AhR活化上调肠上皮源IL-10对DSS诱导的小鼠结肠炎的保护作用及机制研究
发布时间：2018-08-06 12:06
【摘要】：研究背景炎症性肠病(Inflammatory bowel disease,IBD),是一类主要影响人类胃肠道的慢性疾病,包括溃疡性结肠炎和克罗恩病。虽然其具体的病理机制尚未完全明确,但现有的证据表明肠粘膜免疫调控紊乱和上皮屏障功能障碍共同引发了炎症性肠病的发生。芳香烃受体(AhR)是一种胞浆内的转录因子,可由一系列的外界环境中的配体特异性激活。AhR的激活参与肠粘膜免疫的调控。有证据表明,在IBD病人的肠道组织中AhR的表达被抑制,而给予AhR的配体6-甲酰基吲哚并[3,2b]-咔唑(FICZ)和四氯二苯并-p-二恶英(TCDD)可以缓解小鼠结肠炎模型中的炎症反应。虽然现有的一些研究已经证实了AhR在炎症性肠病中的保护作用,但是其作用的具体机制尚未明确。白介素10(IL-10)是一种重要的抗炎因子,可以缓解肠粘膜炎症。本研究通过外源性给予AhR的特异性激动剂FICZ,研究活化的AhR对DSS诱导的小鼠结肠炎的保护作用并探讨其机制是否是通过上调肠上皮源IL-10的表达来实现的。研究方法1.给予小鼠4%的DSS喂养7天诱导小鼠结肠炎模型,两天后腹腔注射AhR激动剂FICZ。每天记录小鼠体重,第8天收集小鼠结肠组织并测量长度,切片并进行HE染色和组织学分析。2.从结肠组织中提取肠上皮细胞,用免疫印迹技术检测肠上皮细胞中IL-10的表达,用实时荧光定量PCR技术检测炎性因子mRNA的表达。3.在体外培养Caco-2细胞株,用FICZ处理细胞,或同时给予脂多糖(LPS)处理,用免疫印迹技术检测IL-10和STAT3/P-STAT3的表达,用用实时荧光定量PCR技术检测炎性因子mRNA的表达。研究结果1.给予FICZ明显缓解了DSS引起的小鼠体重下降;DSS处理后小鼠结肠的长度几乎缩短一半并伴有水肿和淤血,而给予FICZ后这些情况明显改善;组织学分析显示DSS处理后小鼠结肠上皮和隐窝有严重的缺损,而FICZ处理后明显改善了这些损伤。2.与DSS处理组相比,给予FICZ明显上调了肠上皮源IL-10的表达;DSS处理后,促炎的炎性因子的mRNA表达明显增高,而给予FICZ处理后炎性因子明显下调。3.体外培养Caco2细胞,给予LPS处理24小时或LPS和FICZ共同处理24小时,LPS明显上调了IL-1β和IL-6的mRNA的表达,而FICZ明显下调其表达;同时无论是否有LPS的刺激,FICZ均明显促进了IL-10在蛋白水平和mRNA水平的表达。4.FICZ处理Caco-2细胞24小时后,STAT3的表达几乎没有变化而P-STAT3的表达明显上调。结论1.FICZ激活AhR明显缓解DSS诱导的小鼠结肠炎。2.FICZ激活AhR在动物实验和细胞实验均可以降低炎性因子的表达。3.活化的AhR可以上调肠上皮细胞源IL-10和P-STAT3的表达。
[Abstract]:Background: inflammatory bowel disease (Inflammatory bowel) is a class of chronic diseases mainly affecting human gastrointestinal tract, including ulcerative colitis and Crohn's disease. Although its specific pathological mechanism has not been completely clear, the existing evidence shows that intestinal mucosal immune regulation disorder and epithelial barrier dysfunction jointly lead to the occurrence of inflammatory bowel disease. Aromatics receptor (AhR) is a transcriptional factor in the cytoplasm, which can be activated by a series of ligands in external environment. AhR activation is involved in the regulation of intestinal mucosal immunity. It has been shown that the expression of AhR was inhibited in the intestinal tissues of IBD patients, while the ligands 6-formylindoledo [3b2b] -carbazole (FICZ) and tetrachlorodibenzo-pdioxin (TCDD), which were administered with AhR, could attenuate the inflammatory reaction in mice colitis model. Although some existing studies have confirmed the protective role of AhR in inflammatory bowel disease, the specific mechanism of its role has not been clarified. Interleukin 10 (IL-10) is an important anti-inflammatory factor, which can relieve intestinal mucosal inflammation. In this study, the protective effect of activated AhR on DSS induced colitis in mice was studied by exogenous administration of FICZ, a specific agonist of AhR, and the mechanism was investigated by up-regulating the expression of IL-10 in intestinal epithelium. Method 1. Mice were fed with 4% DSS for 7 days to induce colitis. Two days later, AhR agonist FICZ was injected intraperitoneally. The body weight of mice was recorded daily. The colonic tissues were collected and measured on the 8th day, sections were made and HE staining and histological analysis were performed. Intestinal epithelial cells were extracted from colon tissue. The expression of IL-10 in intestinal epithelial cells was detected by Western blotting, and the expression of inflammatory factor mRNA was detected by real-time fluorescence quantitative PCR. Caco-2 cell lines were cultured in vitro. The cells were treated with FICZ or treated with lipopolysaccharide (LPS). The expression of IL-10 and STAT3/P-STAT3 was detected by Western blot and the expression of inflammatory factor mRNA was detected by real-time fluorescence quantitative PCR technique. Results 1. The colonic length of mice treated with FICZ significantly alleviated the weight loss induced by DSS and was accompanied with edema and congestion, but these conditions were significantly improved after FICZ treatment. Histological analysis showed that there were serious defects in colonic epithelium and crypt after DSS treatment in mice, while FICZ treatment significantly improved these lesions. Compared with DSS treatment group, FICZ upregulated the expression of IL-10 in intestinal epithelium and increased the mRNA expression of proinflammatory cytokines after treatment with DSS, while the expression of inflammatory factors decreased significantly after FICZ treatment. Caco2 cells were cultured in vitro, treated with LPS for 24 hours or co-treated with LPS and FICZ for 24 hours. LPs upregulated mRNA expression of IL-1 尾 and IL-6, but FICZ significantly down-regulated the expression of IL-1 尾 and IL-6. FICZ significantly promoted the expression of IL-10 at protein level and mRNA level. 4. After 24 hours of FICZ treatment, the expression of STAT3 in Caco-2 cells was almost unchanged, but the expression of P-STAT3 was significantly up-regulated. Conclusion 1.FICZ activated AhR significantly alleviated DSS induced colitis in mice. 2. FICZ activated AhR could decrease the expression of inflammatory factors in both animal and cell experiments. Activated AhR can up-regulate the expression of IL-10 and P-STAT3 in intestinal epithelial cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574
[Abstract]:Background: inflammatory bowel disease (Inflammatory bowel) is a class of chronic diseases mainly affecting human gastrointestinal tract, including ulcerative colitis and Crohn's disease. Although its specific pathological mechanism has not been completely clear, the existing evidence shows that intestinal mucosal immune regulation disorder and epithelial barrier dysfunction jointly lead to the occurrence of inflammatory bowel disease. Aromatics receptor (AhR) is a transcriptional factor in the cytoplasm, which can be activated by a series of ligands in external environment. AhR activation is involved in the regulation of intestinal mucosal immunity. It has been shown that the expression of AhR was inhibited in the intestinal tissues of IBD patients, while the ligands 6-formylindoledo [3b2b] -carbazole (FICZ) and tetrachlorodibenzo-pdioxin (TCDD), which were administered with AhR, could attenuate the inflammatory reaction in mice colitis model. Although some existing studies have confirmed the protective role of AhR in inflammatory bowel disease, the specific mechanism of its role has not been clarified. Interleukin 10 (IL-10) is an important anti-inflammatory factor, which can relieve intestinal mucosal inflammation. In this study, the protective effect of activated AhR on DSS induced colitis in mice was studied by exogenous administration of FICZ, a specific agonist of AhR, and the mechanism was investigated by up-regulating the expression of IL-10 in intestinal epithelium. Method 1. Mice were fed with 4% DSS for 7 days to induce colitis. Two days later, AhR agonist FICZ was injected intraperitoneally. The body weight of mice was recorded daily. The colonic tissues were collected and measured on the 8th day, sections were made and HE staining and histological analysis were performed. Intestinal epithelial cells were extracted from colon tissue. The expression of IL-10 in intestinal epithelial cells was detected by Western blotting, and the expression of inflammatory factor mRNA was detected by real-time fluorescence quantitative PCR. Caco-2 cell lines were cultured in vitro. The cells were treated with FICZ or treated with lipopolysaccharide (LPS). The expression of IL-10 and STAT3/P-STAT3 was detected by Western blot and the expression of inflammatory factor mRNA was detected by real-time fluorescence quantitative PCR technique. Results 1. The colonic length of mice treated with FICZ significantly alleviated the weight loss induced by DSS and was accompanied with edema and congestion, but these conditions were significantly improved after FICZ treatment. Histological analysis showed that there were serious defects in colonic epithelium and crypt after DSS treatment in mice, while FICZ treatment significantly improved these lesions. Compared with DSS treatment group, FICZ upregulated the expression of IL-10 in intestinal epithelium and increased the mRNA expression of proinflammatory cytokines after treatment with DSS, while the expression of inflammatory factors decreased significantly after FICZ treatment. Caco2 cells were cultured in vitro, treated with LPS for 24 hours or co-treated with LPS and FICZ for 24 hours. LPs upregulated mRNA expression of IL-1 尾 and IL-6, but FICZ significantly down-regulated the expression of IL-1 尾 and IL-6. FICZ significantly promoted the expression of IL-10 at protein level and mRNA level. 4. After 24 hours of FICZ treatment, the expression of STAT3 in Caco-2 cells was almost unchanged, but the expression of P-STAT3 was significantly up-regulated. Conclusion 1.FICZ activated AhR significantly alleviated DSS induced colitis in mice. 2. FICZ activated AhR could decrease the expression of inflammatory factors in both animal and cell experiments. Activated AhR can up-regulate the expression of IL-10 and P-STAT3 in intestinal epithelial cells.
【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R574
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1 张道杰;肠上皮防御微生物及其毒素的机制[J];实用医药杂志;2004年02期
2 石辛甫,高杰英;肠上皮间淋巴细胞的研究进展[J];免疫学杂志;1998年04期
3 曾小川，，汪维伟;胚胎肠上皮的组织发生机制之研究进展[J];重庆医科大学学报;1998年03期
4 Trier J S ,杨希山;肠M细胞的结构和功能[J];国外医学(消化系疾病分册);1992年03期
5 周静,王杉,叶颖江,杨q
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