艾灸通过miR-144-3p抑制HIF-1α调节Th17/Treg细胞失衡治疗类风湿关节炎的机制研究

Rheumatoid arthritis (RA) is one of the major diseases that lead to disability at present. The imbalance of Th17/Treg cells has been shown as the significant features of the onset and development in RA. Moxibustion was proved effective in improving the symptoms of arthritis in patients with RA significantly. Previous study of our research group supported by NSFC indicated that moxibustion therapy on the points of Zusanli and Shenshu is effective in treating RA by regulating Th17/Treg cells differentiation, but the mechanism underline was not clear yet. Gene screening research and bioinformatics prognosis indicated that the keynote mechanism was supposed to be the regulation of miR-144-3p targeting HIF1-α. Therefore, we hypothesize that the therapeutic effect of moxibustion on RA is based on rebalancing Th17/Treg cells by miR-144-3p targeting HIF1-α. From this hypothesis, the collagen induced arthritis (CIA) mice model will be established and the therapeutic effect of moxibustion on arthritis in CIA mice will be evaluated. The molecular interaction will be studied through observing the Th17/Treg cell number in spleen and joint synovial membrane, detecting the related gene expressions. To reprove this discovery, the core role of miR-144-3p and HIF1-α will be proved by inhibitor. This study will be helpful to explain the modern biological connotation of moxibustion treating RA from the aspect of regulating immune imbalance, and provide a new basis for guiding its clinical application.

类风湿性关节炎（RA）是主要的致残性疾病，Th17/Treg 细胞失衡是其发病的关键环节之一。中医艾灸能显著改善RA患者关节炎症，课题组前期研究证实：艾灸足三里、肾俞可改善关节炎模型小鼠关节炎症，其机制与调节Th17/Treg细胞失衡有关，但具体作用靶点尚不明确。进一步靶基因筛选研究提示：艾灸调节上述细胞亚群分化的关键microRNA可能是miR-144-3p，其靶基因为缺氧诱导因子1-α（HIF1-α）。由此我们提出假说：艾灸通过miR-144-3p抑制HIF1-α调节Th17/Treg细胞失衡治疗RA。本研究利用CIA小鼠观察艾灸对关节形态、病理、炎症、T细胞分化、相关信号通路的影响；并通过抗体阻断反证miR-144-3p、HIF1-α在艾灸调节Th17/Treg细胞失衡中的核心作用。研究将从免疫调节角度，在整体、细胞、分子水平阐述艾灸治疗RA的科学内涵，为指导其临床应用提供实验依据。

类风湿关节炎(RA)是一种以滑膜炎症和软骨破坏为特征的慢性自身免疫性疾病。RA的发生与Treg/Th17的平衡有关，但其机制尚不清楚。本研究对艾灸改善RA的作用进行了研究，并探讨了其作用机制。采用胶原诱导关节炎(CIA)小鼠模型，观察艾灸对RA的改善作用。病理评分采用爪水肿和苏木精-伊红(H&E)染色。采用Elisa法检测血清中白细胞介素-1β (IL-1β)、白细胞介素-6 (IL-6)、肿瘤坏死因子- α (TNF-a)、干扰素-γ (IFN-γ)、白细胞介素-17 (IL-17)、白细胞介素-10 (IL-10)的表达。荧光激活细胞分选仪(FACS)检测Treg/Th17平衡。我们的结果证实：艾灸治疗可以减轻病理评分，降低IL-1β、IL-6、TNF-α、IFN-γ和IL-17的表达，而增加IL-10的表达。此外，艾灸治疗改善了Treg/Th17的平衡。通过qRT-PCR检测6个RA相关miRNAs的表达。在这6个miRNAs中，miR-221的表达被艾灸治疗抑制，而不是miR-144-3p。通过靶基因预测、双荧光素酶报告实验和qRT-PCR验证，我们发现SOCS3是miR-221的直接靶标，并通过调节Treg/Th17平衡介导艾灸抑制关节炎症的作用。总的来说，我们的研究表明艾灸通过调节类风湿性关节炎中的miR-221/SOCS3轴来改善Treg/Th17的平衡从而缓解RA关节炎症。