内质网应激相关IRE1/XBP-1信号通路调控关节挛缩的作用机制研究

Joint contracture is a common post-arthritis, trauma and operative complication. The pathogenesis of joint contracture is still obscure. Our preliminary experimental results demonstrated that chaperone CCT-7 and GRP78 from the contractive tissue were increasingly expressed with the relation to the severity of joint contracture, indicating that endoplasmic reticulum stress (ERS) would be involved in the pathogenesis of joint contracture. Furthermore, we figured out that XBP-1 as well as ROS levels were increasingly expressed in joint contractive tissue. Since IRE1/XBP-1 signaling pathway is the crucial pathway in ERS, we infer that IRE1/XBP-1 signaling pathway plays an important role in regulating the joint contracture. Based on our previous study, we will systematically compare the variation of fibrous markers, ERS markers and IRE1/XBP-1 signaling pathway expressions in human joint contractive tissue with technics including immunoprecipitation and plasmid vector transfection. Furthermore, we will discuss the effects of IRE1/XBP-1 signaling pathway on fibroblast function, and the effects of acetylation modification of XBP-1 in joint contracture. The interactions of ERS and ROS will also be analyzed to clarify the mechanisms of regulation in joint contracture by XBP-1, and the effects of ERS in the pathogenesis of joint contracture. New data from these experiments, not only help to elucidate the mechanisms of joint contracture, but also shed new light on the biological treatment for joint contracture.

关节挛缩是关节炎症、创伤及术后常见的并发症，目前发病机制未明确。我们的前期研究表明，分子伴侣CCT-7和GRP78在关节挛缩组织中高表达，提示内质网应激可能参与关节挛缩的发病；进一步研究发现，关节挛缩组织中XBP-1蛋白高表达，且氧化应激也处于高水平，而IRE1/XBP-1信号通路是内质网应激的关键通路。据此，我们提出IRE1/XBP-1信号通路可能参与关节挛缩的调控。本研究拟采用免疫共沉淀、慢病毒转染等方法，探讨IRE1/XBP-1信号通路对成纤维细胞功能的影响，研究XBP-1乙酰化/去乙酰化表观修饰对成纤维细胞活化程度的调控在挛缩进程中发挥的作用，观察在关节挛缩病程中内质网应激与氧化应激的交互作用形式，明确XBP-1通过表观修饰调控关节挛缩的分子机制，阐明内质网应激在关节挛缩发病中的作用模式，为探索治疗关节挛缩的新靶点提供科学依据。

获得性关节挛缩是关节外科常见并发症，其发病机制尚未完全阐明，亦缺乏可行的药物治疗手段。国际研究提示滑膜成纤维细胞(SF)的活化在滑膜关节挛缩进程发挥重要作用，但涉及具体分子机制尚未明确。既往研究发现内质网应激（ERS）相关的未折叠蛋白反应（UPR）参与成纤维细胞的激活过程，但在关节挛缩进程中的具体功能尚未被报道。我们希望通过研究UPR在SF活化过程的具体作用，揭示关节挛缩涉及的分子机制及可能的治疗靶点。为此，本研究课题通过临床标本获取的膝关节挛缩关节囊-分离培养SF，同时利用改进的膝关节挛缩大鼠模型，通过分子生物学方法研究关节挛缩进程中（1）滑膜关节挛缩发生SF活化的UPR具体通路；（2）进一步明确ERS激活的来源分子机制；（3）深入探索SF激活时UPR下游可能的表观修饰调控。本项目主要研究结果包括：（1）利用微创技术改良现有的内置固定物诱导获得性关节挛缩模型；（2）体外实验明确参与SF活化的UPR关键分子ATF4，通过ATF4/NOX4信号通路调控SF的激活；（3）TMT蛋白组学及分子研究提示O-GalNAc糖基化可能是UPR下游的表观修饰调控。本课题为探索内质网应激ERS相关UPR通路参与获得性关节挛缩的SF活化提供基础证据，对可能涉及的表观修饰信号做出探索，为今后的治疗靶点开发提供新的思路。

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