目的:κ阿片受体(KOR)信号参与骨关节炎(OA)的关节发育和炎症,而生化机制仍未阐明。本研究旨在探讨KOR激活的下游分子事件,为OA病理学提供新的观点。
方法:U50,488H,选择性KOR激动剂,作为OA模型,在内侧半月板(DMM)失稳后关节内注射小鼠,以PBS注射作为对照。通过热板试验和红色固体绿色染色评估行为和组织学评估,分别。通过RNA-seq评估mRNA和蛋白质表达的变化,RT-qPCR,用TNF-α或TNF-αU50,488H处理的软骨细胞的免疫组织化学和蛋白质印迹(WB)。与KOR相互作用的蛋白质使用邻近标记然后通过质谱进行探索,然后通过共免疫沉淀(Co-IP)测定和免疫荧光(IF)进行验证。
结果:在DMM小鼠中,当KOR激活时,OA诱导的疼痛减轻并且软骨退化减轻。在软骨细胞中,KOR的激活逆转了MMP的上调,IL-6,IL-1β和磷酸化(p-)STAT3,由TNF-α刺激,而NF-κB的表达,MAPK和AKT信号没有逆转。RNA-seq和IF结果表明,在TNF-α刺激下,KOR激活明显减少了软骨细胞中STAT3的核易位。减少可能是由于质膜中KOR和STAT3的结合,通过邻近标签和Co-IP结果显示。
结论:KOR激活保护软骨免受OA,这种保护作用主要是通过螯合STAT3在质膜上发挥的,导致STAT3依赖性免疫反应失活,否则会导致OA。
OBJECTIVE: Kappa opioid receptor (KOR) signaling is involved in joint development and inflammation in Osteoarthritis (OA), while the biochemical mechanism remains unclarified. This study aims to investigate downstream molecular events of KOR activation, to provide novel perspectives in OA pathology.
METHODS: U50,488H, a selective KOR agonist, was intra-articularly injected in mice upon destabilization of the medial meniscus (DMM) as OA models, with PBS injection as control. The behavioral and histological evaluation was assessed by hot plate test and red solid green staining, respectively. Alterations in mRNA and protein expression were assessed by RNA-seq, RT-qPCR, immunohistochemistry and western blotting (WB) in chondrocytes treated with TNF-α or TNF-α + U50,488H. Proteins interacted with KOR were explored using proximity labeling followed by mass spectrometry and then testified by co-immunoprecipitation (Co-IP) assay and immunofluorescence (IF).
RESULTS: OA-induced pain was reduced and cartilage degeneration was alleviated upon KOR activation in DMM mice. In chondrocytes, activation of KOR reversed the upregulation of MMPs, IL-6, IL-1β and phosphorylated(p-) STAT3, stimulated by TNF-α, while the expression of NF-κB, MAPKs and AKT signaling weren\'t reversed. RNA-seq and IF results presented that KOR activation evidently reduced STAT3 nuclear translocation in chondrocytes upon TNF-α stimuli. The reduction may be resulted from the binding of KOR and STAT3 in the plasma membrane, revealed by proximity labeling and Co-IP results.
CONCLUSIONS: KOR activation protects cartilage from OA, and this protective effect is mainly exerted via sequestering STAT3 on the plasma membrane, resulting in inactivation of STAT3-dependent immune responses which otherwise contributes to OA.