五味消毒饮下调IL-2抑制CD25+CD4+Treg细胞免疫应答在慢性骨髓炎中的作用研究

Chronic osteomyelitis is a common disease, while the treatment is difficult. Study found that the inhibition of CD4+CD25+ Treg cells might be involved in the pathogenesis of chronic osteomyelitis, and IL-2 could mediated the immune response of Treg cells through activation of STAT5. Our preliminary research suggested that wuweixiaoduyin had the possible effect on the treatment of chronic osteomyelitis. Meanwhile, studies have reported that wuweixiaoduyin could down regulate the expression of IL-2, Thus, we hypothesize that the possible effect of wuweixiaoduyin maybe related to the down-regulation of IL-2, which inhibit the STAT5-Foxp3 signal pathways and the immune response of CD4+CD25+ Treg cells. In this study, first, by using the chronic osteomyelitis rabbit model as the reasearch subject，we try to clarify the down-regulation of IL-2 by wuweixiaoduyin could inhibit the immune response of CD4+CD25+ Treg cells, secondly, using the STAT5 knock down rabbit, we try to further elucidate the mechanism that the low expression of IL-2 regulated by wuweixiaoduyin could inhibit the immune response of CD4+CD25+ Treg cells through STAT5-Foxp3 pathway. Therefore，our study aim to futher improve the mechanisms of immune abnormity in chronic osteomyelitis, and the final results of our research might provide new insights for the TCM treatment of Chronic osteomyelitis.

慢性骨髓炎是骨伤科常见疾病，治疗较为困难。研究发现，CD25+CD4+Treg细胞通过抑制T淋巴细胞免疫应答可能参与慢性骨髓炎的发病，而IL-2可以通过STAT5活化调控Treg细胞免疫应答。我们前期研究提示采用五味消毒饮治疗慢性骨髓炎具有优势。已有研究报道五味消毒饮可以下调IL-2表达，因此，我们认为五味消毒饮可能通过下调IL-2的表达抑制STAT5-Foxp3信号通路活化，后者进一步抑制Treg细胞免疫应答，从而在慢性骨髓炎中发挥治疗作用。本研究以慢性骨髓炎兔模型为研究对象，首先明确五味消毒饮下调IL-2抑制Treg细胞免疫应答在慢性骨髓炎中的治疗作用，其次，阐明五味消毒饮下调IL-2通过STAT5-Foxp3信号通路抑制Treg细胞免疫应答的机制。通过本研究将进一步完善慢性骨髓炎免疫调控的机制研究，并为中医药治疗慢性骨髓炎提供新的实验依据。

本研究以慢性骨髓炎免疫调异常为研究切入点,首次提出五味消毒饮是通过下调白细胞介素IL-2 抑制CD25+CD4+Treg 细胞免疫应答,进而在治疗慢性骨髓炎中发挥重要作用;首次提出五味消毒饮的作用机制是调控白细胞介素IL-2,并依赖STAT5-Foxp3 信号通路,进而介导CD25+CD4+Treg 细胞免疫应答..本研究主要内容:1.造模与取材:构建大鼠慢性骨髓炎模型,模型构建成功后五味消毒饮灌胃给药4周,具体如下:A正常组,B模型组,C五味消毒饮低剂量组,D五味消毒饮中剂量组,E五味消毒饮高剂量组;2.病理分析各组病理改变;3.ELISA检测1、2和4周各组血清中IL-2的表达;4.外周血中分离单个核细胞,流式细胞仪检测CD4+CD25+FOXP3 Treg比率;5.外周血中分离单个核细胞,磁珠进一步分选CD25+CD4+Treg细胞,Q-PCR和Western blot检测Foxp3和CTLA-4的表达,Western blot检测STAT5和p-STAT5的表达;6.体外实验部分:(1)慢性骨髓炎模型组大鼠磁珠分选CD25+CD4+Treg细胞,然后使用0、1、10、100U/ml的IL-2处理,Q-PCR和Western blot检测FOXP3的表达;(2)100U/ml的IL-2处理后的CD25+CD4+Treg细胞,分别使用2%、5%和10%的含药血清处理,ELISA检测细胞上清中IL-2、IL-10和TGF-β1的表达,Q-PCR和Western blot检测Foxp3的表达;(3)100U/ml的IL-2处理CD25+CD4+Treg细胞,10%的含药血清和1 μM的STAT5抑制剂处理,Western blot检测Foxp3、CTLA-4、STAT5和p-STAT5表达..通过本研究证实,五味消毒饮可以降低骨组织炎症反应和外周血IL-2浓度;下调Foxp3、CTLA-4和p-STAT5的表达;此外,IL-2对Foxp3表达呈剂量依赖性的促进作用;IL-2通过STAT5调控Foxp3和CTLA-4;含药血清可以通过抑制STAT5磷酸化,抑制IL-2的功能,进而抑制Foxp3和CTLA-4.这就进一步证明五味消毒饮治疗慢性骨髓炎,其机制是通过抑制STAT5磷酸化,抑制IL-2的功能,进而抑制Foxp3和CTLA-4，进而达到治疗慢性骨髓炎的目的。