小乌桂汤对胶原诱导性关节炎模型小鼠脾脏Th17/Treg平衡及血浆炎症因子的影响

Effects of Xiaowugui Decoction(小乌桂汤)on Th17/Treg Balance in the Spleen and Inflammatory Factors in Plasma of Collagen-induced Arthritis Model Mice

目的探讨小乌桂汤治疗类风湿关节炎(RA)的作用机制。方法60只雄性DBA/1小鼠随机分为正常组8只及造模组52只。造模组采用尾根部及背部分3点皮内注射鸡Ⅱ型胶原蛋白乳化液建立胶原诱导性关节炎(CIA)小鼠模型。造模成功的40只小鼠随机分成模型组、甲氨蝶呤组、小乌桂汤低剂量组、小乌桂汤中剂量组、小乌桂汤高剂量组,每组8只。甲氨蝶呤组予0.1mg/ml甲氨蝶呤药液2mg/kg灌胃,每周1次;小乌桂汤低、中、高剂量组分别予含生药19.5、39、75g/(kg·d)小乌桂汤灌胃,每日1次;正常组与模型组给予生理盐水0.2ml/10g灌胃,每日1次。4周后评定关节炎指数评分,测量发病足厚度,观察踝关节组织病理并进行组织病理学评分;检测脾脏辅助性T细胞17(Th17)、调节性T细胞(Treg)及血浆肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)、白细胞介素17(IL-17)、白细胞介素1β(IL-1β)、白细胞介素10(IL-10)的水平。结果与模型组比较,甲氨蝶呤组及小乌桂汤中、高剂量组关节炎指数评分,发病足厚度,脾脏Th17细胞水平,血浆TNF-α、IL-6、IL-17、IL-1β水平,关节病理总评分,血管翳及骨破坏评分均显著降低,Treg细胞水平均显著升高,甲氨蝶呤组及小乌桂汤高剂量组Th17/Treg值、滑膜炎评分显著降低(P<0.05或P<0.01)。与甲氨蝶呤组比较,小乌桂汤低剂量组脾脏Th17细胞水平、Th17/Treg值、关节病理总评分、血管翳及骨破坏评分显著升高,小乌桂汤低、中剂量组Treg细胞水平显著降低(P<0.01)。与小乌桂汤低剂量组比较,小乌桂汤中、高剂量组Th17细胞水平,Th17/Treg值显著降低,Treg细胞水平显著升高,小乌桂汤高剂量组病理总评分、血管翳评分显著降低(P<0.05或P<0.01)。与小乌桂汤中剂量组比较,小乌桂汤高剂量组Treg细胞水平显著升高(P<0.01)。结论小乌桂汤对CIA小鼠具有抗炎及免疫调节作用,以高剂量效果最佳,其作用机制与调节Th17/Treg的平衡,抑制TNF-α、IL-6、IL-17、IL-1β炎症因子的表达相关。

Objective To explore the mechanism of Xiaowugui Decoction(XWGD,小乌桂汤)for the treatment of rheumatoid arthritis.Methods Sixty male DBA/1 mice were randomly divided into normal group(n=8)and model group(n=52).To establish mice model of collagen-induced arthritis(CIA),chicken typeⅡcollagen emulsion was intracutaneously injected into the tail root and back of the mice at 3 points.Forty successful CIA model mice were randomly divided into model group,methotrexate group,XWGD low,medium,and high-dose groups,with 8 mice per group.The mice in methotrexate group were given 0.1 mg/ml methotrexate once a week,and the mice in XWGD low,medium and high-dose groups were respectively given 19.5,39,75 g/(kg·d)XWGD once a day.The mice in the normal group and model group were administered with 0.2 ml/10 g normal saline once a day.All the interventions were given by gavage for 4 weeks.The arthritis index score,thickness of the diseased paws and the histopathology of the ankle joint were evaluated.The ratio of helper T cell 17(Th17)and regulatory T cell(Treg)in spleens,and the expression levels of tumor necrosis factor-α(TNF-α),interleukin-6(IL-6),interleukin-17(IL-17),interleukin-1β(IL-1β)and interleukin-10(IL-10)in plasma were detected.Results Compared to model group,the methotrexate group and XWGD medium and high-dose groups significantly decreased the arthritis index score,the thickness of the diseased paws,the ratio of Th17,the expression levels of plasma TNF-α,IL-6,IL-17,and IL-1β,the total ankle joint pathology score,and the pannus-bone destruction score,as well as increased the ratio of Treg;the methotrexate group and XWGD high-dose group significantly decreased the ratio of Th17/Treg and the synovitis score(P<0.05 or P<0.01).Compared to methotrexate group,XWGD low-dose group had significantly higher Th17 and Th17/Treg in spleen as well as higher total ankle joint pathology score and pannus-bone destruction score;the XWGD low and medium-dose groups had significantly lower ratio of Treg(P<0.05 or P<0.01).Compared to XWGD low-dose group,the Th17 and Th17/Treg ratio in the medium and high groups significantly reduced,Treg level increased;the total ankle joint pathology score and the pannus-bone destruction score in the high-dose group also significantly decreased(P<0.05 or P<0.01).The level of Treg in XWGD high-dose group was significantly higher than that of medium-dose group(P<0.01).Conclusion XWGD has anti-inflammatory and immune regulation effects on CIA model mice,and the high-dose works best.It may reduce the joint inflammation by regulating the balance of Th17/Treg and inhibiting the expression of TNF-α,IL-6,IL-17,and IL-1β.