研究CPB术后HMGB1异常分泌的机制，明确RNS诱导肺泡巨噬细胞焦亡在以上过程中的作用。并通过抑制ROS和NO合成以阻断RNS生成，抑制细胞焦亡，进而减少HMGB1分泌，并改善CPB术后ALI。本课题有望从细胞焦亡的角度进一步探讨CPB术后ALI的发病机制，为该病的有效防治奠定基础。

研究背景: 急性肺损伤（ALI）是体外循环（CPB）术后的严重并发症。我们已发现CPB能引起HMGB1分泌进而诱发ALI，但机制未明。巨噬细胞焦亡能引起HMGB1分泌，并相互促进放大炎性反应。因此本研究聚焦肺泡巨噬细胞焦亡与HMGB1异常分泌的作用机制研究。研究方法: 1) 构建大鼠CPB术后ALI模型及肺泡巨噬细胞系（NR8383）氧糖剥夺模型，观察肺泡巨噬细胞焦亡及HMGB1异常分泌的情况。2) 通过应用ASCsiRNA特异性阻断焦亡的关键蛋白ASC表达，探讨巨噬细胞焦亡导致HMGB1异常释放的机制。3) 采用NLRP3炎性小体抑制剂Glyburide抑制巨噬细胞焦亡中NLRP3炎性小体活化，探讨巨噬细胞焦亡导致HMGB1异常释放的机制。4) NLRP3炎性小体抑制剂Glyburide抑制CPB术后肺组织NLRP3炎性小体活化，探讨巨噬细胞焦亡导致HMGB1释放及CPB术后ALI的机制研究。研究结果：1) 氧糖剥夺导致肺泡巨噬细胞焦亡关键蛋白ASC、 Caspase-1活性体(Caspase-1 P10)、炎性因子IL-1β的蛋白表达显著增高。2) CPB术后肺组织中焦亡关键蛋白ASC、Caspase-1活性体(Caspase-1P10)及炎性因子IL-1β的蛋白表达显著增高和肺泡灌洗液中肺泡巨噬细胞焦亡比例显著增加。3) NLRP3抑制剂Glyburide及ASCsiRNA可以抑制氧糖剥夺导致巨噬细胞焦亡及HMGB1异常释放。4) NLRP3抑制剂Glyburide可以抑制CPB术后肺组织中NLRP3/ASC炎性体活化及HMGB1异常释放减轻ALI。结论: CPB引起肺泡巨噬细胞中NLRP3/ASC炎性小体活化导致肺泡巨噬细胞焦亡放大HMGB1异常释放进而引起CPB术后ALI。本研究为调控肺泡巨噬细胞HMGB1异常释放及减轻CPB术后ALI提供了新的治疗方向，也为该病防治手段的探索奠定基础。