青藤碱对胶原诱导性关节炎小鼠滤泡辅助性T细胞的影响

Effect of sinomenine on follicular helper T cell in collagen-induced arthritis mice

目的研究青藤碱对胶原诱导性关节炎(CIA)小鼠外周血滤泡辅助性T细胞(Tfh)的影响,探讨青藤碱治疗类风湿关节炎的可能机制。方法 35只DBA/1小鼠随机分为正常组(5只),模型组(10只),青藤碱组(10只),地塞米松组(10只)。青藤碱组予青藤碱溶液74 mg/(kg·d),地塞米松组予地塞米松0. 88 mg/(kg·2d),正常组和模型组予0. 9%氯化钠注射液10 m L/(kg·d),造模后1 d即实验第1 d,各组小鼠均后肢肌肉注射给药,连续用药58 d。实验第21 d开始,计算各组小鼠关节炎指数。实验第59 d处死小鼠,观察关节滑膜病理变化。收集眼眶血,应用流式细胞仪测定小鼠Tfh细胞比例,应用酶联免疫吸附法(ELISA)检测白细胞介素21(IL-21)水平,应用实时免疫荧光定量聚合酶链式反应(Real-time PCR)检测脾脏组织B细胞淋巴瘤-6(Bcl-6) mRNA水平,应用蛋白免疫印迹法(WB)检测脾脏组织Bcl-6蛋白水平。结果与正常组比较,模型组的关节炎指数、Tfh细胞比例和IL-21水平均升高(P <0. 01),脾脏组织Bcl-6mRNA表达、Bcl-6蛋白水平增加(P <0. 01);与模型组比较,青藤碱组和地塞米松组关节炎指数下降(P <0. 05,P <0. 01),青藤碱组Tfh细胞比例、IL-21水平、脾脏组织Bcl-6 mRNA表达、Bcl-6蛋白水平降低(P<0. 05,P <0. 01),地塞米松组Tfh细胞比例降低(P <0. 05),脾脏组织Bcl-6 mRNA表达、Bcl-6蛋白水平比较差异均无统计学意义(P> 0. 05)。结论青藤碱能下调CIA小鼠Bcl-6 mRNA和Bcl-6蛋白的表达,降低Tfh细胞比例,抑制IL-21水平,并改善小鼠的关节炎症状。

Objective To investigate the effects of sinomenine on peripheral blood follicular helper T cells(Tfh)in collagen-induced arthritis(CIA)mice,and to explore the possible mechanism of sinomenine in the treatment of rheumatoid arthritis.Methods 35 DBA/1 mice were randomly divided into normal group(5 mice),model group(10 mice),sinomenine group(10 mice),dexamethasone group(10 mice).The sinomenine group was given sinomenine solution 74 mg/(kg·d).The dexamethasone group was given dexamethasone 0.88 mg/(kg·2d).The normal group and model group were given 0.9%sodium chloride injection 10 mL/(kg·d).One day after the modeling,the experiment was performed for the first day,the mice in each group were given intramuscular injection of hind limbs for 58 days.Starting from the 21st day of the experiment,the arthritis index of each group of mice was calculated.The mice were sacrificed on the 59th day of the experiment and the pathological changes of the synovial membrane were observed.The orbital blood was collected,the proportion of Tfh cells in mice was determined by flow cytometry,and the level of interleukin 21(IL-21)was detected by enzyme-linked immunosorbent assay(ELISA).Real-time immunofluorescence quantitative polymerase chain reaction(Real-time PCR)was used to detect B-cell lymphoma-6(Bcl-6)mRNA levels in spleen,and Bcl-6 protein levels in spleen tissues were detected by Western blotting(WB).Results Compared with the normal group,the arthritis index and the Tfh cell ratio and the IL-21 were increased in the model group(P<0.01),the Bcl-6 mRNA expression and Bcl-6 protein level in spleen tissues were increased(P<0.01).Compared with the model group,the arthritis scores of the sinomenine group and the dexamethasone group decreased(P<0.05,P<0.01),the proportion of Tfh cells,IL-21 level,spleen tissue Bcl-6 mRNA expression and Bcl-6 protein level in sinomenine group decreased(P<0.05,P<0.01),the proportion of Tfh cells in dexamethasone group decreased(P<0.05),and there was no significant difference in Bcl-6 mRNA expression and Bcl-6 protein level between spleen tissues(P>0.05).Conclusion Sinomenine can down-regulate the expression of Bcl-6 mRNA and Bcl-6 protein in CIA mice,decrease the proportion of Tfh cells,inhibit the level of IL-21,and improve the arthritis symptoms in mice.