断藤益母汤对类风湿关节炎成纤维样滑膜细胞MEKK2及CIA小鼠关节炎的影响

Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice

目的:为了研究断藤益母汤(DTYMD)在类风湿关节炎中对丝裂原活化蛋白激酶(MAPK)上游激酶的调控作用,阐明DTYMD抗炎的分子机制。方法:培养成纤维样滑膜细胞,将细胞分为空白组,模型组,DTYMD高、中、低质量浓度组(1000,800,600 mg·L^-1),甲氨蝶呤(MTX)组(20μmol·L^-1)组,采用蛋白免疫印迹法(Western blot),实时荧光定量聚合酶链式反应(Real-time PCR)对丝分裂原活化蛋白激酶激酶激酶2(MEKK2)的蛋白和mRNA表达进行分析。将42只雄性DBA/1J小鼠随机分为6组,每组7只,分别为正常组,模型组,MTX组(2 mg·kg-1),DTYMD低、中、高剂量组(6.25,12.5,25 mg·kg-1)。除正常组外,其他5组均采用二次免疫法构建胶原诱导型关节炎(CIA)模型。给药结束后,取小鼠后肢踝关节行苏木素-伊红(HE)染色并进行关节病理评分。结果:与模型组比较,DTYMD以浓度依赖方式抑制成纤维样滑膜细胞活性(P<0.01);与空白组比较,模型组细胞增殖率升高(P<0.01)。与模型组比较,DTYMD高、中质量浓度组降低MEKK2蛋白及mRNA的表达(P<0.05,P<0.01)。与模型组比较,DTYMD不同剂量组均降低细胞基质金属蛋白酶-1(MMP-1),肿瘤坏死因子-α(TNF-α),白细胞介素-6(IL-6)的表达(P<0.01);与空白组比较,模型组MMP-1,IL-6,TNF-α表达明显上升(P<0.05,P<0.01)。在动物实验中,与模型组比较,DTYMD高、中剂量组可以降低CIA小鼠关节肿胀度(P<0.05,P<0.01);与正常组比较,模型组小鼠二次免疫后关节明显肿胀(P<0.05)。在CIA小鼠踝关节HE染色中,与空白组比较,模型组小鼠病理学评分升高(P<0.01);与模型组比较,DTYMD高、中剂量小鼠关节病理评分明显下降(P<0.05,P<0.01)。结论:DTYMD可能通过下调MEKK2对细胞因子IL-6,TNF-α,MMP-1负性调控,从而缓解类风湿关节炎的炎症反应。

Objective:Duanteng Yimu decoction(DTYMD)is effective in treatment of rheumatoid arthritis(RA)by relieving joint inflammation and down-regulating some inflammatory factors in a short period of time,but the mechanism is still unclear.We aimed to investigate upstream kinase of mitogen activated protein kinases(MAPK)and define the anti-inflammatory mechanism of DTYMD.Method:Fibroblasts-like synovial cells(FLSs)were divided into blank group,model group(IL^-1β),high-dose DTYMD group(1000 mg·L^-1),medium-dose DTYMD group(800 mg·L^-1),low-dose DTYMD group(600 mg·L^-1)and armour ammonia butterfly(MTX)group(20μmol·L^-1).The protein and mRNA expressions of mitogen-activated protein kinase kinase kinase 2(MEKK2)were analyzed by real-time fluorescence quantitative PCR(Real-time PCR).Totally 42 male DBA/1 J mice were randomly divided into 6 groups,with 7 mice in each group,namely normal group,model group and MTX group(2 mg·kg-1),low-dose DTYMD group(6.25 mg·kg-1),medium-dose DTYMD group(12.5 mg·kg-1),and high-dose DTYMD group(25 mg·kg-1).Except for the normal group,the other five groups were included in collagen-induced arthritis(CIA)model by secondary immunoassay.After administration,the posterior limbs and ankle joints were stained with htoxylin-eosin(HE),and the pathological scores of the joints were evaluated.Result:Compared with the model group,DTYMD inhibited the activity of FLSs in a concentration-dependent manner(P<0.01).Compared with the blank control group,the cell proliferation rate of the model group increased(P<0.01).Compared with the model group,high and middle-dose DTYMD groups could inhibit protein and mRNA expressions of MEKK2(P<0.01),but there was no significant difference in lowdose group.However,the expression of DTYMD protein in high/medium/low-dose groups was significantly higher than that in blank group(P<0.01),but there was no significant difference in MTX group.Compared with the model group,the expressions of matrix metalloprotease-1(MMP-1),tumor necrosis factor-α(TNF-α)and interleukin(IL)-6 were negatively regulated in different DTYMD groups(P<0.01),and the expressions of MMP-1,IL-6,TNF-αin the model group were significantly higher than those in the blank group(P<0.05,P<0.01).In the animal experiment,compared with the model group,high/middle-dose DTYMD groups could decrease the degree of joint swelling in CIA mice(P<0.01),but there was no significant difference in the low dose group,and the joint swelling in the model group was significantly higher than that in the blank group(P<0.05).In HE staining of ankle joint of CIA mice,the pathological scores of high/small-dose DTYMD groups were significantly lower those of model group(P<0.05,P<0.01),and the pathological score of model group was higher than that of blank group(P<0.01).Conclusion:DTYMD might down-regulate MEKK2 to negatively regulate inflammatory cytokines IL-6,TNF-αand MMP-1,thereby alleviating the inflammatory response in rheumatoid arthritis.