磷酸酶PHLPP1通过调控自噬促进肺缺血再灌注后肺部炎症及纤维化的作用研究

Autophagy plays an important role in ischemia-reperfusion (I/R) injury, while has different effects in different organs, cells and pathological process of I/R injury. Our previous study has found that autophagy can function as a positive regulator to enhance aseptic inflammatory responses induced by lung I/R injury. In view of the aseptic inflammation and fibrosis are two key pathological processes in lung I/R injury, the further research of the regulatory mechanism underlying autophagy can help us find the measures to regulate and prevent lung I/R injury. We found that the expression of serine/threonine phosphatase PHLPP1 was significantly increased in lung tissue after lung I/R injury through microarray analysis. However, the function of PHLPP1 in lung I/R injury remains unclear. Our data from the preliminary experiments showed that PHLPP1 could induce autophagy in lung tissue after lung I/R injury, which in turn promoted aseptic inflammation responses and fibrosis in injured lung，suggesting that PHLPP1 could regulate lung I/R injury by autophagy. In this project, we will use PHLPP1 knock-out mice to investigate the interactions between PHLPP1 and autophagy or autophagy-related genes, and further reveal the role of these above interactions in the regulation of inflammation responses and fibrosis after lung I/R injury and the underlying mechanism. The primary aim of this study is to clarify key points in the regulation of lung I/R injury by autophagy, which will may provide new theoretical basis for the clinical intervention and treatment of diseases related to organ I/R injury.

自噬在器官缺血再灌注损伤中具有重要功能，但在不同器官、不同细胞及不同病理阶段中自噬发挥的调节作用不尽相同。本课题组前期发现自噬在肺缺血再灌注后发生增加并正向调控损伤后无菌性炎症强度，鉴于无菌性炎症和纤维化是肺缺血再灌注损伤后两个关键的病理阶段，进一步研究该疾病模型中自噬的分子调控机制，对于揭示肺缺血再灌注损伤的调控和干预措施具有重要意义。我们通过肺组织的基因芯片筛选出丝/苏氨酸磷酸酶PHLPP1，发现其在肺缺血再灌注后表达显著增加。我们预实验进一步发现PHLPP1可以上调肺缺血再灌注后自噬发生水平，并且促进损伤后肺部无菌性炎症和纤维化，提示PHLPP1通过调控自噬影响肺缺血再灌注损伤。本项目将利用PHLPP1基因敲除小鼠深入研究PHLPP1对自噬或自噬相关基因的作用及在肺缺血再灌注损伤后炎症及纤维化中的调控机制，揭示自噬调控肺缺血再灌注损伤的关键分子环节，为相关疾病临床干预提供理论基础。

肺缺血再灌注损伤经常发生在肺移植、失血性休克等多种情况下，其相关机制和治疗方法尚待进一步深入研究。本项目研究了磷酸酶PHLPP1对肺损伤后炎症反应及纤维化发生发展的影响和作用机制。发现在肺缺血再灌注小鼠模型中，PHLPP1缺陷能够显著抑制肺损伤后炎症反应，在后期促进肺纤维化；进一步研究发现，PHLPP1缺失后M2型巨噬细胞的标志基因表达也显著升高，巨噬细胞向促纤维化的表型转变，同时自噬水平受到抑制。同时，我们还发现PHLPP1缺陷能够上调博来霉素诱导的肺组织和肺泡巨噬细胞中EGR1和CXCL1的表达，而EGR1同样能够参与自噬的抑制和巨噬细胞的极化，这提示PHLPP1可能通过直接或间接的方式调控EGR1的转录水平，进而影响巨噬细胞的自噬水平，从而引发巨噬细胞的表型转换，调节CXCL1的分泌，最终促进肺纤维化的发展。本项目深入阐述了PHLPP1在肺缺血再灌注损伤后炎症及纤维化中的调控作用及其分子机制，为肺缺血再灌注损伤的治疗提供潜在的新靶点。另外，在本项目资助下，我们同期研究发现一系列相关成果，包括研究发现miRNA-21通过直接靶向作用于KBTBD7而抑制TLRs信号通路下游p38和NF-κB的活化，进而减轻小鼠心肌梗死后内源性危险物质DAMPs诱导的TLRs触发的心脏炎症反应（Cell Death & Disease. 2018 Jul 10;9(7):769）；研究发现EC-Foxp1通过调节TGF-β1-内皮素-1通路控制病理性心肌纤维化和心肌肥厚，导致心功能不全（Circulation. 2019 Aug 20;140(8):665-680）；研究发现KDM2B与IL-6启动子结合并且招募BRG1和RNA聚合酶II启动和促进IL-6的转录（Cellular & Molecular Immunology. 2020 Aug;17(8):834-842）；研究发现PPM1L能够直接与IKKβ结合，抑制IKKβ的磷酸化，导致NF-κB信号通路激活受损，从而抑制炎症细胞因子的产生，预防心肌梗死后过度炎症和心功能不全（The Journal of Immunology. 2019 Sep 1;203(5):1338-1347）。综上，本项目发表标注本项目资助的SCI论文4篇，已顺利完成所有研究内容和目标。