基于RANK/RANKL/OPG通路探讨尪痹片对去势后胶原诱导性关节炎大鼠骨吸收的影响

作者	单位
张馨妍;孙龙吉;郑慧兰;陶庆文;周同亮;李鸿;王建明	1.北京中医药大学研究生院（北京 100029）,2.中日友好医院中医风湿病科（北京 100029）,3.免疫炎性疾病北京市重点实验室（北京 100029）,4.中日友好医院临床医学研究所（北京 100029）

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中文摘要:

目的探讨尪痹片（WBT）对肾虚证胶原诱导性关节炎（CIA）大鼠核转录因子-κB受体活化因子（RANK）/RANK配体（RANKL）/骨保护素（OPG）信号通路的影响，探索WBT干预破骨细胞（OC）分化、增殖及骨吸收作用的机制。方法选用雌性SD大鼠，共48只，随机分为空白对照组、CIA 组、肾虚证组、肾虚证CIA 组、CIA+WBT组、肾虚证+WBT组、肾虚证CIA+WBT组、CIA+甲氨蝶呤（MTX）组，每组6只。采用去势法制备肾虚证模型，牛Ⅱ型胶原和不完全弗氏佐剂诱导制备CIA模型。模型制备成功后进行药物灌胃，灌胃周期结束后处死，取血及后肢踝关节。甲苯胺蓝染色检测踝关节骨吸收情况。酶联免疫吸附法（ELISA）检测大鼠血清中白细胞介素4（IL-4）、IL-33、OPG、RANKL水平。实时荧光定量聚合酶链式反应（RT-qPCR）检测大鼠踝关节骨组织中RANKL、OPG mRNA表达。蛋白免疫印迹法（Western Blot）检测大鼠踝关节骨组织中活化T细胞核因子1（NFATc1）、肿瘤坏死因子受体相关因子6（TRAF6）表达。结果大鼠踝关节软骨甲苯胺蓝染色结果显示，经WBT干预后，关节软骨面结构破坏有所改善，关节软骨面相对完整。与空白对照组比较，CIA组、肾虚证CIA组大鼠血清中IL-4、IL-33浓度降低，RANKL浓度、RANKL/OPG比值及NFATc1、TRAF6蛋白表达升高（P＜0.05，P＜0.01），肾虚证CIA组OPG浓度、mRNA表达降低（P＜0.05，P＜0.01），RANKL mRNA表达升高，RANKL/OPG比值升高（P＜0.01）。与肾虚证CIA组比较，肾虚证CIA+WBT组IL-4浓度升高（P＜0.01），RANKL mRNA表达、RANKL/OPG比值及NFATc1、TRAF6蛋白表达降低（P＜0.05，P＜0.01）。与CIA组比较，CIA+WBT组IL-33、OPG浓度升高（P＜0.05，P＜0.01），RANKL/OPG比值降低（P＜0.01）。结论尪痹片能够通过调控RANK/RANKL/OPG信号通路，上调抗破骨因子IL-4、IL-33浓度，抑制炎症反应，直接或间接抑制OC分化、增殖及骨吸收功能。

英文摘要:

Objective To discuss the effect of Wang-bi Tablet （WBT） on receptor activator of NF-κB （RANK）/RANK ligand （RANKL） / osteoprotegerin（OPG）signaling pathway in collagen-induced arthritis（CIA）rats with Shen deficiency syndrome，and to explore the intervention mechanism of WBT in osteoclast（OC）differentiation，proliferation and bone resorption. Methods Totally 48 female SD rats were selected， which were randomly divided into blank control group，CIA group，Shen deficiency syndrome group，CIA with Shen deficiency syndrome group，CIA+WBT group， Shen deficiency syndrome + WBT group，CIA with Shen deficiency syndrome + WBT group and CIA + methotrexate （MTX）group，and 6 rats in each group. The rat model of Shen deficiency syndrome was established by castration，and CIA model was induced by bovine type Ⅱ collagen and incomplete Freund's adjuvant. Intragastric administration was performed after successful model replication. After administration，the rats in each group were sacrificed and materials including blood and ankle joints of hind limbs were taken. Toluidine blue staining was used to detect the bone absorption in ankle joints. The levels of serum interleukin-4（IL-4），interleukin-33（IL-33），OPG and RANKL of rats were detected by enzyme linked immunosorbent assay（ELISA）. The mRNA expression of RANKL and OPG in the ankle bone tissue of rats were determined by real-time fluorescence quantitative polymerase chain reaction（RT-qPCR）. Western Blot was used to determine the expression of nuclear factor of activated T cells 1（NFATc1） and TNF receptor-associated factor 6 （TRAF6） in the ankle bone tissue of rats. Results The results of toluidine blue staining on the ankle cartilage of rats showed that the damage of the articular cartilage surface was improved and the articular cartilage surface was relatively intact after WBT treatment. Compared with the blank control group，the levels of serum IL-4 and IL-33 of rats in CIA group and CIA with Shen deficiency syndrome group decreased，while the level of RANKL，the ratio of RANKL/OPG and the expression of NFATc1 and TRAF6 increased（P＜0.05，P＜0.01）. The level of OPG and OPG mRNA expression in CIA with kidney deficiency syndrome group decreased（P＜0.05，P＜0.01），and RANKL mRNA expression increased，RANKL/OPG ratio increased（P＜0.01）. Compared with CIA with Shen deficiency syndrome group，the level of serum IL-4 in CIA with Shen deficiency syndrome + WBT group increased（P＜0.01），while RANKL mRNA expression，RANKL/OPG ratio，NFATc1 and TRAF6 expression decreased（P＜0.05，P＜0.01）. Compared with CIA group，the level of serum IL-33 and OPG in CIA+WBT group increased（P＜0.05，P＜0.01），and the ratio of RANKL/OPG decreased（P＜0.01）. Conclusion Through regulating the RANK/RANKL/OPG signaling pathway，WBT could up-regulate the levels of anti-osteoclast factors including IL-4 and IL-33，and inhibit the inflammatory response，directly or indirectly inhibit the OC differentiation，proliferation and bone resorption.

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