艾灸调节胶原诱导的关节炎小鼠Treg/Th17细胞及Galectin-9实验研究

Effect of Moxibustion Therapy on Th17/Treg Cells and Galectin-9 in Rheumatoid Arthritis Mice

目的：探讨艾灸对小鼠胶原诱导的关节炎（CIA）炎症、Th17和Treg细胞的影响及Galectin-9在其中的作用。方法：复制Ⅱ型胶原诱导的类风湿关节炎小鼠模型并予以艾灸治疗,观察艾灸治疗及Galectin-9注射对关节肿胀、关节滑膜病理表现的影响,流式细胞术分析Th17、Treg细胞变化,ELISA检测相关细胞因子IL-1β、IL-6、IL-10、IL-17、TGF-β、Galectin-9的含量。结果：CIA模型建立后,小鼠关节明显红肿,关节面粗糙、滑膜层变薄。经治疗,艾灸组、Galectin-9组小鼠关节红肿相对模型组有较明显的消退;而不治疗组、PBS组小鼠关节红肿无明显消退。上述4组小鼠关节评分组间差异经秩和检验比较,有统计学意义（P=0.04）。经艾灸治疗的小鼠病变关节面恢复平整,滑膜层较治疗前变厚,接近正常组。CIA模型小鼠Treg细胞显著下降、Th17细胞明显上升（P〈0.01）;艾灸治疗和Galectin-9注入后,Treg细胞的数量有显著上升（P〈0.01）,且两组无明显差异（P=0.36）;艾灸治疗和Galectin-9注入后,Th17细胞数量显著性下降（P〈0.01）,且两组无明显差异（P=0.71）。细胞因子Galectin-9在CIA模型小鼠脾脏中含量降低（P〈0.01）,艾灸治疗和Galectin-9均能使其增高（P〈0.01）,但艾灸治疗组不及Galectin-9组（P〈0.01）。CIA模型小鼠脾脏中IL-1β、IL-6、IL-17等致炎因子表达增高（P〈0.01）;艾灸治疗及注入Galectin-9后它们的含量相应降低（P〈0.01）,与不治疗组、PBS组比较差异有显著的统计学意义（P〈0.01）;而抑炎因子IL-10、TGF-β在CIA模型小鼠脾脏中表达降低,艾灸治疗及Galectin-9注入后它们的表达明显提高（P〈0.01）,与不治疗组、PBS组比较有显著的统计学差异（P〈0.01）;Galectin-9注入后,Galectin-9组IL-1β、IL-6、IL-17含量提高（P〈0.01）,与PBS组比较有显著的统计学差异（P〈0.01）。艾灸组中IL-1β、IL-6、IL-17、IL-10、TGF-β含量与Galectin-9组比较有显著差异（P〈0.01,P〈0.01,P〈0.01,P〈0.01,P=0.01）。结论：艾灸治疗能改善CIA小鼠关节炎症,调节Th17/Treg细胞失衡;其机制可能与促进Galectin-9分泌有关。

Objective：To observe the effect of moxibustion on arthritis,Treg/Th17 imbalance in mice of collagen induced arthritis（CIA）and the role of Galectin-9. Methods：Mice model of collagen induced arthritis was induced by injection of type Ⅱ collagen. Moxibustion treatment,Galectin-9,PBS were given to CIA mice,compared with none treatment. The ankle swelling was evaluated by RA scale,the pathological changes of articular cavity and synovial membrane were observed by HE analysis. Treg/Th17 imbalance were detected by flow cytometry. The expressions of cytokine IL-1β,IL-6,IL-10,IL-17,TGF-β,Galectin-9 were analyzed by ELISA. Results：Mouse model of collagen induced arthritis was successful established by injection of typeⅡcollagen,in which the swelling joint synovial layer were roughness and thinner than normal mice. The swelling joint of CIA mice partly recovered in moxibustion group and Galectin-9 group,while little such effect were observed in none treatment group and PBS group. Difference were shown between the four groups by rank sum test analysis（P=0.04）. The synovial layer were more flat and thicker in moxibustion treatment group comparing to the model group which was close to the normal group. Regulatory T cells decreased and Th17 cells increased in CIA mice spleen（P〈0.01）by flow cytometry;Treg cells increased in moxibustion group and Galectin-9 group（P〈0.01）,no difference was observed between the two groups groups（P=0.36）;Th17 cells decreased in moxibustion group and Galectin-9 group（P〈0.01）,and no difference was observed between the two groups group（P=0.71）. Galectin-9 decreased in CIA mice,which were up regulated by moxibustion and Galectin-9 injection（P〈0.01）,but differences were observed between the two groups（P〈0.01）. Cytokines IL-1β,IL-6,IL-17 increased in CIA mice which were down regulated by moxibustion and Galectin-9 injection（P〈0.01）,but no differences were observed in none treatment group and PBS group compared with model group. IL-10,TGF-β decreased in CIA mice,which were up regulated by moxibustion and Galectin-9 injection（P〈0.01）,but no differences were observed in none treatment group and PBS group compared with model group;after Galectin-9 injection,the levels of IL-1β,IL-6 and IL-17 in Galectin-9 group were increased（P〈0.01）,and there was a significant difference compared with PBS group（P〈0.01）. The contents of IL-1β,IL-6,IL-17,IL-10,and TGF-βin moxibustion group were significantly different from those in Galectin-9 group（P〈0.01,P〈0.01,P〈0.01,P〈0.01,P=0.01）. Conclusion：Moxibustion could improve the joint swelling and joint synovial pathological changes in CIA mice,and the mechanism might be related to the regulation of Treg and Th17 cells number by promotion of Galectin-9 secretion.