三硝基苯磺酸诱导急性肠纤维化大鼠模型的改良方法

doi:10.12300/j.issn.1674-5817.2021.147

实验动物与比较医学 ›› 2022, Vol. 42 ›› Issue (4): 284-293.DOI: 10.12300/j.issn.1674-5817.2021.147

• 动物实验技术与方法专题 • 上一篇下一篇

三硝基苯磺酸诱导急性肠纤维化大鼠模型的改良方法

王怡如¹()(), 蒋笑影¹, 董若曦¹, 潘一滨¹, 韩向晖², 曹永清¹()()

^1.上海中医药大学附属龙华医院肛肠科, 上海 200032
^2.上海中医药大学附属龙华医院中医外科研究所, 上海 200032

收稿日期:2021-09-02 修回日期:2022-03-20 出版日期:2022-08-25 发布日期:2022-09-01
通讯作者: 曹永清（1961—），男，主任医师，教授，博士生导师，主要从事中医外科学及中医药治疗大肠肛门疾病的研究。E-mail： cyq88tcm@163.com。ORCID：0000-0001-8482-2359
作者简介:王怡如（1991—），女，博士研究生，研究方向：中医外科学及中医药治疗炎症性肠病。E-mail：wangyiru1707@163.com。ORCID：0000-0003-1622-9793
基金资助:
国家自然科学基金资助项目“红萸饮下调chr10：115386962-115390436+/miR-6914-5p/Atg7通路治疗IBD的机制研究”(81874469)

Modified Method for Inducing Acute Intestinal Fibrosis in Rats Using 2,4,6-Trinitrobenzene Sulfonic Acid

Yiru WANG¹()(), Xiaoying JIANG¹, Ruoxi DONG¹, Yibin PAN¹, Xianghui HAN², Yongqing CAO¹()()

^1.Department of Anal-rectal Surgery, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China
^2.Department of Institute of Chinese Traditional Surgery, Shanghai University of Traditional Chinese Medicine, Shanghai 200032, China

Received:2021-09-02 Revised:2022-03-20 Published:2022-08-25 Online:2022-09-01
Contact: CAO Yongqing (ORCID: 0000-0001-8482-2359), E-mail: cyq88tcm@163.com

摘要/Abstract

摘要：

目的探索2，4，6-三硝基苯磺酸（2，4，6-trinitrobenzene sulfonic acid，TNBS）诱导急性肠纤维化大鼠模型的方法及不同药物配比的效果。方法 30只雌性SD大鼠随机分为5组，每组各6只。A～D组为模型组，模型组大鼠采用不同配比的药物溶液通过改良保留灌肠法一次性造模：A组，5%TNBS+ 50%乙醇（体积比1∶1）；B组，5%TNBS+ 75%乙醇（体积比1∶1）；C组，5%TNBS+ 100%乙醇（体积比1∶1）；D组，5%TNBS+ 50%乙醇（体积比2∶1）；对照组用生理盐水（0.9%氯化钠溶液）灌肠；观察造模后1周内动物的症状、体征和体质量变化，并进行疾病活动指数（disease activity index，DAI）评分。分别于造模后7 d和14 d，随机选择每组内一半大鼠进行取材，观察结肠组织大体损伤程度并以结肠炎损伤指数（colon macroscopic damage index，CMDI）评分；结肠组织病理切片以HE染色观察肠炎严重程度，Masson染色观察胶原纤维沉积情况。结果各模型组大鼠均出现轻重不等的肠炎及肠纤维化病变，其中以体积比为1∶1的5%TNBS+75%乙醇溶液造模观察期内未导致大鼠死亡，造模后24 h大鼠体质量明显下降、出现稀便及血便症状，结肠壁纤维化病变明显，体质量下降、DAI评分与CDMI评分升高，与对照组相比差异具有统计学意义（P＜0.05）；光学显微镜下炎症程度较重，呈透壁性；Masson染色显示造模部位的肠壁增厚明显，黏膜下层、肌层及浆膜层均出现弥漫性胶原纤维沉积。结论改良的TNBS保留灌肠法能有效构建肠纤维化大鼠模型，体积比为1∶1的5%TNBS+75%乙醇溶液所建模型可模拟克罗恩病纤维化两大特征，即透壁性炎症和肠壁纤维化。该造模方法简便、高效，动物死亡率低。

关键词: 炎症性肠病, 克罗恩病, 肠道纤维化, 2, 4, 6-三硝基苯磺酸, SD大鼠

Abstract:

Objective To explore 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced acute intestinal fibrosis in rats and the effect of different drug ratios. Methods Thirty female Sprague-Dawley (SD) rats were randomly divided into five groups with six rats in each group. Groups A to D were the model groups, in which the rats were induced by modified retention enemas using solutions with different drug ratios: group A, 5% TNBS + 50% ethanol (1∶1 v/v); group B, 5% TNBS + 75% ethanol (1∶1 v/v); group C, 5% TNBS + 100% ethanol (1∶1 v/v); group D, 5% TNBS + 50% ethanol (2∶1 v/v); and the control group was induced by normal saline (0.9% sodium chloride solution) enema. The symptoms, signs, and body mass changes of the animals were observed within one week after modeling, and scored using the disease activity index (DAI). At 7th d and 14th d after model establishment, half of the rats in each group were randomly selected for sampling to observe the degree of gross damage to the colon tissue and scored using the colon macroscopic damage index (CMDI). Pathological sections of colon tissue were stained with hematoxylin-eosin (HE) to observe the severity of enteritis, and Masson staining was used to observe collagen fiber deposition. Results The rats in each model group showed enteritis and intestinal fibrosis lesions of different severities, of which 5% TNBS + 75% ethanol solution (1∶1 v/v) did not lead to death during the observation period. At 24 h after model establishment, the rats had significantly decreased body weight, loose stool, and bloody stool, significant colonic wall fibrosis lesions, and increased DAI and CDMI scores compared with the control group (P＜0.05). The degree of inflammation was transmural and more severe, as seen under the light microscope, and Masson staining showed that the intestinal wall at the model site was significantly thickened, and diffuse collagen fiber deposition occurred in the submucosa, muscular layer, and serosal layer. Conclusion The modified TNBS retention enema method can effectively construct a rat model of intestinal fibrosis, and the model established using 5% TNBS + 75% ethanol solution (1∶1 v/v) can simulate the two major characteristics of Crohn’s disease fibrosis, namely transmural inflammation and intestinal wall fibrosis. This method is simple and efficient, and the mortality rate of animals is low.

Key words: Inflammatory bowel disease, Crohn's disease, Intestinal fibrosis, 2,4,6-trinitrobenzene sulfonic acid, Sprague-Dawley rats

中图分类号:

R-332

王怡如, 蒋笑影, 董若曦, 潘一滨, 韩向晖, 曹永清. 三硝基苯磺酸诱导急性肠纤维化大鼠模型的改良方法[J]. 实验动物与比较医学, 2022, 42(4): 284-293.

Yiru WANG, Xiaoying JIANG, Ruoxi DONG, Yibin PAN, Xianghui HAN, Yongqing CAO. Modified Method for Inducing Acute Intestinal Fibrosis in Rats Using 2,4,6-Trinitrobenzene Sulfonic Acid[J]. Laboratory Animal and Comparative Medicine, 2022, 42(4): 284-293.

图/表 5

图1 大鼠保留灌肠示意图

Figure 1 Schematic diagram of retention enema

图2 造模后大鼠体质量变化趋势（A）和疾病活动指数评分（B）注：*表示造模当日。对照组用生理盐水灌肠；A～D组为模型组，分别用5%TNBS+50%乙醇溶液（体积比1∶1）、5%TNBS+75%乙醇溶液（体积比1∶1）、5%TNBS+无水乙醇（体积比1∶1）、5%TNBS+50%乙醇溶液（体积比2∶1）灌肠。每组6只大鼠，C组造模后第2天和第6天各有1只大鼠死亡，D组造模后第3天有2只大鼠死亡。

Figure 2 Changes in rat body mass （A） and disease activity index (B) after model establishmentNote：*shows the modeling day. The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema. There're 6 rats contained in each group， but one of which died on days 2 and 6 in group C and two of which died on day 3 in group D after modeling.

图3 模型建立后的大鼠结肠组织注：A图为造模后第7天，B图为造模后第14天，上图为全结肠整体外观，下图为结肠造模部位纵剖面。对照组用生理盐水灌肠；A～D组为模型组，分别用5%TNBS+50%乙醇溶液（体积比1∶1）、5%TNBS+75%乙醇溶液（体积比1∶1）、5%TNBS+无水乙醇（体积比1∶1）、5%TNBS+50%乙醇溶液（体积比2∶1）灌肠。

Figure 3 Colonic tissue of rats after model establishmentNote: (A) Seven days after model establishment. (B) Fourteen days after model establishment. The upper panel shows the overall appearance of the whole colon and the lower panel shows the longitudinal section of the colon model site. The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema.

图4 造模后大鼠结肠长度（A）和结肠炎大体损伤评分（B）注：对照组用生理盐水灌肠；A～D组为模型组，分别用5%TNBS+50%乙醇溶液（体积比1∶1）、5%TNBS+75%乙醇溶液（体积比1∶1）、5%TNBS+无水乙醇（体积比1∶1）、5%TNBS+50%乙醇溶液（体积比2∶1）灌肠。每组6只大鼠，C组造模后第2天和第6天各有1只大鼠死亡，D组造模后第3天有2只大鼠死亡。与对照组比较，*P＜0.05，**P＜0.01。

Figure 4 The length of rat colon after model establishment (A) and colon macroscopic damage index (B) in rats after model establishmentNote: The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema. There're 6 rats contained in each group， but one of which died on days 2 and 6 in group C and two of which died on day 3 in group D after modeling. Compared with the control group, *P < 0.05, **P < 0.01.

图5 造模后大鼠结肠HE（A）和Masson（B）染色结果（×100）注：对照组用生理盐水灌肠；A～D组为模型组，分别用5%TNBS+50%乙醇溶液（体积比1∶1）、5%TNBS+75%乙醇溶液（体积比1∶1）、5%TNBS+无水乙醇（体积比1∶1）、5%TNBS+50%乙醇溶液（体积比2∶1）灌肠。

Figure 5 HE staining （A） and Masson staining （B） of rats’ colons after model establishment (×100)Note：The control group was induced by normal saline enema；Groups A to D were model groups： group A was induced by 5% TNBS + 50% ethanol solution（1∶1 v/v） enema，group B was induced by 5% TNBS+75% ethanol solution （1∶1 v/v） enema，group C was induced by 5% TNBS+100% ethanol solution （1∶1 v/v） enema，group D was induced by 5%TNBS+50% ethanol solution （2∶1 v/v） enema.

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三硝基苯磺酸诱导急性肠纤维化大鼠模型的改良方法

Modified Method for Inducing Acute Intestinal Fibrosis in Rats Using 2,4,6-Trinitrobenzene Sulfonic Acid

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