温经活络方治疗胶原诱导性关节炎小鼠模型实验研究

目的 基于肿瘤坏死因子α（TNF-α）/核因子-κB（NF-κB）及辅助性T细胞17（Th17）细胞分化探讨温经活络方治疗胶原诱导性关节炎（CIA）小鼠作用机制。方法 40只6周雄性DBA/1小鼠采用随机数字表法分为正常对照组、模型组、甲氨蝶呤组和温经活络方组，共4组，每组10只。以胶原注射的方式构建胶原诱导性关节炎模型，在初次免疫后21天起分别以双蒸水、甲氨蝶呤、温经活络方干预35天，期间每周评价各组小鼠的体重，从初次免疫后第24天起每周评价各组足肿胀度、关节炎症评分。HE染色检测足趾及踝关节病理改变情况；酶联免疫吸附（ELISA）法检测小鼠血清中抗环瓜氨酸肽抗体（CCP-Ab）、小鼠白细胞介素-6（IL-6）和TNF-α水平；实时定量聚合酶链式反应（RT-qPCR）检测足关节NF-κB、骨保护素（OPG）和破骨细胞分化因子（RANKL）mRNA表达水平；免疫蛋白质印迹（Western Blot）检测小鼠脾脏及足关节NF-κB、p-NF-κB表达情况；流式细胞术检测小鼠脾脏中Th17细胞表达情况。结果 与正常组比较，模型组小鼠毛发枯槁，进食量减少，体重明显降低（P<0.05，P<0.01）；小鼠血清中抗CCP抗体、IL-6、TNF-α含量升高（P<0.01）；足趾中NF-κB、RANKL mRNA表达亦明显升高（P<0.01），OPG mRNA表达明显下降（P<0.05）；脾脏及足趾关节p-NF-κB蛋白表达明显升高（P<0.01）；脾脏中Th17细胞比值显著升高（P<0.01）。与模型组比较，温经活络方组小鼠体重减轻情况得到缓解（P<0.05）；两干预组脾脏肿大得到显著缓解（P<0.01），足肿胀度显著降低（P<0.01）。温经活络方组关节炎症评分显著下降（P<0.05，P<0.01）；小鼠足趾及踝关节滑膜细胞增生减轻，浸润的炎细胞数量显著减少，血管翳生成减少，骨和软骨破坏明显减轻。两干预组血清抗CCP-Ab、TNF-α、IL-6水平下降（P<0.05，P<0.01）；脾脏和足趾p-NF-κB水平降低（P<0.05，P<0.01）；NF-κB、RANKL mRNA表达水平下降（P<0.01），OPG mRNA表达水平升高（P<0.05，P<0.01）。Th17细胞表达明显抑制（P<0.01）。结论 温经活络方能够通过抑制TNF-α、IL-6等相关炎症因子表达，进一步抑制TNF-α/NF-κB信号通路及Th17细胞的活化，从而减轻RA关节炎症、降低关节炎症评分、抑制骨质破坏、改善其病理变化。

Wenjing Huoluo Recipe Treated Collagen-induced Arthritis Model Mice

Objective To study the therapeutic mechanisms for Wenjing Huoluo Recipe （WHR） in treatment of collagen-induced arthritis （CIA） via inhibition of tumor necrosis factor-α （TNF-α）/nuclear factor kappa B （NF-κB） pathway and regulation of Th17 cells in a murine model. Methods Forty 6-week male DBA/1 mice were randomly divided into four groups： normal control group， model group， methotrexate （MTX） group， and WHR group， 10 in each group. CIA model was established by collagen injection， and intervened with double distilled water， MTX， and WHR for 35 days starting from the 21st day after initial immunization. Body weight of mice in each group was assessed weekly. The degree of foot swelling and joint inflammation score of each group were evaluated weekly from the 24th day. HE staining was used to detect pathological changes in the toes and ankle joints. ELISA was used for detecting anti-cyclic citrullinated peptide （CCP） antibodies， serum TNF-α and IL-6 levels. Real-time quantitative polymerase （RT-qPCR） was used to detect mRNA expression levels of foot joint NF-κB， osteoprotegerin （OPG）， and receptor activator of nuclear factor-κ B ligand （RANKL）. Western Blot was used to detect NF-κB and P-NF-κB expressions in mouse spleen and foot joints. Flow cytometry was used to detect the expression of helper T lymphocyte 17（Th17） cells in the mouse spleen. Results Compared with the normal group， the hair withered， food intake reduced， and body weight obviously decreased in the model group （P<0.05， P<0.01）. Contents of anti-CCP antibody， IL-6， and TNF-α increased （P<0.01）. mRNA expressions of NF-κB and RANKL also increased（P<0.01）， OPG mRNA expression obviously decreased （P<0.05）. p-NF-κB protein expression obviously increased in the spleen and toe joints（P<0.01）. Th17 cell ratio in the spleen significantly increased （P<0.01）. Compared with the model group，the weight loss in WHR was alleviated （P<0.05）. The swollen spleen was significantly alleviated in each treatment group （P<0.01）. The degree of foot swelling was significantly reduced （P<0.01）. The joint inflammation score significantly decreased in mice of WHR group （P<0.05，P<0.01）. The proliferation of synovial cells in the toes and ankle joints of mice was attenuated， and the number of infiltrated inflammatory cells was significantly reduced， the formation of pannus decreased， and the destruction of bone and cartilage were significantly attenuated after treated by WHR. After drug intervention the levels of serum anti-CCP antibodies， IL-6， and TNF-α in each treatment group decreased more significantly （P<0.05，P<0.01）. After drug treatment NF-κB phosphorylation （p-NF-κB） level decreased （P<0.05，P<0.01）， and mRNA expression levels of NF-κB， and RANKL significantly decreased （P<0.01），the mRNA expression level of OPG significantly increased （P<0.05，P<0.01），the expression of Th17 cells was significantly inhibited after treatment with MTX and WHR （P<0.01）. Conclusion WHR inhibited TNF-α/NF-κB signaling pathway and Th17 cells'' activation possibly by further inhibiting expressions of TNF-α， IL-6 and other related inflammatory factors， thus attenuating RA joint inflammation， lowering joint inflammation score inhibiting bone destruction， and improving pathological changes.