不同造模次数对2,4,6-三硝基苯磺酸/乙醇诱导重症溃疡性结肠炎模型大鼠的影响
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摘要
背景:实验采取复合法2,4,6-三硝基苯磺酸(2,4,6-Trinitrobenzenesulfonic acid,TNBS)/乙醇建立溃疡性结肠炎大鼠模型,操作简单,持续时间长且组织学变化与人类溃疡性结肠炎相似,是目前常用的溃疡性结肠炎动物模型之一,但对不同剂量不同造模次数仍存在较大差异。目的:采用相同剂量TNBS/乙醇不同次数诱导重症溃疡性结肠炎大鼠模型,探索重症溃疡性结肠炎适宜的造模次数。方法:选用SPF级SD大鼠80只,购自湖南斯莱克景达实验动物有限公司,实验方案经广西中医药大学第一附属医院动物实验伦理委员会批准[批准号为2013(KF)-E-003]。随机将大鼠分为正常对照组、TNBS+乙醇灌肠1次组、TNBS+乙醇灌肠2次组、TNBS+乙醇灌肠3次组。用药组分别使用灌胃针经大鼠直肠注入相同剂量的100 mg/kg TNBS+等体积乙醇1 d、连续2 d、连续3 d。分别于给药后的3,7 d各处死10只大鼠,观察大鼠生理状态、结肠形态、疾病活动指数及组织病理学改变情况。结果与结论:①造模7 d后大鼠体质量变化,随着造模次数的增加,大鼠体质量呈渐进性下降、症状呈渐进性加重趋势,DAI评分各组呈上升趋势;②TNBS+乙醇灌肠3次组体质量显著低于其他组(P<0.05);DAI评分和大体评分均显著高于其他组(P <0.05);③病理结果示,TNBS+乙醇灌肠3次组病变位于结肠黏膜肌层,杯状细胞丢失,隐窝形态不规则,排列紊乱,大部分可见溃疡面,较TNBS+乙醇灌肠1次组和TNBS+乙醇灌肠2次组更符合重症溃疡性结肠炎的病理表现;④结果说明,TNBS/乙醇造模中,100 mg/kg TNBS+乙醇灌肠3次连续灌肠更符合重症溃疡性结肠炎模型。
BACKGROUND: Establishing the rat model of ulcerative colitis by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ethanol method is easy to operate, has long duration and the histological changes are similar to human ulcerative colitis, which is commonly used animal model.However, the optimal dose and modeling times still remain controversial.OBJECTIVE: To establish the rat model of the severe ulcerative colitis by different times of different doses of TNBS/ethanol, and to explore the optimal modeling times.METHODS: Eighty Sprague-Dawley rats, SPF level, were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., and the study was approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Guangxi University of Chinese Medicine, approval number: 2013(KF)-E-003. The rats were randomly divided into normal control group, and TNBS/ethanol(100 mg/kg once, 100 mg/kg twice,and 100 mg/kg thrice) group. The rats were intragastrically injected with TNBS/ethanol through rectum for 1, 2, and 3 days continuously. Then ten rats were killed at 3 and 7 days after administration, so as to observe the physiological status, colon morphology, disease activity index and histopathological changes.RESULTS AND CONCLUSION:(1) At 7 days after modeling, the body mass of rats progressively decreased and symptoms gradually exacerbated with the modeling times increased. The disease activity index was on a rise.(2) The body mass in the TNBS/ethanol thrice group was significantly lower than that in the other groups(P < 0.05), and the disease activity index and gross scores were significantly higher than those in the other groups(P < 0.05).(3) The pathological results showed that the TNBS/ethanol thrice group caused the lesions located in the colonic mucosa and muscularis, the goblet cells lost, irregular crypts in shape, disordered arrangement and the most of visible ulcers, which were more consistent with the pathological manifestations of severe ulcerative colitis than the other two groups.(4) In summary, in the TNBS/ethanol modeling, using 100 mg/kg TNBS/ethanol thrice is more consistent with the model of severe ulcerative colitis.
BACKGROUND: Establishing the rat model of ulcerative colitis by 2,4,6-trinitrobenzene sulfonic acid(TNBS)/ethanol method is easy to operate, has long duration and the histological changes are similar to human ulcerative colitis, which is commonly used animal model.However, the optimal dose and modeling times still remain controversial.OBJECTIVE: To establish the rat model of the severe ulcerative colitis by different times of different doses of TNBS/ethanol, and to explore the optimal modeling times.METHODS: Eighty Sprague-Dawley rats, SPF level, were provided by Hunan Slack Jingda Experimental Animal Co., Ltd., and the study was approved by the Experimental Animal Ethics Committee of the First Affiliated Hospital of Guangxi University of Chinese Medicine, approval number: 2013(KF)-E-003. The rats were randomly divided into normal control group, and TNBS/ethanol(100 mg/kg once, 100 mg/kg twice,and 100 mg/kg thrice) group. The rats were intragastrically injected with TNBS/ethanol through rectum for 1, 2, and 3 days continuously. Then ten rats were killed at 3 and 7 days after administration, so as to observe the physiological status, colon morphology, disease activity index and histopathological changes.RESULTS AND CONCLUSION:(1) At 7 days after modeling, the body mass of rats progressively decreased and symptoms gradually exacerbated with the modeling times increased. The disease activity index was on a rise.(2) The body mass in the TNBS/ethanol thrice group was significantly lower than that in the other groups(P < 0.05), and the disease activity index and gross scores were significantly higher than those in the other groups(P < 0.05).(3) The pathological results showed that the TNBS/ethanol thrice group caused the lesions located in the colonic mucosa and muscularis, the goblet cells lost, irregular crypts in shape, disordered arrangement and the most of visible ulcers, which were more consistent with the pathological manifestations of severe ulcerative colitis than the other two groups.(4) In summary, in the TNBS/ethanol modeling, using 100 mg/kg TNBS/ethanol thrice is more consistent with the model of severe ulcerative colitis.
引文
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[10]郑文潇,吴翔.溃疡性结肠炎动物模型的研究进展[J].中国病原生物学杂志,2016,11(9):861-864+867.
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[13]Joo M,Kim HS,Kwon TH,et al.Anti-inflammatory effects of flavonoids on TNBS-induced colitis of rat.Korean J Physiol Pharmacol.2015;19(1):43-50.
[14]BrennaФ,Furnes MW,Drozdov I,et al.Relevance of TNBS-colitis in rats:a methodological study with endoscopic,histologic and transcriptomic characterization and correlation to IBD.PloS One.2013;8(1):e54543.
[15]王炳芳,王迎新,王晓蕾,等.已酮可可碱对溃疡性结肠炎治疗作用及其机制[J].第四军医大学学报,2007,28(13):1196-1198.
[16]Korn T,Bettelli E,Gao W,et al.IL-21 initiates an alternativepathway to induce proinflammatory T(H)17cells.Nature.2007;448(7152):484-487.
[17]Butzner JD,Parmar R,Bell CJ,et al.Butyrate enema therapy stimulates mucosal repair in experimental colitis in the rat.Gut.1996;38(4):568-73.
[18]中华医学会病理学分会消化病理学组筹备组.中国炎症性肠病组织病理诊断共识意见[J].中华病理学杂志,2014,43(4):268-274.
[19]李乃卿.中西医结合外科学[M].北京:中国中医药出版社,2005:671-672.
[20]赵平,董蕾,罗金燕,等.DSS与TNBS诱导大鼠实验性结肠炎模型的对比研究[J].胃肠病学,2015,20(11):667-671.
[21]张冰冰,齐越,贾冬,等.2,4,6-三硝基苯磺酸诱导溃疡性结肠炎大鼠模型的建立及评价[J].中华中医药学刊,2015,33(8):1834-1836.
[22]曾于恒,杨芳,何永恒.2,4,6-三硝基苯磺酸与不同浓度乙醇诱导大鼠溃疡性结肠炎模型的建立及其稳定性评价[J].中南药学,2017,15(12):1665-1669.
[23]陈佳奇,陈村龙,王继德,等.2,4,6-三硝基苯磺酸诱导小鼠炎症性肠病模型的建立[J].中国组织工程研究与临床康复,2007,11(21):4174-4177.
[24]钟元帅,韦日娜,孙平良,等.溃疡性结肠炎SD大鼠肠道乙酸、血清内毒素水平变化及安肠汤干预研究[J].时珍国医国药,2018,29(4),780-782.
[25]Antoniou E,Margonis GA,Angelou A,et al.Efstathios Antoniou,Georgios Antonios Margonis,Anastasios Angelou,et al.The TNBS-induced colitis animal model:An overview.Ann Med Surg(Lond).2016;11:9-15.
[2]Torres J,BillioudV,SacharDB,etal.Ulcerative colitis as a progressive disease:the forgotten evidence.Infamm Bowel Dis.2012;18:1356-1363.
[3]Lautenschlaer C,Schmidt C,Fischer D,et al.Drug delivery strategies in the therapy of inflammatory bowel disease.Adv Drug Deliv Rev.2014;71:58-76.
[4]RutterM,SaundersB,Wilkinson K,et al.Severity of infamma-tion is a risk factor for colorectal neoplasia in ulcerative colitis.Gastroenterology.2004;126:451-459.
[5]Gupta RB,HarpazN,ItzkowitzS,etal.Histologic infammation is a risk factor for progression to colorectal neoplasia in ulcerative colitis:a cohort study.Gastroenterology.2007;133:1099-1105.
[6]So J,Tang W,Leung WK,et al.Cancer Risk in 2621 Chinese Patients with Inflammatory Bowel Disease:A Population-based Cohort study.Inflamm Bowel Dis.2017;23(11):2061-2068.
[7]中华医学会消化病学分会炎症性肠病学组.炎症性肠病诊断与治疗的共识意见(2012年广州)[J].中华内科杂志,2012,51(10):818-831.
[8]中国中西医结合学会消化系统疾病专业委员会.溃疡性结肠炎中西医结合诊疗指南(草案)[J].中国中西医结合消化杂志,2011,19(1):61-65.
[9]申睿,刘苗,朱向东,谈望晶.溃疡性结肠炎大鼠实验模型研究进展[J].中华中医药杂志,2018,33(9):3998-4001.
[10]郑文潇,吴翔.溃疡性结肠炎动物模型的研究进展[J].中国病原生物学杂志,2016,11(9):861-864+867.
[11]Morris GP,Beck PL,Herridge MS,et al.Hapten-induced model of chronic inflammation and ulceration in the ratcolon.Gastroenterology.1989;96:795-803.
[12]贺海辉,沈洪,朱宣宣,等.2,4,6-三硝基苯磺酸/乙醇法诱导建立溃疡性结肠炎大鼠模型[J].中国老年学杂志,2015,35(15):4138-4140.
[13]Joo M,Kim HS,Kwon TH,et al.Anti-inflammatory effects of flavonoids on TNBS-induced colitis of rat.Korean J Physiol Pharmacol.2015;19(1):43-50.
[14]BrennaФ,Furnes MW,Drozdov I,et al.Relevance of TNBS-colitis in rats:a methodological study with endoscopic,histologic and transcriptomic characterization and correlation to IBD.PloS One.2013;8(1):e54543.
[15]王炳芳,王迎新,王晓蕾,等.已酮可可碱对溃疡性结肠炎治疗作用及其机制[J].第四军医大学学报,2007,28(13):1196-1198.
[16]Korn T,Bettelli E,Gao W,et al.IL-21 initiates an alternativepathway to induce proinflammatory T(H)17cells.Nature.2007;448(7152):484-487.
[17]Butzner JD,Parmar R,Bell CJ,et al.Butyrate enema therapy stimulates mucosal repair in experimental colitis in the rat.Gut.1996;38(4):568-73.
[18]中华医学会病理学分会消化病理学组筹备组.中国炎症性肠病组织病理诊断共识意见[J].中华病理学杂志,2014,43(4):268-274.
[19]李乃卿.中西医结合外科学[M].北京:中国中医药出版社,2005:671-672.
[20]赵平,董蕾,罗金燕,等.DSS与TNBS诱导大鼠实验性结肠炎模型的对比研究[J].胃肠病学,2015,20(11):667-671.
[21]张冰冰,齐越,贾冬,等.2,4,6-三硝基苯磺酸诱导溃疡性结肠炎大鼠模型的建立及评价[J].中华中医药学刊,2015,33(8):1834-1836.
[22]曾于恒,杨芳,何永恒.2,4,6-三硝基苯磺酸与不同浓度乙醇诱导大鼠溃疡性结肠炎模型的建立及其稳定性评价[J].中南药学,2017,15(12):1665-1669.
[23]陈佳奇,陈村龙,王继德,等.2,4,6-三硝基苯磺酸诱导小鼠炎症性肠病模型的建立[J].中国组织工程研究与临床康复,2007,11(21):4174-4177.
[24]钟元帅,韦日娜,孙平良,等.溃疡性结肠炎SD大鼠肠道乙酸、血清内毒素水平变化及安肠汤干预研究[J].时珍国医国药,2018,29(4),780-782.
[25]Antoniou E,Margonis GA,Angelou A,et al.Efstathios Antoniou,Georgios Antonios Margonis,Anastasios Angelou,et al.The TNBS-induced colitis animal model:An overview.Ann Med Surg(Lond).2016;11:9-15.