基于Caspase-1/GSDMD通路探讨雷公藤甲素抗胶原诱导性关节炎大鼠骨破坏作用

Discussion on the effects and mechanism of triptolide in bone destruction in collagen-induced arthritis rats based on Caspase-1/GSDMD pathway

目的探讨雷公藤甲素调控Caspase-1/Gasdermins D蛋白(GSDMD)通路干预胶原诱导性关节炎大鼠骨破坏的作用及机制。方法将大鼠按随机数字表法分为空白组、模型组、雷公藤甲素组、甲氨蝶呤组,每组5只。除空白组外,其余各组大鼠尾根部注射牛Ⅱ型胶原建立关节炎模型。加强免疫7 d后,雷公藤甲素组灌胃雷公藤甲素18μg/kg,1 d/次;甲氨蝶呤组腹腔注射甲氨蝶呤0.3 mg/kg,3 d/次,连续干预15 d。记录大鼠关节炎指数(AI)评分与足趾容积变化,采用HE染色观察大鼠踝关节组织形态学改变,番红固绿染色观察大鼠踝关节软骨及骨改变;ELISA法检测大鼠血清IL-18、IL-1β水平;实时荧光定量PCR法检测大鼠踝关节GSDMD、Caspase-1、骨骼保护因子(OPG)、NF-κB受体活化因子配体(RANKL)mRNA水平;Western blot法检测踝关节组织中GSDMD、Caspase-1、OPG、RANKL蛋白表达。结果给药1、2周,与模型组比较,雷公藤甲素组、甲氨蝶呤组AI评分及足趾容积数值降低(P<0.01);模型组大鼠踝关节组织可见大量炎性细胞浸润、滑膜血管翳生成,软骨及骨染色潮线模糊缺损,各给药组均不同程度改善大鼠关节炎性浸润、滑膜血管翳生成、关节软骨及骨破坏。与模型组比较,雷公藤甲素组与甲氨蝶呤组大鼠血清IL-18、IL-1β水平降低(P<0.01),GSDMD、Caspase-1、RANKL mRNA及蛋白表达降低(P<0.01),OPG mRNA及蛋白表达升高(P<0.01)。结论雷公藤甲素可有效改善胶原诱导性关节炎大鼠关节炎症与骨破坏表现,其作用机制可能与下调GSDMD、Caspase-1、RANKL表达,上调OPG表达相关。

Objective To discuss the effects and mechanism of triptolide regulating Caspase1/GSDMD pathway in bone destruction in collagen-induced arthritis rats.Methods The rats were divided into blank group,model group,triptolide group,and methotrexate group using a random number table method,with 5 rats in each group.Except for the blank group,all other groups were injected with bovine typeⅡcollagen at the tail root to establish an arthritis model.After 7 days of strengthening immunity,the dosage of triptolide A was calculated based on the body surface area in rats,and the triptolide A group was orally administered with triptolide A for 18μg/kg,1 d/time;the methotrexate group received intraperitoneal injection of 0.3 mg/kg methotrexate for 3 days per dose,with continuous intervention for 15 days.The arthritis index(AI)score and toe volume changes of the rats were recorded.The ankle joint histological changes were observed with HE staining,and the ankle joint cartilage and bone changes were observed with ferruginine solid green staining.The contents of IL-18 and IL-1βin serum were determined by ELISA.The mRNA levels of GSDMD,Caspase-1,OPG and RANKL in ankle joints were detected by real-time quantitative PCR.The expressions of GSDMD,Caspase-1,OPG and RANKL in ankle tissues were detected by Western blot.Results After 1 and 2 weeks of administration,compared with the model group,the AI scores and toe volume values of the triptolide group,and methotrexate group decreased(P<0.01);in the model group,a large number of inflammatory cell infiltration,synovial pannus formation,and blurred defects of the tide line of cartilage and bone staining were observed.The inflammatory infiltration,synovial pannus formation,articular cartilage and bone destruction were improved to varying degrees in each administration group.Compared with model group,serum IL-18 and IL-1βcontents in triptolide group and methotrexate group significantly decreased(P<0.01),mRNA and protein expressions of GSDMD,Caspase-1 and RANKL decreased(P<0.01),and mRNA and protein expression of OPG increased(P<0.01).Conclusion Triptolide can effectively improve joint inflammation and bone destruction in collagen-induced arthritis rats,and its mechanism may be related to down-regulating the expressions of GSDMD,Caspase-1 and RANKL,and up-regulating the expression of OPG.