基于TLR2/p38 MAPK/NF-κB信号通路探讨独活寄生汤对类风湿性关节炎大鼠的抗炎作用及机制

Anti-inflammatory Effect and Mechanism of Duhuo Jishengtang on Rheumatoid Arthritis Rats Based on TLR2/p38 MAPK/NF-κB Signaling Pathway

目的:探讨独活寄生汤对胶原诱导性关节炎(CIA)模型大鼠的抗炎作用及其对Toll样受体2(TLR2)/p38丝裂原活化蛋白激酶(p38 MAPK)/核转录因子-κB(NF-κB)信号通路的影响。方法:将48只雄性SD大鼠,随机分为以下6组(n=8),正常组、模型组、甲氨蝶呤组、独活寄生汤低、中、高剂量组。采用胶原抗体诱导法于大鼠尾根部注射牛Ⅱ型胶原蛋白建立CIA大鼠模型,模型建立成功后进行灌胃给药。甲氨蝶呤组每次给予2.0 mg·kg^(-1)甲氨蝶呤,每周3次;独活寄生汤低、中、高剂量组每次给予3.8、7.6、15.2 g·kg^(-1)·d^(-1),连续灌胃治疗28 d;正常组和模型组给予等体积生理盐水。通过记录大鼠体质量,观察大鼠足肿胀度,测定关节炎指数、免疫器官指数,微循环检测仪检测大鼠微循环指标变化,苏木素-伊红(HE)染色检测大鼠滑膜组织病理形态的改变及流式细胞术检测滑膜细胞凋亡率以明确独活寄生汤对类风湿性关节炎的治疗作用,采用酶联免疫吸附测定法(ELISA)检测血清中肿瘤坏死因子-α(TNF-α)、白细胞介素(IL)-1β、IL-17A、γ干扰素(IFN-γ)水平变化,蛋白免疫印迹法(Western blot)检测TLR2、NF-κB p65、磷酸化NF-κB p65(p-NF-κB p65)、p38 MAPK、p-p38 MAPK蛋白表达。结果:与正常组比较,模型组大鼠体质量显著降低(P<0.01),足肿胀度、关节炎指数及免疫器官指数显著升高(P<0.01),微血管综合评分及血管阻力显著提高(P<0.01),病理切片可观察到滑膜组织增生明显、炎性细胞大量浸润,血清中TNF-α、IL-1β、IL-17A和IFN-γ表达及滑膜组织TLR2、p-NF-κB p65/NF-κB p65、p-p38 MAPK/p38 MAPK的表达水平均显著升高(P<0.01)。与模型组比较,独活寄生汤低、中、高剂量组大鼠体质量显著升高(P<0.01),足肿胀度、关节炎指数及免疫器官指数明显下降(P<0.05,P<0.01),微血管综合评分及血管阻力明显下降(P<0.05,P<0.01),滑膜组织病理学损伤情况明显好转,滑膜细胞凋亡率显著升高(P<0.01),血清中TNF-α、IL-1β、IL-17A和IFN-γ水平明显下降(P<0.05,P<0.01),滑膜组织TLR2、p-NF-κB p65/NF-κB p65、p-p38MAPK/p38 MAPK的表达水平均明显下降(P<0.05,P<0.01)。结论:独活寄生汤可能通过调节TLR2/p38 MAPK/NF-κB信号通路缓解CIA大鼠体内炎症反应,从而发挥其抗类风湿性关节炎作用。

Objective:To explore the anti-inflammatory effect of Duhuo Jishengtang(DHJST)on collagen-induced arthritis(CIA)model rats and its effect on the Toll-like receptor 2(TLR2)/p38 mitogenactivated protein kinase(MAPK)/nuclear factor-κB(NF-κB)signaling pathway.Method:Forty-eight male SD rats were randomly divided into the following six groups(n=8):normal group,model group,methotrexate(MTX)group,low-dose DHJST(DHJST-L)group,medium-dose DHJST(DHJST-M)group,and high-dose DHJST(DHJST-H)group.The CIA model was established by injecting bovine typeⅡcollagen into the rat tail root with the collagen antibody induction method.After model induction,rats were treated with drugs by gavage.The rats in the MTX group received MTX at 2.0 mg·kg^(-1),three times a week,and those in the DHJST groups received DHJST at 3.8,7.6,15.2 g·kg^(-1)·d^(-1) for 28 days.The rats in the normal group and the model group were given the same dose of normal saline.The weight of the rats was recorded,and the paw swelling degree was observed.The arthritis index and immune organ index were measured,and the changes in the microcirculation indexes of the rats were detected with a microcirculation detector.Hematoxylin-eosin(HE)staining was used to detect the pathological morphologic changes in rat synovial tissues and the apoptosis rate of synovial cells was detected by flow cytometry to determine the therapeutic effect of DHJST on rheumatoid arthritis.Enzyme-linked immunosorbent assay(ELISA)was used to detect the changes in serum levels of tumor necrosis factor-α(TNF-α),interleukin(IL)-1β,IL-17A,and interferon-γ(IFN-γ).The protein expression of TLR2,NF-κB p65,phosphorylated NF-κB p65(p-NF-κB p65),p38 MAPK,and p-p38 MAPK was detected by Western blot.Result:Compared with the normal group,the model group showed reduced body weight(P<0.01),increased paw swelling degree,arthritis index,and immune organ index(P<0.01),increased comprehensive microvascular score and vascular resistance(P<0.01),significant hyperplasia of synovial tissues and massive infiltration of inflammatory cells as revealed by pathological sections,and up-regulated expression levels of TNF-α,IL-1β,IL-17A,and IFN-γin serum,and TLR2,p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in synovial tissues(P<0.01).Compared with the model group,the DHJST groups showed increased body weight of rats(P<0.01),decreased paw swelling degree,arthritis index,and immune organ index(P<0.05,P<0.01),reduced comprehensive microvascular score and vascular resistance(P<0.05,P<0.01),improved synovial histopathological injury,increased apoptosis rate of synovial cells(P<0.01),and downregulated levels of TNF-α,IL-1β,IL-17A,and IFN-γin serum(P<0.05,P<0.01)and TLR2,p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in synovial tissues(P<0.05,P<0.01).Conclusion:DHJST may alleviate the inflammatory reaction in CIA rats by regulating the TLR2/p38 MAPK/NF-κB signaling pathway,thus exerting its anti-rheumatoid arthritis effect.