三叶青藤总黄酮干预Th17细胞增殖对小鼠胶原性关节炎作用及机制研究

The excessive proliferation of Th17 cells is an important pathogenesis of rheumatoid arthritis，which is induced by interleukin-23 and enhanced by prostaglandin E2（PGE2）through signal pathway IL-23R/JAK2/STAT3 and EP2-EP4/CAMP/PKA respectively. The herb of Illigera rhodantha Hance. is characteristic crude drugs of guangxi zhuang nationality, and widely used clinically. Our project group found total flavonoids in Illigera rhodantha Hance.（TFIR ）could decrease the amounts of PGE2 in inflammatory tissue, and alleviate the symptom of collagen-induced arthritis （CIA）in rats significantly，speculates it may inhibit Th17 cells proliferation, but it is unclear in the molecular mechanism. This project will establish Th17 cells proliferation model in vivo and in vitro，deeply reveal the intervention effects of TFIR on proliferation of Th17 cells ,as well as the signal transduction modulating mechanism for with aspect of Th17 cells quantity/proportion、dynamics of proliferation、differentiation stage 、cell cycle and apoptosis, by using technology such as flow cytometry and carboxyfluorescein diacetate succinimidyl ester( CFSE) labeling assay、western-blot、RT-PCR and so on, provide a scientific basis for application of the herb of Illigera rhodantha Hance..

Th17细胞过度增殖是类风湿性关节炎重要发病机制，IL-23和PGE2分别通过IL-23R/JAK2/STAT3和EP2-EP4/CAMP/PKA信号通路诱导及增强Th17细胞增殖。三叶青藤是广西壮族特色抗风湿药材，临床应用广泛。本项目组前期研究发现三叶青藤总黄酮（TFIR）显著降低炎性组织中PGE2含量，抑制胶原性关节炎大鼠关节肿胀率，推测其具有抑制Th17细胞增殖作用，但分子机制不详。本项目通过建立Th17细胞体内外增殖模型，运用CFSE荧光标记及胞内抗原染色结合流式细胞术、western-blot、RT-PCR等技术，从Th17细胞数量/比率、增殖动力学、分化阶段、细胞周期、凋亡等角度深入揭示TFIR对Th17细胞增殖的干预作用及信号转导调节机制，为三叶青藤应用提供科学依据。

Th17 细胞过度增殖是类风湿性关节炎重要发病机制，IL-23通过IL-23R/JAK2/STAT3信号通路诱导Th17 细胞增殖。三叶青藤是广西壮族特色抗风湿药材，临床应用广泛。本项目通过建立 Th17 细胞体内外增殖模型，运用胞内抗原染色结合流式细胞术、ELISA、RT-PCR 等技术，深入揭示三叶青藤正丁醇部位（nbuIR）对 Th17 细胞增殖的干预作用及信号转导调节机制。本项目证实了nbuIR显著下调CIA小鼠血清TNF-α、PGE2、IL-6、IL-1β、IL-23和IL-17水平，减轻CIA小鼠关节滑膜细胞增生与炎症细胞浸润程度，抑制体内外Th17细胞增殖，上调Treg细胞含量，抑制IL-17A、RORγt、IL-23R、JAK2、STAT3 mRNA表达水平，上调Foxp3 mRNA表达，说明nbuIR可能通过抑制IL-23R/JAK2/STAT3轴的作用从而抑制Th17细胞增殖；并通过调节Th17/Treg比率来调控机体免疫平衡。此外，项目组对nbuIR的化学成分进行了研究,从nbuIR中首次分离到8个已知化合物：赤式-紫丁香酰甘油（1）、黑风藤苷A（2），黑风藤苷（3）、阿江榄仁树葡糖苷Ⅱ（4）、β-谷甾醇（5）、没食子酸 (6)、没食子酸甲酯 (7)、没食子酸乙酯 (8)。其中化合物1～4和6～8为首次从该属植物中分离到。为三叶青藤的进一步开发利用提供了科学依据。