基于PPARγ和Th17细胞探讨槲皮素抗类风湿关节炎的机制

Quercetin (QU) remarkably attenuates rheumatoid arthritis (RA). However, the underlying mechanism has not been revealed. Th17 cell differentiation plays a crucial role in the pathogenesis of RA. Our previous research found that QU dramatically inhibited the differentiation of Th17 cells in collagen-induced arthritis rats, and the inhibitory effect was probably related to peroxisome proliferator-activated receptor γ (PPARγ). It was reported that there is a close correspondence between the the inhibitory effect of PPARγ on Th17 cells and the activation of signal transducer and activator of transcription 3 (STAT3). We propose a hypothesis: the mechanism of the antiarthritic efficacy of QU is that QU selectively activates PPARγ, then the activation of PPARγ encumbers the differentiation of Th17 cells via dual supression of STAT3. In this study, antibody array, flow cytometry and conditional gene knockout technology will be employed to investigate the relationship between the suppressive effect of QU on RA and inhibition of Th17 cell differentiation and find out the central target. Additionally, we also take the small molecule-DNA interaction and small molecule-protein interaction as breakthrough points to discuss, from the perspective of dual supression of STAT3, to probe the explicit mechanism of the inhibition of Th17 cell differentiation induced by QU based on PPARγ activation by using point mutation, ChIP, IP and luciferase reporter assay. This research would provide a bedrock to develop high efficient and low toxic new drugs of Traditional Chinese Medicine for the treatment of RA .

中药活性成分槲皮素（QU）对类风湿关节炎(RA)疗效显著且安全性高，但其作用机制尚不明确。Th17细胞分化在RA发病过程中起着关键作用。课题组前期研究表明QU可抑制胶原诱导性关节炎大鼠Th17细胞分化，且该作用或依赖于激活过氧化物酶体增殖物活化受体γ（PPARγ）；另文献证实，PPARγ对Th17细胞的抑制作用与转录因子STAT3密切相关。因此我们提出假说：“靶向激活PPARγ-双重抑制STAT3-抑制Th17细胞分化”是QU干预RA的重要途径。本项目拟运用抗体芯片、流式细胞术、条件基因敲除等方法探究QU抗RA作用与抑制Th17细胞分化的相关性并寻找其关键作用靶点；以小分子与DNA及蛋白互作为切入点，运用点突变、CHIP、IP、荧光素酶报告基因系统等方法，从双重抑制STAT3的角度探讨QU基于激活PPARγ抑制Th17细胞分化的确切机制，为研制高效低毒的抗RA中药新药奠定理论基础。

本课题通过流式细胞检测技术、激光共聚焦免疫荧光等技术手段分别在动物和细胞层面明确了槲皮素抗类风湿关节炎的药效作用、抗Th17细胞分化效应以及二者之间的关系。利用western blot、PCR、转染、免疫共沉淀等实验技术揭示了槲皮素作为PPARγ激动剂的抗Th17细胞分化的机制：一方面可激活PPARγ转录活性从而上调Socs3表达直接抑制STAT3通路，同时PPARγ的激活可导致转录抑制因子SMRT 与STAT3结合的增加，发挥双重阻断STAT3通路作用。基于荧光素酶报告基因系统、EMSA和ChIP等实验技术验证了PPARγ对Socs3基因启动子的调控位点。为槲皮素作为高效低毒的抗RA中药新药研究奠定理论基础。