氧化苦参碱治疗小鼠胶原诱导性关节炎的作用机制

Mechanism of oxymatrine in treatment of collagen-induced arthritis in mice

苦参为豆科槐属植物苦参Sophorae flavescenstis的干燥根,具有抗炎、镇痛、抑菌、抗病毒、抗肿瘤等药理活性,在医药领域有广泛应用。该实验以苦参中的有效成分氧化苦参碱(oxymatrine,OMT)为研究对象,探究氧化苦参碱(oxymatrine,OMT)经“TLR9-MyD88-STAT3”通路对胶原诱导性关节炎(collagen-induced arthritis,CIA)小鼠B细胞的功能影响从而发挥抗类风湿关节炎的作用。构建牛Ⅱ型胶原和完全弗氏佐剂诱导的DBA/1J胶原诱导性关节炎(collagen-induced arthritis,CIA)小鼠模型,首次免疫后第15天腹腔注射OMT,持续30 d,每隔3 d监测CIA小鼠的关节肿胀度及关节评分;HE染色法观察关节组织病理学情况;采用免疫磁珠分离法(MACS)分选出CIA小鼠脾脏B淋巴细胞;运用ELISA(enzyme-linked immunosorbent assay)法检测小鼠血清IL-21的表达水平;Western blot检测B淋巴细胞上Toll样受体9(Toll-like receptors 9,TLR9)、信号转导及转录激活因子3(signal transducers and activators of transcription 3,STAT3)、磷酸化信号转导及转录激活因子3(phosphorylated signal transducers and activators of transcription3,p-STAT3)和白细胞介素21(interleukin-21,IL-21)的表达水平;实时荧光定量PCR(qRT-PCR)法检测B淋巴细胞上TLR9、STAT3、IL-21 mRNA的表达水平。结果表明OMT缓解CIA小鼠足爪肿胀度、降低关节炎指数、减轻滑膜炎性细胞浸润及增生程度,降低炎症因子水平,同时抑制B淋巴细胞中TLR9、STAT3、p-STAT3、IL-21的表达。因此,OMT可能通过调节B淋巴细胞上TLR9-STAT3通路从而缓解类风湿关节炎,为临床应用氧化苦参碱治疗类风湿关节炎提供有价值的依据。

Sophorae Flavescentis Radix,the root of Sophora flavescens Ait.,has been widely applied in the medical field due to its anti-inflammatory,analgesic,bacteriostatic,antiviral,antitumor,and other pharmacological effects.The present study investigated the anti-rheumatoid arthritis effect of oxymatrine(OMT),the active component of Sophorae Flavescentis Radix by observing its effect on the function of B lymphocytes in collagen-induced arthritis(CIA)mice through the Toll-like receptor 9(TLR9)/myeloid differentiation factor 88(MyD88)/signal transducer and activator of transcription 3(STAT3)pathway.The CIA model in DBA/1J mice was induced by bovine typeⅡcollagen and complete Freund's adjuvant(CFA).Fifteen days after the primary immunization,mice were treated with OMT for 30 days by intraperitoneal injection.Paw swelling and arthritis index(AI)score were evaluated every 3 days.Joint histopathologic changes were observed by HE staining.Magnetic-activated cell sorting(MACS)was used to isolate B lymphocytes from the spleen of CIA mice spleen.The serum expression level of interleukin(IL)-21 was examined by the enzyme-linked immunosorbent assay(ELISA).The expression of TLR9,STAT3,p-STAT3,and IL-21 in B lymphocytes was detected by Western blot.The mRNA expression of TLR9,STAT3,and IL-21 in B lymphocytes was detected by real-time fluorescence-based quantitative PCR(qRT-PCR).The results showed that OMT could significantly alleviate the paw swelling,decrease the AI score,relieve synovial inflammatory cell infiltration and hyperplasia,reduce the level of inflammatory cytokines,and inhibit the expression of TLR9,STAT3,p-STAT3,and IL-21 of B lymphocytes in CIA mice.Therefore,OMT may alleviate rheumatoid arthritis by regulating TLR9/MyD88/STAT3 pathway in B lymphocytes,providing a valuable reference for the application of OMT in the clinical treatment of rheumatoid arthritis.