克罗恩病(CD)是一种病因不明的胃肠道慢性非特异性炎症性疾病。本病不同程度地影响患者的消化吸收和营养状况，并易造成肠梗阻、穿孔以及肠瘘等严重并发症。虽然国际上已对其进行了多年的深入研究，但尚无令人满意的治疗手段，治疗药物有限，副作用大，效果不满意。 卫矛科植物雷公藤是祖国传统医学中一种常用的中草药，对多种免疫性疾病有显著的疗效，如类风湿性关节炎、系统性红斑狼疮等。多年来，我科应用雷公藤复合物-雷公藤多甙治疗CD患者，取得了令人满意的临床疗效，但目前国内外尚罕见雷公藤用于CD治疗的作用机理研究。 雷公藤甲素(Triptolide，PG490)是从雷公藤中分离出的活性最高的环氧化二萜内酯化合物，具有明显的抗炎和免疫抑制活性。IL-10基因敲除(IL-10〓)小鼠是公认较好的CD动物模型。本实验以IL-10〓小鼠为动物模型，观察并探讨雷公藤甲素对IL-10〓小鼠慢性肠炎的治疗作用，并进一步研究雷公藤甲素对于IL-10〓小鼠肠粘膜免疫的调节作用，为中药雷公藤及其有效成分在CD治疗的临床应用提供理论依据。 第一章 雷公藤甲素对白介素10基因敲除小鼠慢性肠炎发生发展的影响 背景：IL-10〓小鼠出生4周后可自发慢性肠炎，其发病机制、病变特征接近人类CD。我们观察雷公藤甲素给药后，对IL-10〓小鼠慢性肠炎的发生发展是否有抑制作用。 方法：建立IL-10〓小鼠慢性肠炎模型。选取4-5周龄雄性IL-10〓小鼠，等量分为给药组和对照组，给药组腹腔注射雷公藤甲素，对照组腹腔注射相同剂量生理盐水，连续8周，观察小鼠体重变化、结肠形态学和组织病理学改变，测定血清SAA水平，评估炎性活动度。 结果：雷公藤甲素可明显提高IL-10〓小鼠体重增长率，显著改善结肠慢性炎症损伤，降低组织学评分(4.625±1.061 vs7.250±1.669，P＜0.01)及血清SAA水平(17.845±3.667 vs 34.470±5.645，P＜0.01)。 结论：雷公藤甲素能够抑制 IL-10〓小鼠慢性的发生发展，有一定的治疗作用。 第二章 雷公藤甲素对白介素10基因敲除小鼠肠粘膜免疫的调节 第一部分：雷公藤甲素对IL-10〓小鼠Th1肠粘膜免疫的影响 背景：研究发现，肠粘膜树突状细胞及巨噬细胞(CD11c+)异常聚集，过度激活CD4+T细胞，导致Th1炎症因子(IL-12及IL-23、IFN-γ)过度释放是其发病的核心机制。本实验设计观察雷公藤甲素对于IL-10〓小鼠肠粘膜Th1免疫反应的调节作用。 方法：选取4-5周龄雄性IL-10〓小鼠，等量分为给药组和对照组，给药组腹腔注射雷公藤甲素0.07㎎/㎏，对照组腹腔注射相同剂量生理盐水，连续8周，取结肠组织，免疫荧光法观察雷公藤甲素对肠粘膜局部CD4+T细胞、CD11c+细胞浸润的调控，酶联免疫吸附测定(Elisa)结肠组织IFN-γ水平，实时定量聚合酶链反应实验(RT-PCR)测定结肠组织 IL-12 p35、IL-12/23 p40、IL-23 p19 mRNA基因表达水平。 结果：雷公藤甲素减少了 IL-10〓小鼠肠粘膜CD4+T细胞、CD11c+细胞的浸润分布，显著降低组织 IFN-γ 蛋白水平(17.106±3.391 vs24.68±5.539， P＜0.01)、 IL-12及IL-23基因表达。 结论：雷公藤甲素能够抑制Th1肠粘膜免疫的过度活化，抑制肠道慢性炎症的发生发展。 第二部分：雷公藤甲素对IL-10〓小鼠肠道TNF-α受体通路的影响 背景：TNF-α是参与CD炎症发生、发展的重要细胞因了，其主要通过细胞膜上的受休TNFR1和TNFR2发挥生物效应，大量实验证实：TNFR1信号通路在CD炎性反应中主要起保护作用，而TNFR2 - NF-κB信号通路在CD发病机制里起着关键作用。本实验通过对IL-10〓小鼠肠道TNFR1/2信号通路的研究，进一步探讨雷公藤甲素用于治疗慢性肠炎的作用机理。 方法：动物模型、给药方法同第一部分，Elisa法测定结肠组织TNF-α水平， RT-PCR法测定结肠组织TNFR1、TNFR2 mRNA基因表达水平，电泳迁移率变动分析实验(EMSA)测定结肠组织NF-κB活性。 结果：雷公藤甲素显著降低结肠组织TNF-α蛋白水平(TNF-α 38.474±8.767 vs 19.048±5.473，P＜0.01)及TNFR2 mRNA基因表达，并抑制结肠组织NF-κB活性。雷公藤甲素对TNFR1基因表达亦有抑制作用，仍活性明显低于对TNFR2。 结论：雷公藤甲素能够通过干扰TNF-α/TNFR2 - NF-κB信号通路，抑制肠道炎症的发生发展。根据其对TNFR的不同活性，我们推测雷公藤甲素在安全性方面具有潜在优势。 第三章 雷公藤甲素对成年白介素10基因敲除小鼠已确定性肠炎治疗及相关机制研究 背景：研究发现，CD患者的肠粘膜免疫随时间发展存在Th1向Th2转变的情况。肠道Toll样受体(TLR)是连接肠粘膜固有免疫及适应性免疫的重要因子。其中TLR4在CD发病机理中起着关键作用。本实验研究雷公藤甲素对成年IL-10〓小鼠已确定性肠炎的治疗作用，评估其用于克罗恩病诱导缓解治疗的可能性，并进一步探讨相关作用机理。 方法：选取15-16周龄，雄性IL-10〓小鼠，随机等量分为给药组及对照组。给药组每日腹腔注射雷公藤甲素，对照组每日腹腔注射等量生理盐水，共给药2周，通过临床评分、组织病理学评估结肠炎症程度改变，Elisa法测定结肠组织IL-4水平，Western blot方法测定结肠组织Toll样受体4 (TLR4)的蛋白表达， EMSA法测定结肠组织NF-κB活性。 结果：雷公藤甲素能明显减少成年IL-10〓小鼠临床评分、减轻肠道炎症损伤，停药后可维持至少3周时间。雷公藤甲素治疗可明显降低成年IL-10〓小鼠肠道IL-4 (101.993±7.176 vs 60.417±8.424，**p＜0.01)及 TLR4 蛋白表达水平，并抑制结肠组织NF-κB活性。 结论：雷公藤甲素对成年IL-10〓小鼠已确定肠炎具有一定的治疗效果。雷公藤甲素对诱导CD缓解，可能有一定疗效，且针对CD患者肠粘膜免疫类型的转换，雷公藤甲素也有着较好的适应性 本实验结果提示雷公藤甲素针对IL-10〓小鼠慢性肠炎具有良好的治疗效果。其主要通过减少巨噬细胞、树突状细胞及CD4+T细胞的聚集，干预IL-12/INF-γ、 IL-23、TNF-α/TNFR2 -NF-κB， TLR4- NF-κB 信号通路多种途径调节 IL-10〓小鼠异常的肠粘膜免疫，抑制炎症反应。我们推测雷公藤甲素具有用于CD临床治疗的价值。此外研究还发现雷公藤甲素对TNF-α/TNFR1信号通路影响小，且对Th2类炎症因子亦具有抑制作用，提示我们雷公藤甲素用于CD的治疗具有潜在的优势，这有待进一步证实。 关键词：雷公藤甲素；IL-10〓小鼠；肠粘膜免疫；Th1细胞因子；TNFR；IL-4； TLR4

 Crohn's disease (CD) is a chronic, idiopathic, and relapsing inflammatory bowel disease of the human gastrointestinal tract accompanied with many serious complications such as bowel obstruction and fistula. The etiology and pathogenesis of CD remain unknown. However, most therapies are still not satisfactory or entirely effective for the complex disorders of CD and new approaches are needed. Triptolide, a diterpene triepoxide, is an active component of extracts derived from a traditional Chinese herb Tripterygium wilfordii Hook, which has anti-inflammatory and immunosuppressive activities. Tripterygium extracts have been used to treat CD in Jinling hospital for many years with satisfactory effect. However, the therapeutic effect and mechanisms of triptolide on IBD is still unclear. In this study, we studied the possible therapeutic effects and intestinal mucosal immune regulation of triptolide on Interleukin-10 deficient mice, a well characterized murine model of CD. It may conduce to the application of tripterygium extracts on CD. Chapter 1: Effects of triptolide on the development of chronic colitis in IL-10 deficient mice Background: Since 4-5 weeks old, IL-10〓 mice would develop progressive chronic colitis with many similarities to CD. We observed the effect of triptolide on the development of colitis in IL-10〓 mice. Methods: Triptolide was administered to IL-10〓 mice, 4-5 weeks old, intraperitoneally every other day for 8 weeks. Control mice were similarly given an equivalent quantity of normal saline. The weight changes, the gross and histological appearances of the colon, the level of SAA were evaluated and compared with the control group. Results: The triptolide-treated IL-10〓 mice grew more steadily in contrast to the control mice. After administration, the inflammation of the colon in IL-10〓 mice was significantly improved. And the histological score (4.625±1.061 vs 7.250±1.669, P＜0.01) and the level of SAA (17.845±3.667 vs 34.470±5.645, P＜0.01) were also decreased. Conclusion: Treatment with triptolide protects against chronic colitis development in IL-10〓 mice. Chapter 2: Regulation of triptolide on intestinal mucosal immune response in IL-10 deficient mice Section Ⅰ: Effects of triptolide on the Th1 intestinal mucosal immune response in IL-10 deficient mice Background: Th1 cytokines (that is, IL-12, IFN-γ and IL-23), produced by CD4+ T cells, resident macrophages and dendritic cells, play a critical role in the pathogenesis of CD. This study was designed to investigate the effect of triptolide on the activated Th1 intestinal mucosal immune response in IL-10〓 mice. Methods: Triptolide was administered to IL-10〓mice, 4-5 weeks old, intraperitoneally every other day for 8 weeks. Control mice were similarly given an equivalent quantity of normal saline. The distributions of CD4+ T cells and CD11c+ cells in mucosa were measured by immunohistochemistry. The level of IFN-γ in colon was evaluated by ELISA. mRNA expressions of IL-12 and IL-23 in colon were measured by RT-PCR and compared with the control group. Results: The numbers of CD4+ T cells and CD11c+ cells in lamina propria were obviously reduced after triptolide treatment. Triptolide also significantly decreased the production of IFN-γ (17.106±3.391 vs 24.68±5.539, P＜0.01) in mucosa. Furthermore, triptolide suppressed IL-12 and Ⅱ-23 mRNA expressions in colon of IL-10〓 mice. Conclusion: Treatment with triptolide suppressed the activated Th1 intestinal mucosal immune response in IL-10 deficient mice. Section Ⅱ: Effects of triptolide on the TNFR signal pathway in IL-10 deficient mice Background: TNF-α is a multifunctional cytokine known to play a key role in the pathogenesis of CD. TNF-α exerts its effects via two TNF-α specific membrane bound receptors TNFR1 and TNFR2. More evidence proved that TNF-α/TNFR2-NF-κB signal pathway plays an important role in the pathogenesis of CD while TNF-signaling through TNFR1, perhaps, is protective in inflammatory disease states. This study was designed to investigate the effect of triptolide on the TNFR signal pathway in IL-10〓 mice. Methods: Drug and administration protocol was the same as the section Ⅰ. The level of TNF-α, mRNA expressions of TNFR and NF-κB activation in colon were measured and compared with the control group. Results: Triptolide significantly decreased the production of TNF-α (TNF-α 38.474±8.767 vs 19.048±5.473, P＜0.01) in mucosa. The mRNA expressions of TNFR1 and TNFR2 were both decreased after triptolide treatment. But the reduction of TNFR1 was much milder compared with that of TNFR2. Furthermore, NF-κB activation was significantly suppressed in treated IL-10〓 mice. Conclusion: Triptolide could ameliorate Th1-mediated chronic colitis in IL-10〓 mice by inhibiting TNF-α/TNFR2 signal pathway. We hypothesized that triptolide could be a safer and more effective therapeutic strategy with less suppression of TNFR1 under inflammatory conditions. Chapter 3: Effects and Mechanisms of triptolide on established colitis in elder IL-10 deficient mice Background: Present studies indicated changes in overall immunologic patterns over time, including a shift from predominantly Th1 to Th2 responses in the aging intestinal mucosa of CD. And evidence for a relevance of Toll-like receptors (TLR) , especially TLR4, in the development of colitis has also been derived mostly from clinical and experimental studies. In this study, we evaluated its therapeutic effects for established colitis in IL-10〓 mice. Methods: 16 weeks old interleukin-10 deficient mice with established colitis were intraperitoneally administrated for 2 weeks with triptolide. The clinical score, the histological appearances of the colon, the expressions of IL-4 and TLR4 and NF-κB activation were evaluated and compared with the control group. Results: The triptolide treatment reduced the severity of colitis in elder IL-10〓 mice. The administration also reduced expression of IL-4(101.993±7.176 vs 60.417±8.424, **p＜0.01) and TLR 4 in colon. Furthermore, the NF-κB activation in colon was also reduced after treatment. Conclusion: Administration of triptolide resulted in the reduction of the inflammatory conditions characteristic of colitis in elder IL-10〓 mice. Triptolide may hold potential therapeutic effect for inducing remission of the symptoms of human CD. In addition, triptolide might be effective for possible immunological changes of an aging mucosa. Summary Our data suggested that triptolide could ameliorate Th1-mediated chronic colitis and disordered immune state in IL-10〓 mice. The possible mechanisms could be reducing the numbers of CD4+ T cells and CD11c+ cells in lamina propria and inhibiting IL-12, IL-23, TNF-α/TNFR2 and TLR4 signal pathways. We conclude that triptolide may be applicable for maintenance therapy for the control or reduction of the symptoms in CD. Furthermore, triptolide could be a safer and more effective therapeutic strategy with the effect for inhibiting Th2-mediated inflammation and less suppression on TNFR1. It needs further studies to certify. Key words: triptolide; IL-10〓 mice; intestinal mucosal immune; Th1 cytokines; TNFR; IL-4; TLR4