积雪草苷对DBA/1小鼠胶原诱导型关节炎中Th17/Treg细胞表达的影响

Effect of Asiaticoside on Expression of Th17/Treg Cells in DBA/1 Mice with Collagen-Induced Arthritis

目的:观察积雪草苷对DBA/1小鼠胶原诱导型关节炎(CIA)中辅助性T细胞17(Th17)和调节性T细胞(Treg)表达的影响。方法:将SPF级DBA/1雄性小鼠按体质量随机分为6组:正常组,CIA组,甲氨蝶呤组(MTX组,0.5 mg·kg^(-1)),积雪草苷低、中、高剂量(5,15,45 mg·kg^(-1))组。除正常组外,其余组小鼠均构建CIA模型,分别于第1天给予牛Ⅱ型胶原和完全弗氏佐剂进行一次免疫,第21天给予牛Ⅱ型胶原和不完全弗氏佐剂进行二次免疫,并在二次免疫当天开始给药,MTX腹腔注射方式给药,积雪草苷灌胃方式给药,每天1次,共给药28 d。第49天小鼠取材后,小鼠关节组织进行苏木素-伊红(HE)染色观察病理改变;免疫组化检测Th17细胞标志物IL-17和Treg细胞标志物叉头样转录因子3(FoxP3)的表达情况;免疫荧光双染法检测小鼠关节组织CD4+T细胞中IL-17和FoxP3的表达情况;流式细胞术检测小鼠淋巴结中Th17和Treg细胞比例。结果:与正常组比较,CIA组小鼠关节结构严重紊乱,关节软骨及骨破坏明显,骨侵蚀严重(P<0.01);小鼠关节组织CD4及IL-17阳性染色明显增多,且积分吸光度IA值显著升高(P<0.01),FoxP3阳性染色减少且IA值显著降低(P<0.01),Th17/Treg值显著升高(P<0.01);小鼠淋巴结中Th17细胞表达比例明显增高(P<0.01),Treg细胞的表达比例显著减少(P<0.01);与CIA组比较,MTX组和积雪草苷各剂量组小鼠关节结构均相对正常,骨侵蚀、骨破坏较轻,关节面相对完整光滑;MTX组和积雪草苷中、高剂量组小鼠关节组织中CD4阳性染色减少且IA值明显降低(P<0.05);MTX组和积雪草苷各剂量组IL-17阳性染色明显减少且IA值明显降低(P<0.05,P<0.01);积雪草苷中、高剂量组FoxP3阳性表达明显增加且IA值明显升高(P<0.05,P<0.01);MTX组和积雪草苷中、高剂量组Th17/Treg值明显降低(P<0.05,P<0.01);小鼠淋巴结组织中,积雪草苷各剂量组Th17细胞表达比例明显降低(P<0.05,P<0.01),Treg细胞的表达比例明显增加(P<0.05,P<0.01)。结论:积雪草苷可抑制CIA小鼠Th17细胞的表达,促进Treg细胞的表达,从而调节Th17/Treg平衡。

Objective:To observe the effect of asiaticoside(AC)on the expression of T helper 17(Th17)cells and regulatory T(Treg)cells in DBA/1 mice with collagen-induced arthritis(CIA).Method:Male SPF DBA/1 mice were randomized into six groups according to body weight:control group,CIA group,methotrexate group(MTX group,ip,0.5 mg·kg^(-1)),and AC low-,medium-,and high-dose groups(ig,5,15,45 mg·kg^(-1),respectively).Modeling was performed in rats other than the control group.To be specific,they were immunized with bovine typeⅡcollagen and complete Freund’s adjuvant on the first day and with bovine typeⅡcollagen and incomplete Freund’s adjuvant on the 21 stday.Administration began on the day of the second immunization,once a day for 28 days.On the 49 thday,related tissues were collected.Then,hematoxylin-eosin(HE)staining was performed to observe the pathological changes of the joints.Immunohistochemical method was used to detect the expression of interleukin-17(IL-17)and forkhead box protein-3(FoxP3),the markers of Th17 and Treg cells,respectively,immunofluorescence double staining the expression of IL-17 and FoxP3 in CD4+T cells of mouse joint tissue,and flow cytometry the proportions of Th17 and Treg cells in mouse lymph nodes.Result:Compared with the control group,CIA group demonstrated joint disorder,damage of articular cartilage and bone,severe bone erosion(P<0.01),increase in stained CD4 and IL-17 and the integral absorbance(IA)(P<0.01),decrease in stained FoxP3 and the IA(P<0.01),rise of Th17/Treg ratio(P<0.01),elevation of Th17 expression in mouse lymph nodes(P<0.01),and reduction in Treg expression(P<0.01).Compared with CIA group,MTX group and three AC groups showed normal joints,alleviated bone erosion and damage,intact and smooth joint surface,and decrease in stained IL-17 and IA(P<0.05,P<0.01),and MTX group and AC medium-dose and high-dose groups registered decrease in stained CD4 and IA(P<0.01)and reduction in Th17/Treg ratio(P<0.05,P<0.01).Moreover,AC medium-dose and high-dose groups showed rise in stained FoxP3 and IA(P<0.05,P<0.01).In the lymph nodes of mice,decrease in expression of Th17 cells(P<0.05,P<0.01)and the increase in expression of Treg cells(P<0.05,P<0.01)were observed in all the three AC group.Conclusion:AC can regulate Th17/Treg balance by inhibiting the expression of Th17 cells and promoting the expression of Treg cells in CIA mice.