羟基积雪草甙介导NLRP3信号通路治疗痛风性关节炎的机制

Mechanism of madecassoside mediated NLRP3 signaling pathway in the treatment of gouty arthritis

[目的]探讨羟基积雪草甙介导NLRP3信号通路治疗痛风性关节炎的分子机制。[方法]8周龄SPF级Wistar大鼠依次分为对照组(10只,注射等量生理盐水后再经右踝关节注入PBS溶液)、模型组(10只,注射等量生理盐水后再经关节腔注入浓度为80 mg/m L的MSU混悬液)、实验组(分别注射10、20、40 mg/kg/d羟基积雪草甙溶液分别作为低剂量组、中剂量组、高剂量组各10只,经关节腔注入浓度为80 mg/m L的MSU混悬液)。处死大鼠后获取关节液采用酶联免疫吸附法(ELISA)检测IL-1β、TNF-ɑ浓度;收集关节周围软组织,HE染色观察关节组织病理学特点;实时荧光定量聚合酶链反应(RT-q PCR)检测关节周围软组织NLRP3、ASC、caspase-1 mRNA的表达;免疫印迹法(WB)检测NF-κB、ASC、caspase-1相对表达量。[结果]模型组大鼠关节组织病理学图显示关节组织损坏,羟基积雪草甙可减轻该损伤,且具有一定的剂量依赖性。模型组IL-1β、TNF-ɑ浓度及NLRP3、ASC、caspase-1、NF-κB表达量均高于空白组(P<0.05);低剂量组、中剂量组、高剂量组IL-1β、TNF-ɑ浓度及NLRP3、ASC、caspase-1、NF-κB表达量均低于模型组(P<0.05),羟基积雪草甙对IL-1β、TNF-ɑ、NLRP3、ASC、caspase-1、NF-κB升高的抑制作用具有一定的剂量依赖性。[结论]羟基积雪草甙可降低炎性因子IL-1β、TNF-ɑ浓度和NLRP3信号分子表达量,NLRP3信号通路的激活与痛风性关节炎相关,说明羟基积雪草甙可通过抑制NLRP3炎性体轴及信号通路相关分子来抑制炎性因子释放而发挥治疗痛风性关节炎的作用机制。

[Objective]To investigate the molecular mechanism of madecassoside mediated NLRP3 signaling pathway in the treatment of gouty arthritis.[Method]Eight-week old SPF Wistar rats were divided into three groups.Control group(n=10)received the same amount of normal saline injection and intra-articular injection of PBS solution.Model group(n=10)received same amount of normal saline injection and intra-articular injection of MSU suspension with a concentration of 80 mg/m L.Experimental group(n=30)received intra-articular injection of MSU suspension with a concentration of 80 mg/m L,and equally divided into three subgroups for different madecassoside treatment,low dose group(10 mg/kg/d madecassoside),middle dose group(20 mg/kg/d madecassoside)and high dose group(40 mg/kg/d madecassoside).Enzyme-linked immunosorbent assay(ELISA)was used to detect the concentration of IL-1βand TNF-ɑin synovial fluid.HE staining was used to observe the histopathological characteristics of the joint;Real-time fluorescent quantitative polymerase chain reaction(RT-q PCR)was used to detect the mRNA expression of NLRP3,ASC,and caspase-1 in soft tissues around joints;Western Blotting(WB)was used to detect the relative expression of NF-κB,ASC,and caspase-1.[Result]HE staining showed that the joint tissues of model group were pathologically damaged,while madecassoside had an alleviation effect on that damage in a dose-dependent manner.The concentration of IL-1β,TNF-ɑand expression of NLRP3,ASC,caspase-1 and NF-κB in model group were higher than those in control group(P<0.05);The concentration of IL-1β,TNF-ɑand the expression of NLRP3,ASC,caspase-1,and NF-κB in three dose groups were lower than those in model group(P<0.05),moreover,the inhibitory effect of madecassoside on the elevation of IL-1β,TNF-ɑ,NLRP3,ASC,caspase-1 and NF-κB had a dose-dependent manner.[Conclusion]Madecassoside can decrease the levels of IL-1β,TNF-αand NLRP3,activation of NLRP3 signaling pathway is associated with gouty arthritis.Madecassoside have the treatment of gouty arthritis,the related molecular mechanism may be related to its mediation effect on NLRP3 inflammatory body,axissignaling pathway and inhibit the release of inflammatory factors.