HIF-1α调控PD-1介导肺泡巨噬细胞极化在肺缺血再灌注损伤中的作用机制研究

Lung ischemia-reperfusion injury (LIRI) has a high incidence, and the mechanism remains unclear. Our previous studies had shown that the hypoxia-ischemia could cause severe lung tissues injury after a long period of single-lung ventilation, during which process the expression of HIF-1α and inflammatory mediators had increased. PD-1 was screened out by iTRAQ to be associated with perioperative LIRI. In order to further explore the mechanisms of LIRI, we propose the hypothesis that PD-1 might induce the polarization of alveolar macrophages regulated by HIF-1α, which can lead to the release of inflammatory mediators with different functions, and ultimately affect the extent and prognosis of lung tissues damage in LIRI. To verify the hypothesis, we will investigate at the molecular, cellular, tissue and animal level to clarify the effects of PD-1-mediated the polarization of alveolar macrophages regulated by HIF-1α in the LIRI through the establishment of I / R animal and cell models, the preparation of overexpression and silencing PD-1, HIF-1αof alveolar macrophages. We will also explore in depth the molecular mechanisms of PD-1-mediated the polarization of alveolar macrophages in LIRI. This study will reveal the new mechanisms of LIRI and provide new ways of prevention and treatment of LIRI.

肺缺血再灌注损伤的发病率高，且机制尚未明确。我们前期研究显示长时间单肺通气时缺氧缺血肺组织损伤严重、HIF-1α及炎症介质表达增高，通过iTRAQ技术筛选并验证了PD-1与围术期肺I/R损伤相关；为进一步探讨其损伤机制，我们提出假说：HIF-1α调控PD-1介导肺泡巨噬细胞极化，导致不同功能的炎性介质释放，最终影响肺I/R损伤的程度和转归。为验证假说，本项目通过建立I/R的动物和细胞模型、制备过表达和沉默PD-1、HIF-1α的肺泡巨噬细胞，从分子、细胞、组织及动物整体水平等多层次阐明PD-1介导的肺泡巨噬细胞极化在肺I/R损伤及修复过程中的作用，以及此过程中HIF-1α对PD-1的调控作用，并深入探讨PD-1介导肺泡巨噬细胞极化的上下游分子调控机制。本研究将从HIF-1α调控PD-1介导肺泡巨噬细胞M1/M2极化这一新视点为揭示肺I/R损伤的新机制奠定基础，为肺I/R损伤防治提供新思路。

肺缺血再灌注损伤（LIRI）的发病率高，危害大，其发生机制与炎症反应爆发密切相关，但是具体调控机制尚未明确。我们前期研究显示长时间单肺通气时缺氧缺血肺组织损伤严重、HIF-1α及炎症介质表达增高，通过iTRAQ技术筛选并验证了PD-1与围术期肺I/R损伤相关；本项目旨在探讨PD-1参与LIRI的具体机制及其干预手段。我们构建了缺血再灌注损伤的动物及肺泡巨噬细胞模型；首先，应用PD-1抑制剂、SHP1 / 2抑制剂、PI3K/AKT通路抑制剂、构建shRNA-PD1慢病毒载体等，明确PD-1参与了大鼠肺缺血再灌注损伤，并进一步证实了PD-1通过激活SHP1和SHP2抑制PI3K/AKT通路的激活，进而促进巨噬细胞向M1极化，从而在肺缺血再灌注损伤中发挥重要作用；其次，明确肺缺血再灌注后，HIF-1α介导Keap1/Nrf-2/HO-1通路调控 PD-1的表达，rHMGB1预处理通过逆转此过程，从而起到保护LIRI的作用；此外，我们探讨了不同药物对肺缺血再灌注损伤发生机制的干预和治疗效果及其机制：（1）乌司他丁可以缓解肺缺血再灌注损伤，降低肺血气屏障通透性，其机制可能与减弱p38 MAPK信号通路的激活有关；（2）地塞米松可能通过激活PI3K/AKT通路介导巨噬细胞极化来降低肺组织中的炎症反应来减轻大鼠肺缺血再灌注后的肺损伤；（3）甘草素预处理可以减轻缺血再灌注肺损伤，其保护作用的机制可能是通过下调肺泡巨噬细胞TLR2 信号级联进而抑制肺缺血再灌注诱导的炎症反应；（4）眼镜蛇毒因子（CVF）诱导的补体耗竭可以抑制I/R诱导的炎性反应，减轻LIRI，其保护作用的机制可能是通过改善血-气屏障损伤。本研究从HIF-1α调控PD-1介导肺泡巨噬细胞M1/M2极化这一新视点揭示了LIRI损伤的新机制，并为LIRI的防治手段和药物进行了有益的探索，为临床转化奠定了基础。