摘要
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.
作者
马茹
郭子嘉
陶庆文
鄢泽然
刘振权
徐愿
MA Ru;GUO Zi-jia;TAO Qing-wen;YAN Ze-ran;LIU Zhen-quan;XU Yuan(Xi'an Public Health Center(Xi'an Emergency Medical Center),Xi'an 710200,China;College of Traditional Chinese Medicine(TCM),Inner Mongolia Medical University,Hohhot 010110,China;Beijing Key Laboratory of Immune Inflammatory Diseases,Rheumatology Department of TCM,China-Japan Friendship Hospital,Beijing 100029,China;National Institute for TCM Constitution and Preventive Medicine,Beijing University of Chinese Medicine,Beijing 100029,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2022年第5期46-54,共9页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81603588)
北京市中医药科技发展项目(QN-2020-32)。