背景:骨关节炎(OA)是一种以软骨破坏和炎症为特征的退行性关节疾病。CC趋化因子受体1(CCR1),趋化因子家族及其受体家族的成员,在自身免疫反应中起作用。CCR1的特异性小分子抑制剂BX471对软骨中CCR1表达的影响及其对OA的影响仍未充分研究。
方法:本研究使用免疫组织化学(IHC)评估IL-1β诱导的小鼠软骨细胞和内侧半月板失稳(DMM)的内侧半月板小鼠模型中CCR1的表达。用不同浓度的BX471处理24小时的软骨细胞进行IL-1β(10ng/ml)处理。通过蛋白质印迹分析衰老相关基因P16INK4a和P21CIP1的水平,并测量衰老相关的β-半乳糖苷酶(SA-β-gal)活性。诱导型一氧化氮合酶(iNOS)的表达水平,环氧合酶-2(COX-2),aggrecan(AGG),通过蛋白质印迹和RT-qPCR检测转录因子SOX9。胶原蛋白II,基质金属蛋白酶13(MMP13),通过蛋白质印迹分析过氧化物酶体增殖物激活受体(PPAR)-γ的表达,RT-qPCR,和免疫荧光。在PPAR-γ抑制条件下(使用GW-9662)BX471对炎症代谢相关蛋白的影响通过蛋白质印迹法进行检查。通过蛋白质印迹法评估MAPK信号通路相关分子的表达。在体内,将各种浓度的BX471或等效培养基注入DMM模型关节。通过SafraninO/固绿和苏木精-曙红(H&E)染色评价软骨破坏。
结果:这项研究表明,抑制CCR1可以减轻IL-1β诱导的衰老,下调iNOS的表达,COX-2和MMP13,并减轻IL-1β诱导的合成代谢指数下降。机械上,MAPK信号通路和PPAR-γ可能参与抑制CCR1对软骨细胞的保护作用。在体内,BX471在DMM模型中保护软骨。
结论:本研究证实了CCR1在软骨细胞中的表达。抑制CCR1减少炎症反应,缓解软骨老化,通过MAPK信号通路和PPAR-γ,提示其对OA的潜在治疗价值。
BACKGROUND: Osteoarthritis (OA) is a degenerative joint disease characterized by cartilage destruction and inflammation. CC chemokine receptor 1 (CCR1), a member of the chemokine family and its receptor family, plays a role in the autoimmune response. The impact of BX471, a specific small molecule inhibitor of CCR1, on CCR1 expression in cartilage and its effects on OA remain underexplored.
METHODS: This study used immunohistochemistry (IHC) to assess CCR1 expression in IL-1β-induced mouse chondrocytes and a medial meniscus mouse model of destabilization of the medial meniscus (DMM). Chondrocytes treated with varying concentrations of BX471 for 24 h were subjected to IL-1β (10 ng/ml) treatment. The levels of the aging-related genes P16INK4a and P21CIP1 were analyzed via western blotting, and senescence-associated β-galactosidase (SA-β-gal) activity was measured. The expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), aggrecan (AGG), and the transcription factor SOX9 were determined through western blotting and RT‒qPCR. Collagen II, matrix metalloproteinase 13 (MMP13), and peroxisome proliferator-activated receptor (PPAR)-γ expression was analyzed via western blot, RT‒qPCR, and immunofluorescence. The impact of BX471 on inflammatory metabolism-related proteins under PPAR-γ inhibition conditions (using GW-9662) was examined through western blotting. The expression of MAPK signaling pathway-related molecules was assessed through western blotting. In vivo, various concentrations of BX471 or an equivalent medium were injected into DMM model joints. Cartilage destruction was evaluated through Safranin O/Fast green and hematoxylin-eosin (H&E) staining.
RESULTS: This study revealed that inhibiting CCR1 mitigates IL-1β-induced aging, downregulates the expression of iNOS, COX-2, and MMP13, and alleviates the IL-1β-induced decrease in anabolic indices. Mechanistically, the MAPK signaling pathway and PPAR-γ may be involved in inhibiting the protective effect of CCR1 on chondrocytes. In vivo, BX471 protected cartilage in a DMM model.
CONCLUSIONS: This study demonstrated the expression of CCR1 in chondrocytes. Inhibiting CCR1 reduced the inflammatory response, alleviated cartilage aging, and retarded degeneration through the MAPK signaling pathway and PPAR-γ, suggesting its potential therapeutic value for OA.