【作者】 翟金海；

【导师】 沈洪；

【作者基本信息】 南京中医药大学 ， 中医内科学， 2013， 博士

【摘要】 目的通过观察清肠化湿方对三硝基苯磺酸(TNBS)诱导的溃疡性结肠炎模型大鼠的治疗效果,以NF-κB为中心分别探讨其抑制NF-κB过度活化,减少肠黏膜促炎细胞因子分泌,保护肠上皮紧密连接(tight junction, TJ)蛋白,减少树状突细胞(dendritic cells, DCs)数目并抑制其活化程度,以及抑制细胞间黏附分子ICAM-1表达的疗效机制,为临床用药提供客观依据,为开发治疗UC的中药新药打下科学基础。方法将40只雄性清洁型Wistar大鼠,体重(200±20)g,6-7w鼠龄,随机分为四组,每组10只；空白对照组,常规饲养,自由饮水进食。采用TNBS造模后分为模型对照组：造模后每日给予等体积生理盐水灌胃。清肠化湿方组：按成人10倍剂量(12.8g生药.kg-1.d)一次性灌胃给药。美沙拉嗪组,按按成人10倍剂量(0.67g.kg-1.d)一次性灌胃给药；于造模后第2天给药,连续10d。最后1次给药后,禁食24h处死大鼠,留取新鲜结肠标本和肠系膜淋巴结组织,观察各组大鼠一般情况和结肠组织病理形态：ELISA法检测肠黏膜组织促炎细胞因子IL-6、IL-1β、TNF-α的表达水平；免疫组化法检测结肠黏膜组织和MLN DCs数目,流式细胞仪检测肠系膜淋巴结大鼠DC特异标志分子OX-62百分比和DCs表面分子MHC-Ⅱ和CD86的表达；免疫组化法检测大鼠结肠粘膜ICAM-1的表达情况,ELISA法检测sICAM-1水平；免疫组化SP法检测大鼠结肠粘膜上皮occludin、 claudin-1蛋白的表达,实时荧光定量PCR (real-time PCR)方法检测结肠黏膜组织(?) occludin mRNA、claudin-1mRNA表达情况；(?)eal-time PCR法检测NF-κBp65mRNA的表达水平,免疫组化SP法和蛋白免疫印迹法检测大鼠结肠粘膜NF-κB p65的表达水平；采用相关分析探讨NF-κB p65与炎性积分、IL-1β, TNF-α, IL-6, Claudin-1、occludin, DCs, ICAM-1等指标的相关性。结果造模后,模型组大鼠比空白组大鼠结肠黏膜的病理炎性积分明显升高(P<0.01)；促炎细胞因子IL-6、IL-1β、TNF-α的表达水平明显升高(P<0.01)；结肠粘膜DCs和MLNDCs计数明显增多(P<0.01),MLN DCs表面分子MHC-Ⅱ表达水平明显增高(P<0.01)；ICAM-1和sIC AM-1水平明显增高(P<0.01)；occludin mRNA、claudin-1mRNA, occludin、 claudin-1蛋白表达水平下降(P<0.05)；NF-κB p65mRNA和NF-κB p65蛋白表达水平增高(P<0.05)。经清肠化湿方和美沙拉嗪治疗后,病理炎性积分明显降低(P<0.01)；IL-6、IL-1β、TNF-α的表达水平明显降低(P<0.01)；结肠粘膜DCs和MLN DCs计数明显减少(P<0.01),MLN DCs表面分子MHC-Ⅱ表达水平明显降低(P<0.01)；ICAM-1和sICAM-1水平明显降低(P<0.01);occludin mRNA、claudin-1mRNA, occludin、claudin-1蛋白表达水平下降(P<0.05);NF-κB p65mRNA和NF-κB p65蛋白表达水平降低(P<0.05)。NF-κB p65与炎性积分、IL-1β, TNF-α, IL-6, Claudin-1、occludin, DCs, ICAM-1等指标呈高度相关(P<0.01)。治疗前后各组大鼠MLN DCs的比例和其表面分子CD86表达水平无统计学差异(P>0.05)；清肠化湿方和美沙拉嗪对大鼠各观察指标的影响无统计学差异(P>0.05)。结论清肠化湿方可以改善TNBS模型大鼠结肠的一般情况和炎症病变,其机制与其可以抑制结肠黏膜NF-κB的表达,从而降低促炎细胞因子IL-6、IL-1β、TNF-α的分泌水平,减少结肠黏膜及MLN DCs的数目,抑制DCs的活化,降低结肠黏膜黏附分子ICAM-1的表达水平,增加UC结肠黏膜紧密连接蛋白Claudin-1、Occludin的表达,恢复肠粘膜屏障功能等有关。更多还原

【Abstract】 Objective The purpose of the research is to supply the objective evidences for clinical prescription and to pave the way for developing the new Chinese herbal drugs through exploring the mechanism of QingChangHuaShi decoction treating Ulcerative colitis. The observing targets include the treatment effectiveness on TNBS-induced model rats colitis, proinflammatory cytokines, intercellular adhesion molecule-1, dendritic cells tight junction protein, nuclear factor-KB of colonic mucous membrane and dendritic cells of mesenteric lymph nodes.Methods Forty male depuratory Wistar rats were randomly divided into four groups, and each group has ten rats.The weight of one rat is about200±20gram,and the age is about six to seven weeks. The blank group ten rats were raised commonly,eat and drink freedomly; The other thirty rats were divided into model group、QingChangHuaShi decoction group and Mesalazine group after successfully building the model induced by TNBS. The model group rats were gave equivalent volume physiological saline though intragastric administrations at one time everyday. The dosage of the QingChangHuaShi decoction and Mesalazine was12.8g dried medicinal herb·kg-1·d and0.67g·kg-1·d respectively. The administration methods were the same as the blank group. The administration time was ten days and the drugs were gave from the second day successively after building the models. Fasting for24h form the last administration, and then all of the rats were killed. The fresh colonic tissues and the MLNs were resected for detection. The general conditions and the colonic pathology morphology were observed. The proinflammatory cytokines including IL-6、IL-β、TNF-α were measured by the method of ELISA. The numbers of the DCs in the colonic mucous membrane and mesenteric lymph nodes were measured by the method of IHC. The ratio of the DCs in the MLNs and the expression of superficial molecule CD86and MHC-Ⅱwere checked out by the flow cytometer.ICAM-1and s ICAM-1were checked out by IHC and ELISA respectively. And the expression levels of the occludin mRNA、 claudin-1mRNA、NF-κB p65mRNA were measured by the method of the real-time PCR, and the occludin、claudin-1、NF-κB p65proteins by IHC. The NF-κB p65protein was also checked out by WB.At last,the correlation between the NF-κB p65and the inflammatory accumulated points、IL-1β, TNF-α, IL-6, Claudin-1、occludin, DCs and ICAM-1were evaluated by the correlation analysis.Results After modeling, the pathology inflammatory accumulated points of the colonic membrane and the expression level of proinflammatory cytokines including IL-6、IL-1β、 TNF-a,ICAM-1、sICAM-1, NF-κB p65mRNA and NF-κB p65protein increase significantly (P<0.01). The numbers of the DCs in the colonic mucous membrane and mesenteric lymph nodes and the expression of the superficial molecule MHC-Ⅱ of the DCs in the mesenteric lymph nodes increase significantly (P<0.01).And the expression levels of the occludin mRNA, claudin-1mRNA, occludin、claudin-1proteins decrease (P<0.05).After treatment, the pathology inflammatory accumulated points of the colonic membrane and the expression level of proinflammatory cytokines including IL-6、IL-1β、TNF-α,ICAM-1、sICAM-1, NF-κB p65mRNA and NF-κB p65protein decrease significantly (P<0.01). The numbers of the DCs in the colonic mucous membrane and mesenteric lymph nodes and the expression of the superficial molecule MHC-Ⅱ of the DCs in the mesenteric lymph nodes decrease significantly (P<0.01) And the expression levels of the occludin mRNA、claudin-1mRNA, occludin、claudin-1proteins increase (P<0.05). The expression of NF-κB p65intimately correlates with inflammatory accumulated points, IL-1β、TNF-α、IL-6, Claudin-1、occludin, DCs, ICAM-1(P<0.01).The ratio of the DCs in the MLNs and the expression of superficial molecule CD86have no statistical difference (P>0.05) between pretherapy and prior treatment among four groups,and the influences on all of the indices have no statistical difference (P>0.05) between QingChangHuaShi decoction and Mesalazine.Conclusion QingChangHuaShi decoction can improve general conditions and colonic inflammation of the TNBS-induced model rats. The concrete mechanism are as follows: suppressing the secretion level of proinflammatory cytokines such as IL-6、IL-1β、TNF-α, decreasing the numbers of the colonic membrane and the mesenteric lymph nodes, suppressing the activating of the DCs, reducing the expression level of ICAM-1,and increasing the expression of TJ proteins, thus to retrieve the function of intestine barrier, possibly through suppressing the expression of the nuclear factor-KB.更多还原