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    基于RANKL/RANK/NF-κB通路探讨益气活络方对气虚血瘀证类风湿关节炎大鼠的干预效应

    Exploring the Interventional Effect of Yiqi Huoluo Formula on Qi Deficiency and Blood Stasis Syndrome in Rheumatoid Arthritis Rats Based on RANKL/RANK/NF-κB Pathway

      摘要
    • 摘要:
      目的  基于核因子κB配体(receptor activator of nuclear factor-κB ligand，RANKL)/核因子NF-κB受体激活因子(receptor activator of nuclear factor kappa-B，RANK)/核因子κB(nuclear factor kappa-B，NF-κB)通路探讨益气活络方对气虚血瘀证类风湿关节炎大鼠的干预效应。
      方法  采用复合因素法和胶原诱导复制气虚血瘀证胶原诱导性关节炎(collagen-induced arthritis，CIA)大鼠模型，分为模型组，甲氨蝶呤组(0.21 mg·kg−1，1周给药2次)，益气活络方低(9.45 g·kg−1·d−1)、中(18.90 g·kg−1·d−1)、高(37.80 g·kg−1·d−1)剂量组(n=8)，持续治疗28 d，另设空白对照组。观察并记录大鼠体质量、脾指数、关节炎指数评分以及踝关节肿胀度。HE染色和SO/FG染色观察踝关节病理变化；Micro-CT扫描观察踝关节骨破坏情况；透射电镜观察脾脏超微结构变化；全自动血凝分析仪分析血液流变学变化；ELISA检测血清中肿瘤坏死因子-α(tumor necrosis factor-α，TNF-α)、白细胞介素-6(interleukin-6，IL-6)、白细胞介素-1β(interleukin-1β，IL-1β)水平变化；免疫组化检测滑膜组织中组织蛋白酶K(recombinant cathepsin K，CTSK)、基质金属蛋白酶-9(matrix metalloproteinase 9，MMP-9)蛋白表达；Western blotting检测大鼠滑膜组织中受体活化的RANKL、RANK、NF-κB、丝裂原激活蛋白激酶(mitogen-activated protein kinase，MAPK)蛋白表达。
      结果 与空白对照组相比，模型组大鼠体质量显著降低，关节炎指数(arthritis index，AI)评分和关节肿胀度显著升高，踝关节滑膜增生以及关节软骨与软骨下骨破坏严重，红细胞聚集指数、卡松黏度及全血黏度显著升高，脾指数显著升高及脾脏线粒体破坏严重，血清中TNF-α、IL-6、IL-1β水平显著升高，踝关节及滑膜组织中CTSK、MMP-9、RANKL、RANK、NF-κB、MAPK蛋白表达显著升高；与模型组相比，大鼠体质量升高，AI评分和关节肿胀度显著降低，踝关节病理损伤显著改善，红细胞聚集指数、卡松黏度及全血黏度显著降低，脾指数显著降低，血清中TNF-α、IL-6、IL-1β水平显著降低，踝关节及滑膜组织中CTSK、MMP-9、RANKL、RANK、NF-κB、MAPK蛋白表达降低，且益气活络方高剂量组治疗效果显著。
      结论  益气活络方能够抑制气虚血瘀证CIA大鼠滑膜炎症，降低AI评分并抑制骨组织破坏，其机制可能与抑制RANKL/RANK/NF-κB通路的激活有关。

       

      Abstract:
      OBJECTIVE  To investigate the intervention effect of Yiqi Huoluo(YQHL) formula on rheumatoid arthritis rats with Qi deficiency and blood stasis syndrome based on the nuclear factor-κB ligand(RANKL)/receptor activator of nuclear factor kappa-B(RANK)/nuclear factor kappa-B(NF-κB) pathway.
      METHODS Using the compound factor method combined with collagen induction to replicate collagen-induced arthritis(CIA) rat model of Qi deficiency and blood stasis syndrome, the rats were divided into the model group, methotrexate group(0.21 mg·kg−1, twice a week), and YQHL formula low(9.45 g·kg−1·d−1), medium(18.90 g·kg−1·d−1), and high(37.80 g·kg−1·d−1) dose groups(n=8), with treatment lasting for 28 d, a blank control group was set additionally. The body weight, spleen index, arthritis index score and ankle swelling degree of rats were observed and recorded. HE staining and SO/FG staining were used to observe pathological changes in ankle joint; Micro-CT scanning was used to observe bone destruction in the ankle joint; transmission electron microscopy was used to observe ultrastructural changes in the spleen; a fully automated coagulation analyzer was used to analyze changes in blood rheology; ELISA was used to detect changes in tumor necrosis factor-α(TNF-α), interleukin-6(IL-6), and interleukin-1β(IL-1β) levels in serum; immunohistochemistry was used to detect recombinant cathepsin K(CTSK) and matrix metalloproteinase 9(MMP-9) protein expression in rat synovial tissues; Western blotting was used to detect receptor activator of RANKL, RANK, NF-κB, and mitogen-activated protein kinase(MAPK) protein expression in rat synovial tissues.
      RESULTS Compared with the blank control group, the body weight of rats in the model group was significantly reduced, arthritis index(AI) scores and joint swelling were significantly increased, ankle joint synovial hyperplasia and damage to articular cartilage and subchondral bone were severe, red blood cell aggregation index, Casson viscosity, and whole blood viscosity were significantly increased, spleen index was significantly increased, and splenic mitochondrial damage was severe. Levels of TNF-α, IL-6, and IL-1β in serum were significantly increased, and the expression of CTSK, MMP-9, RANKL, RANK, NF-κB, and MAPK proteins in ankle joint and synovial tissue was significantly increased; compared with the model group, the rats showed increased body mass, significantly reduced AI scores and joint swelling, significantly improved pathological damage in ankle joint , significantly reduced red blood cell aggregation index, Casson viscosity, and whole blood viscosity, significantly reduced spleen index, significantly decreased levels of TNF-α, IL-6, and IL-1β in serum, and reduced expression of CTSK, MMP-9, RANKL, RANK, NF-κB, and MAPK proteins in ankle joint and synovial tissues. Moreover, the high dose group of the YQHL formula showed a significant therapeutic effect.
      CONCLUSION YQHL formula can inhibit synovial inflammation in CIA rats with Qi deficiency and blood stasis syndrome, reduce AI scores, and suppress bone tissue destruction. Its possible mechanism is related to inhibiting the activation of the RANKL/RANK/NF-κB pathway.

       

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