S100钙结合蛋白B在骨性关节炎软骨损伤修复中的作用及机制研究

The role and mechanism of S100 calcium binding protein B in osteoarthritis cartilage damage repair

目的探讨S100钙结合蛋白B(S100B)在骨性关节炎(osteoarthritis,OA)软骨损伤修复中的作用及机制。方法取20只新西兰兔随机分为对照组和模型组,每组10只。模型组兔右膝关节制动法制备软骨损伤模型,对照组不作任何处理。4周后采用ELISA法检测关节液IL-1β、TNF-α水平,实时荧光定量PCR(real-time fluorescence quantitative PCR,qRT-PCR)和Western blot检测软骨组织S100B、FGF-2、FGF受体1(FGF receptor 1,FGFR1)基因及蛋白表达。分离培养人滑膜成纤维细胞(synovial fibroblasts,SF),观察过表达、干扰S100B以及拮抗FGFR1对细胞IL-1β和TNF-α水平(ELISA法)以及FGF-2和FGFR1基因(qRT-PCR检测)和蛋白(Western blot检测)表达的影响。结果 ELISA检测示模型组兔关节液中IL-1β和TNF-α表达水平均明显高于对照组(P<0.05);qRT-PCR和Western blot检测示,模型组兔软骨组织S100B、FGF-2、FGFR1 mRNA和蛋白表达量均显著高于对照组(P<0.05)。过表达和干扰S100能够分别显著升高和降低脂多糖(lipopolysaccharides,LPS)诱导的IL-1β和TNF-α水平及FGF-2和FGFR1 mRNA和蛋白表达,差异均有统计学意义(P<0.05)。而拮抗FGFR1能够显著降低LPS诱导的IL-1β和TNF-α水平及FGF-2和FGFR1 mRNA和蛋白表达,差异均有统计学意义(P<0.05)。结论 S100B能够调节SF炎性反应并可能影响OA软骨损伤修复,其机制可能与激活FGF-2/FGFR1信号通路有关。

Objective To investigate the role and mechanism of S100 calcium binding protein B（S100 B） in osteoarthritis（OA） cartilage damage repair. Methods Twenty New Zealand rabbits were randomly divided into control group and model group, with 10 rabbits in each group. Rabbits in the model group were injured by the right knee joint immobilization method to make the artilage injury model, while the control group did not deal with any injury. After 4 weeks, the levels of interleukin-1β（IL-1β） and tumor necrosis factor α（TNF-α） in synovial fluid were detected by ELISA method; the mRNA and protein expressions of S100 B, fibroblast growth factor 2（FGF-2）, and FGF receptor 1（FGFR1） in cartilage tissue were examined by real-time fluorescence quantitative PCR（qRT-PCR） and Western blot assay. Human synovial fibroblasts（SF） were isolated and cultured in vitro. The effects of S100 B overexpression and knockdown on the levels of IL-1β and TNF-α（ELISA method） and the expressions of FGF-2 and FGFR1 gene（qRT-PCR） and protein（Western blot） were observed. Moreover, the effects of FGFR1 knockdown in above S100 overexpression system on the levels of IL-1β and TNF-α（ELISA method） and the expressions of FGF-2 and FGFR1 gene（qRT-PCR） and protein（Western blot） were observed. Results ELISA detection showed that the expressions of IL-1β and TNF-α in the synovial fluid of the model group were significantly higher than those of the control group（P0.05）; qRT-PCR and Western blotdetection showed that the mRNA and protein expressions of S100 B, FGF-2, and FGFR1 in cartilage tissue were significantly higher than those of the control group（P0.05）. Overexpression and knockdown S100 could respectively significantly increase and decrease lipopolysaccharides（LPS） induced IL-1β and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1（P0.05）; whereas FGFR1 knockdown could significantly decrease LPS induced IL-1β and TNF-α levels elevation and the mRNA and protein expressions of FGF-2 and FGFR1（P0.05）.Conclusion S100 B protein can regulate the inflammatory response of SF and may affect the repair of cartilage damage in OA, and the mechanism may be related to the activation of FGF-2/FGFR1 signaling pathway.