选择性CB2受体激动剂修复Th17/Treg细胞失衡治疗类风湿关节炎

Th17/Treg细胞失衡在RA的病理机制中起重要作用。我们已证明CB2受体激动剂治疗可有效减轻CIA鼠的关节炎。已获初步结果基础上，我们拟采用免疫组化、定量PCR、蛋白印迹、流式细胞术、RNA测序，检测CIA鼠经CB2受体激动剂慢性治疗后滑膜组织、淋巴结/脾淋巴细胞中Th17和Treg细胞的数量与功能、特异性转录因子的表达以及miRNA表达谱的变化；体外诱导Th17、Treg细胞分化，分析CB2受体激动剂对细胞的分化、扩增、功能的影响；通过病毒质粒转染抑制STAT3活化、增高STAT3活化、敲低SOCS3、上调和下调候选miRNA表达，研究SOCS3/STAT3、miRNA是否介导CB2受体激动剂对Th17/Treg的调节作用，双荧光素酶报告基因检测SOCS3、STAT3是否为候选miRNA的靶基因。进一步证明CB2受体激动剂治疗可纠正RA的Th17/Treg细胞失衡，阐明相关分子机制。

Th17/Treg cell imbalance plays a pivotal role in the pathogenesis of rheumatoid arthritis (RA). Our previous studies have demonstrated that selective agonists of canabinoid receptor 2 (CB2) effectively suppressed synovitis and joint destruction in mice with collagen II induced arthritis (CIA). Based on our preliminary findings, the samples of synovium, spleens and lymph nodes were isolated from CIA mice after chronic treatment with a CB2 agonist. Compared with vehicle treated mice, the changes in the amount of Th17 cells, Treg cells, specific transcription factors, cellular products and expression profile of microRNAs (miRNA) in the samples from CB2-agonist treated mice were analyzed with immunohistochemistry, quantitative PCR, Western blotting, flow cytometry or RNA sequencing. In vitro, differentiation of Th17 cells and Treg cells were induced under polarizing conditions, respectively. The potential effects of a CB2 agonist on differentiation, expansion and functions of Th17 cells and Treg cells are determined. Inhibition of STAT3 activation, over-activation of STAT3, down-regulation and up-regulation of candidate miRNAs, or knock-down of SOCS3 were performed by various plasmid transfection to study whether the SOCS3/STAT3 pathway or candidate miRNA mediates the modulatory effects of the CB2 agonist. Dual-luciferase reporter assay verified whether SOCS3 or STAT3 is the target of candidate miRNAs. By completing this study, it would be clarified whether CB2 agonists restore Th17/Treg cell imbalance in RA, and whether the miRNA/SOCS3/STAT3 pathway mediates the therapeutic effects.