目的 探讨山茱萸-白芍酒炙配伍前后对类风湿性关节炎小鼠关节评分、足趾红肿度、胫骨关节与踝关节损伤度及膝关节滑膜组织中骨保护素(osteoprotegerin,OPG)/核因子-κB(nuclear factor-κB,NF-κB)受体激活因子配体(receptor activator of NF-κB ligand,RANKL)/NF-κB受体激活因子(receptor activator of NF-κB,RANK)信号通路的影响。方法 尾根部sc牛II型胶原和完全弗氏佐剂的混合乳状液建立类风湿性关节炎(rheumatoid arthritis,RA)小鼠模型。将DBA/1雄性小鼠按照体质量分布随机分为9组,即对照组、模型组、来氟米特阳性药组、生山茱萸组、酒山茱萸组、生白芍组、酒白芍组、生山茱萸-生白芍配伍组、酒山茱萸-酒白芍配伍组,每组8只。除对照组外,首次免疫造模7 d后进行第2次加强免疫,第30天造模完成后,各给药组第31天起每天ig相应剂量的75%乙醇提取物无菌水溶液,阳性药组给予来氟米特无菌水溶液(6 mg/kg),对照组、模型组小鼠ig相应体积的无菌水,连续给药30 d。显微镜下观察小鼠踝关节HE病理变化,断层扫描(Micro-CT)观察小鼠胫骨关节及踝关节损伤程度,RT-qPCR法测定膝关节滑膜组织中OPG、RANKL、肿瘤坏死因子受体相关因子6(tumor necrosis factor receptor-associated factor 6,TRAF6)、酪氨酸蛋白激酶(sarcoma,Src)、核因子-p65(nuclear factor-p65,P65)基因表达水平及Western blotting法测定OPG、RANKL、RANK、TRAF6、Src、磷酸化NF-kB抑制蛋白α(phosphorylation inhibitory κBα,p-IκBα)、磷酸化P65(p-P65)蛋白的表达。结果 与对照组相比,模型组小鼠踝关节组织有明显的滑膜成纤维细胞及破骨细胞等炎性浸润,足趾关节及踝关节发生变形,OPG蛋白和基因表达水平明显降低(P<0.001),RANKL、TRAF6、Src、P65基因表达水平及RANKL、RANK、TRAF6、Src、p-IκBα/IκBα、p-P65/P65蛋白表达水平显著升高(P<0.001);与模型组相比,山茱萸和白芍酒炙配伍前后各组小鼠踝关节炎症及损伤均有改善的趋势,OPG蛋白和基因表达水平升高,RANKL、TRAF6、Src、P65基因表达水平及RANKL、RANK、TRAF6、Src、p-IκBα/IκBα、p-P65/P65蛋白表达水平降低,且酒山茱萸-酒白芍配伍组改善效果最为明显(P<0.001),生山茱萸-生白芍配伍组和酒山茱萸组次之,生白芍组、酒白芍组和生山茱萸组虽有一定改善作用,但关节炎评分相对较高。结论 山茱萸和白芍酒炙配伍前后均可一定程度上改善小鼠膝关节及踝关节损伤,其中二者的酒炙品配伍或生品配伍抗RA效果更佳,可能是通过调控OPG/RANKL/RANK信号通路来发挥抗RA的作用。

Objective To investigate the effects of Shanzhuyu (Corni Fructus)-Baishao (Paeoniae Radix Alba) herbal pair with wine before and after processing on joint scores, toe swelling degree, tibial articular and ankle joint injury degree, and osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL)/receptor activator of NF-κB (RANK) pathway of knee synovial tissue in rheumatoid arthritis mice. Methods The emulsion of bovine type II collagen and complete Freund's adjuvant were injected subcutaneously into the tail root of mice to establish the rheumatoid arthritis model. According to the weight distribution, the DBA/1 male mice were randomly divided into nine groups (eight per group). Namely, the control group, the model group, the leflunomide positive drug group, the raw Corni Fructus group, the wine-processed Corni Fructus group, the raw Paeoniae Radix Alba group, the wine-processed Paeoniae Radix Alba group, the raw Corni Fructus and Paeoniae Radix Alba compatibility group, and the wine-processed Corni Fructus and Paeoniae Radix Alba compatibility group, respectively. The second booster immunization was performed 7 d after the first immunization and then the rheumatoid arthritis model was established on the 30th d. Furthermore, each administration group was given a corresponding dose of 75% ethanol extract sterile aqueous solution, the positive drug group was given a sterile aqueous solution of leflunomide (6 mg/kg), and mice in the control group and model group were given a corresponding volume of sterile water for the following 30 consecutive days. Finally, the pathological changes of HE in the mouse ankle joint were observed under microscope, and the injury degree of the mouse tibial articular and ankle joint were observed by Micro-CT tomography, respectively. Moreover, the OPG, RANKL, tumor necrosis factor receptor-associated factor 6 (TRAF6), sarcoma (Src), nuclear factor-p65 (P65) genes and the OPG, RANKL, RANK, TRAF6, Src, phosphorylation inhibitory κBα (p-IκBα), p-P65 protein in the synovial tissue of the knee joint were determined by RT-qPCR and Western blotting, respectively. Results Compared with the control group, mice in the model group had obvious inflammatory infiltration of fibroblast synovial cells and osteoclasts in ankle joints, deformed knee and ankle joints. In addition, the OPG gene levels and protein expression were reduced significantly (P < 0.001) while the RANKL, TRAF6, Src, P65 gene levels and RANKL, RANK, TRAF6, Src, p-IκBα/IκBα, p-P65/P65 protein expression were significantly increased (P < 0.001). Besides, all the groups of Corni Fructus-Paeoniae Radix Alba herbal pair with wine before and after processing have a trend of improvement compared with the model group, especially in the wine-processed Corni Fructus-Paeoniae Radix Alba herbal pair group (P < 0.001) followed by the raw Corni Fructus-Paeoniae Radix Alba herbal pair group and the wine-processed Corni Fructus group, and the OPG gene level and protein expression increased. Moreover, RANKL, TRAF6, Src, P65 gene level and RANKL, RANK, TRAF6, Src, p-IκBα/IκBα, p-P65/P65 protein expression were reduced. In addition, the administration group of the raw Paeoniae Radix Alba, wine-processed Paeoniae Radix Alba, and raw Corni Fructus also showed a significant improvement trend, however, the arthritis score was relatively higher. Conclusion Corni Fructus-Paeoniae Radix Alba herbal pair can reduce the knee and ankle joint injury in rheumatoid arthritis mice through regulating the OPG/RANKL/RANK signaling pathway to play an anti-rheumatoid arthritis effect.

R285

国家自然科学基金项目（81773900）