治尪汤联合甲氨蝶呤调节RANKL/OPG通路改善胶原诱导关节炎大鼠骨破坏的作用机制

Mechanism of Zhiwang Decoction Combined with Methotrexate Against Bone Destruction Through Regulating RANKL/OPG Pathway in Rats with Collagen-induced Arthritis

目的:探究治尪汤联合甲氨蝶呤改善胶原诱导关节炎模型(CIA)大鼠的骨破坏作用及机制。方法:SD雄性大鼠随机分为正常组,模型组,甲氨蝶呤组(1.5 mg·kg^(-1)),甲氨蝶呤+治尪汤低(40.8 g·kg^(-1)),高(102.0 g·kg^(-1))剂量组。除正常组外,其余大鼠分别于第1,7天尾部注射浓度为2 g·L^(-1)的Ⅱ型胶原乳剂50μL建立CIA模型;第8天开始连续给药21 d,每7 d检测1次体质量、足趾厚度,进行关节炎指数(AI)评分,酶联免疫吸附测定法检测血清肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6),白细胞介素-1β(IL-1β)含量,抗酒石酸酸性磷酸酶染色法检测踝关节破骨细胞的数量,免疫组化(IHC),实时荧光定量聚合酶链式反应(Real-time PCR),蛋白免疫印迹法(Western blot)检测踝关节核转录因子-κB受体活化因子配体(RANKL),骨保护素(OPG)的表达。结果:与正常组比较,模型组大鼠体质量显著降低(P<0.01),足趾厚度显著增加(P<0.01),踝关节AI评分显著增加(P<0.01),血清TNF-α,IL-6,IL-1β含量显著升高(P<0.01),踝关节破骨细胞数量显著增加(P<0.01),踝关节RANKL mRNA和蛋白表达显著升高(P<0.01),OPG mRNA和蛋白表达明显降低(P<0.05),RANKL/OPG显著升高(P<0.01)。与模型组比较,甲氨蝶呤+治尪汤低、高剂量可改善大鼠体质量,降低足趾厚度,AI评分,RANKL mRNA和蛋白表达并增加OPG mRNA和蛋白表达,使RANKL/OPG降低(P<0.05,P<0.01),且甲氨蝶呤+治尪汤高剂量效果最好,甲氨蝶呤+治尪汤低剂量次之,甲氨蝶呤再次之。结论:治尪汤联合甲氨蝶呤能更好地改善类风湿关节炎的骨破坏,可能是通过抑制RANKL/OPG通路实现的,本研究为中西医结合方案治疗类风湿关节炎骨破坏提供了实验基础。

Objective: To explore the effect and mechanism of Zhiwang decoction combined with methotrexate(MTX)against bone destruction in rats with collagen-induced arthritis(CIA). Method: Male SD rats were randomly divided into a normal group,a model group,an MTX group(1.5 mg·kg^(-1)),and low-(40.8 g·kg^(-1))and high-(102.0 g·kg^(-1))combination groups(MTX+ Zhiwang decoction). The CIA model was induced in rats except for those in the normal group by tail injection of 50 μL of 2 g·L^(-1)type Ⅱ collagen on the1 st and 7 th days. From the 8 th day,the rats were treated correspondingly for 21 days. The body weight,toe thickness, and arthritis index(AI)were measured every seven days. Enzyme-linked immunosorbent assay(ELISA)was used to detect the content of inflammatory factors in the serum,such as tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),and interleukin-1β(IL-1β). Tartrate-resistant acid phosphatase staining was used to detect the number of osteoclasts in the ankle joint. The expression of receptor activator of nuclear factor-κB ligand(RANKL)and osteoprotegerin(OPG)in ankle tissues was detected by immunohistochemistry(IHC),real-time polymerase chain reaction(Real-time PCR),and Western blot. Result: Compared with the normal group,the model group showed decreased body weight(P<0.01),increased toe thickness and AI score(P<0.01),up-regulated serum levels of TNF-α,IL-6,and IL-1β(P<0.01),elevated number of osteoclasts(P<0.01),increased mRNA and protein expression of RANKL(P<0.01),reduced mRNA and protein expression of OPG(P<0.05),and increased RANKL/OPG ratio(P<0.01). Compared with the model group,the high-dose combination group showed increased body weight,decreased toe thickness and AI score,reduced mRNA and protein expression of RANKL,up-regulated mRNA and protein expression of OPG,and declining RANKL/OPG ratio(P<0.05,P<0.01). The therapeutic effect was optimal in the high-dose combination group,followed by the low-dose combination group and the MTX group. Conclusion: Zhiwang decoction combined with MTX can improve bone destruction induced by rheumatoid arthritis,which may be achieved by inhibiting the RANKL/OPG pathway. This study provides an experimental basis for the treatment of bone destruction induced by rheumatoid arthritis with integrated traditional Chinese and western medicine.