未折叠蛋白反应关键分子ATF6α在类风湿关节炎中的功能及分子机制研究

The abnormal apoptosis and inflammation are the characteristics of rheumatoid arthritis fibroblast-like synoviocytes (RA FLS). Unfolded protein response (UPR) induced by endoplasmic reticulum stress has been shown to regulate these cell characteristics. Our previous studies have confirmed that molecular chaperone TXNDC5 is a pathogenic factor of RA. ATF6α, the component of UPR, is the upstream regulator of TXNDC5. However, the relationship between ATF6α and RA is unknown. Recently, we found that the expression and localization of ATF6α in RA FLS were regulated by inflammatory factors; silencing ATF6α inhibited the proliferation of RA FLS and increased the apoptosis; RNA-seq and bioinformatics analysis showed that ATF6α activity was significantly correlated with classical signaling pathways such as NF-κB. Therefore, we proposed a scientific hypothesis that ATF6α regulates the abnormal apoptosis and inflammatory response of RA FLS through NF-κB and other signaling pathways, and thus participates in the pathological process of RA. In this study, RA FLS and ATF6α knockout mice were used to clarify the effect of ATF6α on the pathological process of RA; NF-κB was used as a breakthrough to clarify its mechanism; and evaluate the effect of ATF6α inhibitor on the occurrence and development of arthritis. The study will provide a new target and basis for the treatment of RA from the point of view of UPR.

类风湿关节炎成纤维样滑膜细胞（RA FLS）具有凋亡异常和炎性反应过度的特点，而内质网应激引起的未折叠蛋白反应（UPR）可调控其上述特性。我们前期研究证实内质网分子伴侣TXNDC5是RA致病因子，而UPR组分ATF6α正是其上游调控分子，但ATF6α与RA关系未知。最近，我们发现ATF6α在RA FLS中的表达和定位受炎性因子调控；沉默ATF6α可导致RA FLS增殖受阻和凋亡增加；RNA-seq与生物信息学分析显示，ATF6α活性与NF-κB等经典信号通路显著相关。因此，我们提出“ATF6α通过NF-κB等信号通路调控RA FLS凋亡异常和炎性反应，进而参与RA病变过程”的科学假说。本课题拟以RA FLS与ATF6α敲除小鼠为模型明确ATF6α对RA病变过程的影响；以NF-κB为突破口阐明其作用机制；评价ATF6α抑制剂对关节炎发生发展的影响，为从UPR角度治疗RA提供新的靶点和依据。

在类风湿关节炎(RA)病理过程中，滑膜成纤维细胞(FLSs)表现出炎性反应、侵袭和凋亡异常的活性表型，是导致滑膜增生及软骨破坏的关键效应细胞。鉴于未折叠蛋白反应（UPR）在细胞活性调控中发挥重要作用，本项目聚焦UPR关键组分ATF6α在RA病变中的功能特点展开了研究。本项目研究发现，ATF6α在RA FLSs中的表达显著高于骨关节炎(OA)FLSs,通过沉默ATF6α的表达或者采用抑制剂抑制其活性可以显著降低RA FLSs炎性因子的表达并促进内质网应激下RA FLSs凋亡，表明ATF6α在RA FLSs发挥促炎和抗凋亡的作用。通过构建ATF6敲除小鼠以及胶原诱导的关节炎 (CIA) 模型，发现ATF6α敲除后可明显缓解关节炎的发生和发展。采用ATF6α抑制剂干预CIA后，其滑膜增生及骨侵蚀破坏得到改善，并表现出与TNF阻断剂-依那西普协同治疗效果。转录学测序及CHIP实验证实，BIRC3为ATF6α的直接靶点，BIRC3作为凋亡抑制因子也参与炎症反应，沉默BIRC3的表达可明显削弱ATF6α对RA FLSs的促炎及抗凋亡作用。因此，本项目证实了ATF6α是RA病变过程中的关键致病分子，并可作为RA潜在的治疗靶点。另外，本项目还进行了研究内容的扩展。通过靶向GRP78调控UPR活性可作为RA的治疗靶点干预CIA小鼠关节炎进展，另外，鉴于RA FLSs活化在RA进展中的重要性，本项目开发了可无创体内监测RA FLSs活化的同位素示踪剂，并利用中药小分子异补骨脂素进一步研究了抑制RA FLSs活性在干预RA进展中的潜力。