青蒿素衍生物治疗小鼠结肠炎功能靶点探究

Crohn's disease (CD) is a chronic gastrointestinal transmural inflammatory disease, which tends to relapse frequently. Because the etiopathogenesis of CD remains unknown, the current therapeutic strategies are target at controlling the disease activity by inducing remission and maintaining remission for long-term. At present the effective medicines in practice are limited in kinds and the toxicity of immunomodulators and biologics are significant. More safer and affordable alternatives are urgently needed. Although the mechanisms of effective medicines are different, all of them show the same effect on promoting apoptosis of T cells, and the most powerful agent for CD treatment anti-tumor necrosis factor-a mononuclear antibody promotes apoptosis of macrophages as well. To the report, derivatives of artemisinin in vitro directly inhibited Th1 cell and macrophage in producing inflammatory cytokines, and induced spleen T cells apoptosis. Our preliminary experiments showed that artesunate alleviated TNBS and DSS colitis but not oxazolone colitis, the described effects of artesunate are similar to the effects of anti-tumor necrosis factor-a mononuclear antibody. The facts that artesunate has been clinically used in the treatment of systemic lupus erythematosus and rheumatoid arthritis patients with few side-effects for long term, and that increasing numbers of powerful water-soluble artemisinin derivatives are synthesized, we believe artemisinin derivatives have therapeutic potential for CD and may provide a new alternative for patients. Our project is to explore whether macrophage and/or dendritic cell is one of the functional target of derivatives of artemisinin, and confirm that derivatives of artemisinin promoting the apoptosis of intestinal mucosal T cells and/or macrophages.

克罗恩病病因未明，不能被根治，需要长期服用药物维持缓解；现有有效药物种类少，且免疫调节剂和生物制剂副作用不可忽视，更多高效低毒药物亟待发现。虽然有效药物的确切作用机制不同，但都能促进T细胞凋亡；其中疗效最强的抗肿瘤坏死因子-a单克隆抗体还能促进巨噬细胞凋亡。青蒿素衍生物体外直接抑制Th1细胞和巨噬细胞产生相应细胞因子，促进小鼠脾T细胞凋亡，我们的前期工作发现：青蒿琥酯显著减轻TNBS和DSS结肠炎却对oxazolone结肠炎无效，此结果与抗肿瘤坏死因子-a单克隆抗体一致。青蒿素衍生物曾被用于治疗类风湿性关节炎和系统性红斑狼疮，长期口服副作用少耐受性好。综合已有结果我们相信青蒿素衍生物从机制上能治疗克罗恩病。本课题将继续探讨青蒿素衍生物的功能靶点是否在巨噬细胞和/或树突状细胞上，并促进肠粘膜T细胞和/或巨噬细胞凋亡，为青蒿素衍生物临床治疗克罗恩病提供更多可信依据。

研究按计划进行，野生型C57BL/6小鼠三硝基苯磺酸和葡聚糖急性结肠炎结果均显示：青蒿琥酯的治疗降低炎症因子表达水平，组织学上肠炎显著减轻。 . 机制上我们进一步研究青蒿素对炎性细胞的作用，探讨作用靶点。虽然克罗恩病的发病原因尚不清楚，但抗TNF-a单抗能高效控制克罗恩病活动性，说明了TNF-a所起的关键作用。而TNF-a主要由巨噬细胞和树突状细胞产生。树突状细胞还产生IL-12，其单克隆抗体也能有效减轻克罗恩病活动性。因此我们重点观察了青蒿素对巨噬细胞和树突状细胞的作用（未见以往报道）。. 体外，分离骨髓细胞诱导分化成树突状细胞的实验发现，青蒿琥酯通过caspase-9介导的内源性凋亡途径（不是外源性途径），促进树突状细胞凋亡。然而腹腔巨噬细胞的凋亡没有受影响。改用人巨噬细胞白血病细胞株THP-1，得到的结果与树突状细胞一致。这两种细胞都具有分裂繁殖的特点。因此，青蒿素是通过促进分裂细胞的凋亡，减少新增炎症细胞参与而减轻炎症。. 体内，采用没有T细胞的RAG-1-/- C57BL/6小鼠（SCID只有BALB/c小鼠）葡聚糖结肠炎模型，观察到青蒿琥酯促进结肠黏膜中巨噬细胞和树突状细胞的凋亡，显著减少结肠炎巨噬细胞和树突状细胞的数量，减轻结肠炎症。. 综合体内外实验结果，我们证实了巨噬细胞和树突状细胞是青蒿琥酯的直接作用靶点。利用THP-1细胞株，我们还观察到青蒿琥酯与甲氨蝶呤/硫唑嘌呤/雷公藤有协同促进细胞凋亡的作用。青蒿素促进T细胞凋亡已有报道。 . 机制上我们还观察了青蒿琥酯对肠粘膜通透性的影响。青蒿琥酯治疗的葡聚糖急性结肠炎粘膜损伤显著减轻，提示了保护作用。除了组织学完整性，结肠粘膜通透性还受到多种细胞因子的调控，如TNF-a增加肠炎粘膜通透性，导致异体蛋白质不断进入机体，免疫应答持续，炎症不愈。体外实验中青蒿琥酯直接抑制腹腔巨噬细胞产生TNF-a。我们用FITC标记的右旋糖酐检测葡聚糖急性结肠炎，结果显示青蒿琥酯没有降低肠炎的粘膜通透性，提示有其他调节因素参与而不能被青蒿素抑制。虽然不是药物有效性的评判标准，但目前用于治疗克罗恩病的有效药物都能降低肠粘膜通透性。因此，未再进行深入实验研究。