PGC1β通过线粒体氧化磷酸化/ROS通路调控类风湿关节炎成纤维样滑膜细胞促炎及关节侵蚀的分子机制

Fibroblast-like synoviocytes (FLS) are critical for joint destruction of rheumatoid arthritis (RA) through mediating inflammation, cartilage degradation and bone erosion. However, little was known about related regulating mechanism. We previously reported that peroxisome proliferator-activated receptor gamma coactivator (PGC)1β, a transcription factor, promoted pro-inflammatory effect, migration, invasion of RA-FLS and inhibited their apoptosis. Preliminary experiments of microarray analysis and KEGG pathway analysis on lentiviral shRNA-PGC1β transfected RA-FLS identified mitochondrial oxidative phosphorylation (OXPHOS) as the most differentially regulated gene set of PGC1β. We also found that PGC1β can increase the production of reactive oxygen (ROS) and activate NF-κB pathway in RA-FLS. Therefore, our hypothesis is that elevated PGC1β in RA-FLS will promote mitochondrial OXPHOS and increase ROS level, leading to pro-inflammatory effect and joint destruction by activating NF-κB pathway. In this project, we plan to perform cross-sectional and prospective clinical studies, up or down regulating PGC1β in RA-FLS for mitochondrial OXPHOS/ROS analysis, drug intervention of ROS level for NF-κB phosphorylation and RA-FLS functional experiments, and intra-articular injection of lentiviral shRNA suppressing PGC1β in CIA mice for treatment. We aim to determine whether PGC1β promotes RA-FLS pro-inflammatory effect and joint destruction by regulating mitochondrial OXPHOS and increasing ROS to activate NF-κB pathway, and whether blockage of PGC1β can inhibit joint inflammation and destruction in RA, in order to develop a new therapeutic target for RA.

成纤维样滑膜细胞（FLS）促炎及关节侵蚀是造成类风湿关节炎（RA）关节炎症及破坏的重要原因，但调控机制不清。我们前期报道转录因子PGC1β促进RA-FLS促炎、迁移和侵袭且抑制凋亡。预实验表达谱芯片分析发现PGC1β主要调控线粒体氧化磷酸化（OXPHOS），且发现PGC1β促进RA-FLS活性氧（ROS）产生，激活NF-κB通路。因此我们提出RA-FLS中上调的PGC1β通过调控线粒体OXPHOS增加ROS水平激活NF-κB通路从而促进RA-FLS促炎及关节侵蚀的假说。本项目拟通过临床横断面及前瞻性研究、RA-FLS体外实验基因调控PGC1β、药物干预ROS水平及动物实验CIA小鼠关节腔注射慢病毒shRNA抑制PGC1β，明确PGC1β是否通过线粒体OXPHOS/ROS激活NF-κB通路促进RA-FLS促炎及关节侵蚀及阻断PGC1β能否抑制RA关节炎症及破坏，为开发RA治疗药提供新靶点。

成纤维样滑膜细胞（FLS）促炎及关节侵蚀是造成类风湿关节炎（RA）关节炎症及破坏的重要原因，但调控机制不清。新近研究发现物质和能量代谢可影响胞内信号的传递，调控细胞活化及功能改变。线粒体氧化磷酸化是细胞内物质转化为能量的主要环节，其产生的活性氧（ROS）是细胞信号传导的重要物质。我们前期报道调控代谢的转录因子PGC-1β促进RA-FLS促炎、迁移和侵袭且抑制凋亡。预实验表达谱芯片分析发现PGC-1β主要调控线粒体氧化磷酸化，且发现PGC-1β促进RA-FLS产生ROS，激活NF-κB通路。因此，本项目拟明确RA-FLS中上调的PGC-1β是否通过调控线粒体氧化磷酸化增加ROS水平激活NF-κB通路，从而促进RA-FLS促炎及关节侵蚀，阐明PGC-1β调控RA-FLS的具体分子作用机制，明确阻断PGC-1β能否有效抑制RA关节破坏。..本项目首先通过临床研究纳入确诊RA患者，发现RA患者膝关节滑膜PGC-1β表达升高并与滑膜局部及全身炎症水平、关节破坏程度呈正相关，提示PGC-1β可能参与RA滑膜炎及关节破坏过程。进一步体外实验通过慢病毒转染基因上调或下调RA-FLS中PGC-1β的表达，发现PGC-1β可上调RA-FLS呼吸链复合体I-V的活性及ATP水平，同时促进ROS生成。体外细胞功能实验结合补救实验发现PGC-1β通过调控RA-FLS中ROS水平，激活NF-κB信号通路，促进RA-FLS迁移和侵袭。动物实验构建免疫缺陷NCG小鼠模型，通过植入慢病毒shRNA-PGC-1β转染RA-FLS的软骨-海绵“三明治”移植物，组织病理学检查分析PGC-1β对RA-FLS侵袭软骨的调控作用，发现下调PGC-1β可抑制RA-FLS的软骨侵袭作用。研究结果明确了PGC-1β通过调控线粒体氧化磷酸化/ROS激活NF-κB通路促进RA-FLS的关节侵蚀作用，阐明了PGC-1β调控RA-FLS的具体分子作用机制，并初步探讨了阻断PGC-1β抑制RA关节破坏的作用。..本项目按研究计划完成，解决了提出的关键科学问题，达到了研究目标。从免疫代谢的角度阐明了RA-FLS 线粒体能量代谢的特点及其调控机制，结合临床研究、体外实验及动物实验全面验证了转录因子PGC-1β通过调控ROS激活NF-κB信号通路促进RA-FLS迁移、侵袭，最终导致关节破坏，为今后开发RA 的治疗药物提供新靶点。