【作者】 高永健；

【导师】 钱家鸣； 朱峰； 何桂珍；

【作者基本信息】 北京协和医学院 ， 内科消化， 2010， 博士

【摘要】 研究背景炎症性肠病(inflammatory bowel disease, IBD)是常见且危害严重的消化系统疾病,近年来我国发病率呈明显上升。免疫调节异常在其发病机制中起了重要的作用。传统药物治疗如5—氨基水杨酸、糖皮质激素、免疫抑制剂以及新型生物制剂如英夫利昔(Infliximab)疗效肯定,但仍有小部分患者疗效不好,尤其是克罗恩病(Crohn’s disease,CD)的长期维持缓解疗效较差。近年来,中医基础和临床研究发展迅速,在IBD治疗方面取得了较好的疗效。黄芪为最常用的几种中药之一,被誉为“补气之最”、“疮家圣药”。已有研究发现黄芪提取物可以预防和治疗实验性结肠炎,但关于其主要生物活性成份—黄芪多糖(Astragalus polysaccarides, APS),对实验性结肠炎的治疗效果及免疫机制目前尚无研究。研究目的探讨APS对2,4,6-三硝基苯磺酸(TNBS)诱导的大鼠结肠炎的疗效及免疫机制。研究方法1.建立大鼠TNBS实验性结肠炎模型。40只SD雄性大鼠随机分为5组(n=8),分别为空白对照组、TNBS造模组、APS小剂量组、APS大剂量组和激素组。,空白对照组给予生理盐水灌肠,余4组给予TNBS灌肠。建模24小时后开始给予药物干预,APS小剂量组给予APS (0.5g/Kg.d)灌胃,APS大剂量组给予APS1.0g/Kg.d)灌胃,激素组给予泼尼松1.0mg/Kg.d)灌胃,造模组给予相同容积饮用水灌胃,疗程14天。每天观察大鼠体重、腹泻及便血情况,给药结束后处死动物。2.疗效评价：观察结肠大体病变及镜下病理标本炎症情况,测定肠道组织MPO活性。3.免疫机制研究：提取结肠组织总蛋白,应用ELISA方法检测IL-4、IL-10的水平,并用免疫组化和免疫印迹方法检测肠道组织中转录因子T-bet和GATA-3的表达。实验结果1.造模组大鼠结肠大体病变及镜下病理炎症评分,MPO活性均显著高于对照组(P=0.01)。APS小剂量治疗可减轻结肠大体及镜下炎症病变,但和对照组及造模组相比无统计学差异；可显著降低MPO活性(P=0.03)。APS大剂量组大体及镜下炎症病变评分与造模组接近,MPO活性显著高于对照组和APS小剂量组(P=0.003,0.008)。激素组大体、镜下炎症病变评分以及MPO活性均较造模组下降,但差异无统计学意义。2.造模组大鼠较对照组IL-4、IL-10水平下降,其中IL-10与对照组相比有统计学差异(P=0.007), APS小剂量治疗可升高IL-4、IL-10水平,但与造模组相比无统计学差异；APS大剂量治疗使IL-4进一步下降,但IL-10略有升高,与对照组相比有统计学差异(P=0.046,0.035)；激素组IL-4、IL-10均较造模组进一步下降,与对照组相比有统计学差异(P=0.049,0.001)。3.对照组大鼠肠道GATA-3表达高于T-bet表达,造模组T-bet和GATA-3较对照组均有升高,以T-bet升高较明显,使GATA-3/T-bet比值显著下降(P=0.025)。APS小剂量治疗可同时促进T-bet和GATA-3表达,与造模组相比均有统计学差异(P=0.04,0.019),但GATA-3表达升高更为显著,使GATA-3/T-bet比值升高。激素组T-bet和GATA-3表达较造模组均下降,T-bet下降较为明显,使GATA-3/Tbet比值较造模组升高。激素组GATA-3/Tbet比值高于APS小剂量组。实验小结1.不同剂量APS对TNBS诱导的大鼠结肠炎疗效不同,小剂量(0.5g/Kg.d)灌胃治疗2周可缓解TNBS诱导的大鼠结肠炎,疗效与泼尼松接近；而大剂量1.Og/Kg.d)治疗则轻度加重大鼠结肠炎。2.APS小剂量治疗可促进抗炎细胞因子IL-4和IL-10的表达,大剂量则抑制IL-4表达,但IL-10略有升高。小剂量APS免疫调节作用与促进转录因子GATA-3及T-bet的表达增加有关,其对GATA-3促进作用更明显。3.泼尼松具有免疫抑制作用,可降低IL-4和IL-10的水平,并可抑制转录因子GATA-3和T-bet的表达,但对T-bet抑制作用更明显。4.小剂量APS和泼尼松治疗均可提高GATA-3/T-bet比值,有助于恢复Th1/Th2平衡,以激素的免疫调节作用更为显著。结论APS对大鼠实验性结肠炎具有双向免疫调节作用,大剂量治疗可加重肠道炎症,小剂量APS治疗通过促进抗炎性细胞因子和转录因子的表达,使异常的Th1/Th2漂移趋于平衡,可能是其发挥抗炎、肠道保护作用的免疫机制。更多还原

【Abstract】 Background:Inflammatory bowel disease (IBD) is a major chronic inflammatory disease of the gastrointestinal tract in human。Its incidence has been increasing for the past several decades in Chinese people. The etiology of IBD is considered to be multifactorial, which includes genetic factors, dysfunction of immunoregulation, disruption of intestinal mucosal barrier and intestinal flora. Immune system is thought to play a critical role in the development and persistence of IBD. The traditional therapeutic agents such as 5-ASA and glucocorticoid as well as new biotherapeutic ones such as Infliximab have good therapeutic effect, but there is a small portion of patients who can not benefit from these therapies. In addition, to remain remission is another tough problem. Recently, basic and clinical research in the field of traditional Chinese medicine have developed dramatically. Studies showed that traditional Chinese medicine was a promising therapy in IBD. Astragalus membranaceus is one of the most frequently used traditional Chinese medicine, which is famous for" bu qi " and "ulcer cure". Studies showed that the extract of astragalus membranaceus possessed both preventive and therapeutic potential in experimental colitis. But the therapeutic and immunoregulatory effects of Astragalus polysaccarides (APS) on TNBS-induced colitis in rats has not been reported.Objective:To investigate the therapeutic and immunoregulatory effects of APS on TNBS-induced colitis in rats.Methods: 1. Establishment of experimental colitis. A total of 40 SD rats were randomly divided into five groups (n=8, each group):normal control, TNBS group (treated with water, the same volume with the intervention groups), lower dosage group (treated with APS 0.5g/kg.d), higher dosage group (treated with APS 1.0g/kg.d) and prednisolone group. Experimental colitis was induced in rats by enema administration of TNBS. Rats were treated by lavage everyday for the following 14 days.2. Evaluation of therapeutic effect. Rats were sacrificed on d16. Macroscopic lesion and histological damage were determined, and the activity of myeloperoxidase (MPO) was measured in the excised colonic tissues.3. Investigation of immunoregulatory mechanism. Cytokine levels (IL-4, IL-10) were determined by enzyme-linked immunosorbent assay. T-box transcription factor (T-bet) and GATA-binding protein-3 (GATA-3) expression were determined by immunohistochemisty and immunoblot analysis.Results:1. Both macroscopic lesion and histological colonic damage scores were elevated in TNBS group, the activity of MPO was higher than normal control significantly (P=0.01). APS (0.5g/Kg.d) treatment reduced both macroscopic lesion and histological colonic damage, and this was accompanied by reduction of MPO activity significantly (P=0.03). The macroscopic lesion and histological colonic damage scores of APS (1.0g/Kg.d) group were near to TNBS group, but the activity of MPO was significantly higher than normal control and APS (0.5g/Kg.d) group (P=0.003,0.008). There was no significant difference between Prednisone group and the other groups.2. The levels of IL-4 and IL-10 decreased in TNBS group comparing with normal control, especially the level of IL-10 decreased significantly (P=0.007). APS (0.5g/Kg.d)treatment increased the levels of IL-4 and IL-10 with no significance. APS (1.0g/Kg.d) treatment further reduced the level of IL-4 but not IL-10. Prednisone treatment also decreased the levels of these two kinds of cytokines. The levels of IL-4 and IL-10 in higher dosage group and Prednisone group were lower than normal control significantly (APS 1.0g vs normal control, P=0.046, 0.035; PREDNISONE group vs normal control, P=0.049,0.001).3. GATA-3 expression was higher than T-bet in normal control. Both GATA-3 and T-bet expression increased in TNBS group comparing with normal control, but T-bet increased more, resulting in significant reduction of GATA-3/T-bet ratio (P=0.025). APS (0.5g/Kg.d) treatment encouraged the expression of both T-bet and GATA-3 with significance (P=0.04,0.019), but GATA-3 increased more, resulting in raise of GATA-3/T-bet ratio. PREDNISONE inhibited the expression of both two transcriptions, and T-bet decreased more, resulting in raise of the GATA-3/T-bet ratio too. GATA-3/T-bet ratio was higher in Prednisone group than APS lower dosage group.Summary:1. Treatment with different dosage of APS showed different effects on TNBS-induced colitis. Lower dosage treatment possesed therapeutic potential in experimental colitis, while higher dosage treatment might aggravate the colitis.2. Treatment of lower dosage APS increased the levels of Th2 type cytokines IL-4 and IL-10, while higher dosage treatment decreased the level of IL-4, but increased that of IL-10. The immunoregulation mechanism of lower dosage APS was associated with encouragement of both GATA-3 and T-bet expression, especially for GATA-3.3. Prednisone possesed immunosuppression effect. It decreased the levels of IL-4 and IL-10, and inhibited the expression of both GATA-3 and T-bet, especially for T-bet.4. Treatment with lower dosage APS and prednisone could raise GATA-3/T-bet ratio, and help to restore Th1/Th2 balance.This immunoregulatory effect of prednisone was better than that of lower dosage APS. Conclusion:APS possesed bilateral immunoregulatory effects on TNBS-induced colitis. Lower dosage APS could restore Th1/Th2 balance by encouraging the expression of anti-inflammatory cytokines and transcriptions. This may explain the immunoregulation mechanism of its anti-inflammatory and intestinal protective effects.更多还原