Stephanthraniline A 对CD4+ T细胞活化的抑制作用及其分子机制

Recent findings suggest that Hedgehog signaling pathway serves as a co-stimulatory signaling in T cell activation. Our previous results indicated that the inhibitory effects of Stephanthraniline A (STA) on the activation of CD4+ T cells was mainly related to Hedgehog signaling pathway. Thus, in this project, we plan to investigate the inhibitory effect of STA on the activation of CD4+ T cells through Hedgehog pathway and its molecular mechanism. CD4+ T cells activated by TCR/Hedgehog co-stimulation were treated with STA. The inhibitory effects of STA on the activation of CD4+ T cells were evaluated by determining cell proliferation, cell cycle, expression of surface antigens and the elaboration of cytokines. Then, to elucidate the potential molecular targets of STA, the Mouse Hedgehog Signaling Pathway RT2 Profiler? PCR Array and Western blot were performed to profile the expression of 84 genes related to Hedgehog pathway and responsive key protein, respectively. The significantly, differentially expressed genes and proteins were further confirmed using gene-loss or overexpressing cell, signal molecular inhibitors, agonist or antagonist, etc.

Hedgehog通路系T细胞活化一种新发现的非特异性协同刺激信号通路。前期研究推测Stephanthraniline A (STA)主要通过Hedgehog (Hh)通路抑制CD4+ T细胞的活化发挥免疫抑制作用。本项目拟针对Hh通路深入探讨STA对CD4+ T细胞活化的抑制作用及分子机制。首先，以Hh协同TCR信号激活CD4+ T细胞，观察STA抑制活化CD4+ T细胞的作用特点，并采用定量PCR芯片结合蛋白分析筛选STA调控CD4+ T细胞Hh通路的潜在靶分子。在此基础上，采用基因缺失或过表达细胞、分子特异性抑制剂或激动剂等手段研究潜在靶分子在STA调控Hh通路中的作用，确定STA调控的信号靶分子，揭示STA抑制CD4+ T细胞活化的分子机制，为免疫抑制剂分子机制研究开辟新途径和奠定理论基础。

Stephanthraniline A(STA)是我们课题组前期从中药中经化学分离和活性筛选发现的具有较好免疫抑制活性的天然C21甾体化合物。本研究的目的是进一步对STA抑制T细胞免疫反应的作用进行评价，并对其机制进行深入研究。.本项目我们发现1. STA体外能抑制T细胞的激活和增殖，且对CD4+ T细胞亚型的抑制作用比CD8+ T细胞敏感。.2. STA可通过阻止细胞进入分裂周期抑制T细胞的增殖，但不会促进激活诱导的T细胞凋亡。.3. 区别于传统甾体类抗炎药物，STA与GR无竞争性结合，不依赖于GR起免疫抑制作用。.4. 区别于钙调蛋白酶抑制剂(calcineur ininhibitors, CNIs)，STA抑制T细胞激活不是通过TCR近端信号和Ca2+信号。.5. PKCθ极可能是STA作用的分子靶点。STA可通过氢键结合的方式连接于PKCθ的ATP口袋域，从而抑制PKCθ的磷酸化和活性，进而抑制PKCθ下游NFAT、NFκB和MAPK信号级联传递，最终抑制T细胞的活化。.6. STA抑制T细胞活化和增殖还可能与其调控多重信号通路的传导有关。.7. 腹腔注射或口服给予STA在体内对T细胞介导的免疫性疾病模型均显示较好的治疗作用。STA通过抑制CD4+ T细胞活化和在肝脏聚集，从而缓解Con A诱导的爆发性肝炎。.8. STA与CsA可通过互补的分子机制，或可能提高CsA的体内生物利用度，在体内外发挥协同作用。.以上结果为STA作为用于治疗T细胞介导的炎症和自身免疫性疾病的免疫抑制先导化合物的研究奠定了基础。