C57BL/6小鼠炎症性肠病模型的建立与指标评价

doi:10.16431/j.cnki.1671-7236.2024.09.042

摘要/Abstract

摘要： 【目的】通过葡聚糖硫酸钠(dextran sulfate sodium，DSS)或脂多糖(lipopolysaccharide，LPS)刺激构建小鼠炎症性肠病(inflammatory bowel disease，IBD)模型，为IBD研究提供高效经济的模型参考，并为IBD抗炎药物筛选与药效评价提供可靠的方法支撑。【方法】本试验以C57BL/6雄性小鼠为研究对象，分别暴露于不同浓度的DSS(3%、4%和5%)7 d或LPS(5、10和20 mg/kg)4 h。通过测量小鼠结肠长度，HE染色观察肠道形态结构及实时荧光定量PCR检测肠道炎性因子的mRNA表达等对模型进行评价。【结果】 DSS诱导小鼠结肠变短、病理出血、腺体萎缩、结构排列絮乱、炎性细胞浸润以及结肠中炎性因子白细胞介素-1β(interleukin-1β，IL-1β)、IL-6、IL-10和肿瘤坏死因子-α(tumour necrosis factor-α，TNF-α)的mRNA表达显著上升(P＜0.05)，4% DSS即可高效建立IBD模型。同时，LPS引起小鼠肠系膜出血、肠道肿胀和充血，肠绒毛高度降低，隐窝深度升高，小肠中IL-10和IL-1β mRNA水平升高、IL-6 mRNA水平下降，十二指肠和回肠TNF-α mRNA水平上升，空肠TNF-α mRNA水平降低，且以5 mg/kg LPS综合影响最为显著。【结论】 4% DSS和5 mg/kg LPS是C57BL/6小鼠IBD模型建立的最适浓度，肠道病理形态改变、肠道炎性细胞浸润及炎性因子IL-1β、IL-6、IL-10和TNF-α的mRNA表达异常是评价IBD模型的重要指标。

关键词: 葡聚糖硫酸钠(DSS); 脂多糖(LPS); 炎症性肠病; 肠道炎症; C57BL/6小鼠

Abstract: 【Objective】 The study was aimed to construct a mouse inflammatory bowel disease (IBD) model by stimulating with dextran sulfate sodium (DSS) or lipopolysaccharide (LPS), providing an efficient and economical model reference for IBD research, and providing reliable methodological support for the screening and efficacy evaluation of IBD anti-inflammatory drugs. 【Method】 C57BL/6 male mice were chosen and exposed to different concentrations of DSS (3%, 4% and 5%) for 7 d or LPS (5, 10 and 20 mg/kg) for 4 h in the current study.The model was evaluated by measuring colon length, observing intestinal morphology and structure with HE staining, and detecting mRNA expression of intestinal inflammatory factors by Real-time quantitative PCR. 【Result】 DSS could decrease the colon length of mice, led to pathological bleeding, atrophy of glands and disordered structure in colon.Moreover, DSS significantly induced inflammatory cell infiltration and the mRNA expression of the inflammatory cytokines interleukin-1β (IL-1β), IL-6, IL-10 and tumor necrosis factor-α (TNF-α) in the colon of mice, which implied that 4% DSS could establish the IBD efficiently.Meanwhile, LPS could cause mesenteric hemorrhage, intestinal swelling and congestion of mice.Moreover, LPS could decrease the villus height and increased the crypt depth in the intestine.In addition, LPS could also upregulate mRNA levels of intestinal inflammatory cytokines IL-10 and IL-1β, downregulate IL-6 mRNA level and elevate TNF-α mRNA level in the duodenum and ileum, and reduce TNF-α mRNA level in the jejunum.It was found that the most significant effect of intestinal damage was 5 mg/kg LPS treatment. 【Conclusion】 4% DSS or 5 mg/kg LPS treatment were the optimal concentrations to establish IBD model in C57BL/6 mice.Intestinal pathomorphological changes, intestinal inflammatory cell infiltration and inflammatory factors disorder such as the mRNA expression of IL-1β, IL-6, IL-10 and TNF-α were important indicators to evaluate the models of IBD.

Key words: dextran sodium sulfate (DSS); lipopolysaccharide (LPS); inflammatory bowel disease; intestinal inflammation; C57BL/6 mice

中图分类号:

S856.4

何家宇, 杨茗淇, 伍娇, 杨宇, 姚欢, 易金娥, 孔丽. C57BL/6小鼠炎症性肠病模型的建立与指标评价[J]. 中国畜牧兽医, 2024, 51(9): 4153-4163.

HE Jiayu, YANG Mingqi, WU Jiao, YANG Yu, YAO Huan, YI Jine, KONG Li. Establishment and Index Evaluation of Inflammatory Bowel Disease Model in C57BL/6 Mice[J]. China Animal Husbandry and Veterinary Medicine, 2024, 51(9): 4153-4163.

参考文献

[1] ZHU H,WANG H,WANG S,et al.Flaxseed oil attenuates intestinal damage and inflammation by regulating necroptosis and TLR4/NOD signaling pathways following lipopolysaccharide challenge in a piglet model[J].Molecular Nutrition and Food Research,2018,62(9):e1700814.
[2] 高楠,窦秀静,杨洋,等.氨基酸在炎症性肠病中的作用及其信号通路[J].畜牧兽医学报,2020,51(10):2349-2358. GAO N,DOU X J,YANG Y,et al.Effects of amino acids on inflammatory bowel disease and its signaling pathways[J]. Acta Veterinaria et Zootechnica Sinica,2020,51(10):2349-2358.(in Chinese)
[3] 李时杰,牛俊坤,缪应雷.炎症性肠病动物模型研究进展[J].中华炎性肠病杂志,2019,1:21-24. LI S J,NIU J K,MIU Y L.Advances in animal models of inflammatory bowel disease[J].Chinese Journal of Inflammatory Bowel Diseases,2019,1:21-24.(in Chinese)
[4] 陈珠,杨彩霞,倪婉晔,等.炎症性肠病动物模型的研究进展[J].实验动物科学,2016,33(5):61-63. CHEN Z,YANG C X,NI W Y,et al.Research progress in animal models of inflammatory bowel disease[J].Laboratory Animal Science,2016,33(5):61-63.(in Chinese)
[5] MOHAMMAD S,THIEMERMANN C.Role of metabolic endotoxemia in systemic inflammation and potential interventions[J].Frontiers in Immunology,2020,11:594150.
[6] RANGAN P,CHOI I,WEI M,et al.Fasting-mimicking diet modulates microbiota and promotes intestinal regeneration to reduce inflammatory bowel disease pathology[J].Cell Reports,2019,26(10):2704-2719.e6.
[7] WANG L,HOU Y,YI D,et al.Beneficial roles of dietary oleum cinnamomi in alleviating intestinal injury[J].Frontiers in Bioscience-landmark,2015,20(5):814-828.
[8] 陈素傲,金世柱.葡聚糖硫酸钠诱导鼠溃疡性结肠炎模型研究进展[J].中国比较医学杂志,2020,30(4):142-146. CHEN S A,JIN S Z.Murine model of dextran sodium sulfate-induced ulcerative colitis[J].Chinese Journal of Comparative Medicine,2020,30(4):142-146.(in Chinese)
[9] 王梦竹,贾军峰,乔新月,等.不同浓度脂多糖对小鼠免疫功能及小肠组织形态的影响[J].中国生物制品学杂志,2019,32(4):380-385. WANG M Z,JIA J F,QIAO X Y,et al.Effect of lipopolysaccharide at various concentrations on immune function and intestinal tissue morphology of mice[J].Chinese Journal of Biologicals,2019,32(4):380-385.(in Chinese)
[10] LIU Y,XU Q,WANG Y,et al.Necroptosis is active and contributes to intestinal injury in a piglet model with lipopolysaccharide challenge[J].Cell Death & Disease,2021,12(1):62.
[11] 席芳.改良HE染色技术在病理诊断中的应用效果[J].中国民康医学,2023,35(13):127-129. XI F.Application effects of modified HE staining technique in pathological diagnosis[J].Medical Journal of Chinese People’s Health,2023,35(13):127-129.(in Chinese)
[12] CHASSAING B,AITKEN J D,MALLESHAPPA M,et al.Dextran sulfate sodium (DSS)-induced colitis in mice[J].Current Protocols in Immunology,2014,104:15.25.1-15.25.14.
[13] NUNES N S,CHANDRAN P,SUNDBY M,et al.Therapeutic ultrasound attenuates DSS-induced colitis through the cholinergic anti-inflammatory pathway[J].EBioMedicine,2019,45:495-510.
[14] 李雨芯.IL-22对DSS诱导的溃疡性结肠炎小鼠模型肠上皮屏障的作用及机制研究[D].南宁:广西医科大学,2021. LI Y X.Effects of IL-22 on intestinal epithelial barrier in mice with DSS-induced ulcerative colitis and its mechanism[D].Nanning:Guangxi Medical University,2021.(in Chinese)
[15] 高威,张昱,张伟,等.DSS模型小鼠结肠组织变化及葛根芩连汤干预的实验研究[J].时珍国医国药,2019,30(2):510-512. GAO W,ZHANG Y,ZHANG W,et al.Experiment study on the change of colon tissue in DSS model mice and the intervention of Gegenqinlian decoction[J].Lishizhen Medicine and Materia Medica Research,2019,30(2):510-512.(in Chinese)
[16] 衡宇,李晰,孙涛,等.葡聚糖硫酸钠自由饮用与灌胃诱导小鼠溃疡性结肠炎模型的对比研究[J].中国药师,2017,20(4):603-606. HENG Y,LI X,SUN T,et al.Comparison of ulcerative colitis models respectively induced by free drinking and intragastric administration of dextran sodium sulfate in mice[J].China Pharmacist,2017,20(4):603-606.(in Chinese)
[17] 顾思臻,薛艳,王宇,等.3种浓度葡聚糖硫酸钠诱导小鼠溃疡性结肠炎模型的比较[J].现代中西医结合杂志,2019,28(19):2053-2056. GU S Z,XUE Y,WANG Y,et al.Comparison of the models of ulcerative colitis induced by three kind of concentration of dextran sulfate sodium in mice[J].Modern Journal of Integrated Traditional Chinese and Western Medicine,2019,28(19):2053-2056.(in Chinese)
[18] 牛鹏飞,王延召,曾庆敏,等.肠黏膜屏障功能及损伤机制研究进展[J].中华临床医师杂志(电子版),2020,14(9):735-739. NIU P F,WANG Y Z,ZENG Q M,et al.Progress in research of intestinal mucosal barrier function and its dysfunction mechanisms[J].Chinese Journal of Clinicians (Electronic Edition),2020,14(9):735-739.(in Chinese)
[19] CHELAKKOT C,GHIM J,RYU S H.Mechanisms regulating intestinal barrier integrity and its pathological implications[J].Exxperimental and Molecular Medicine,2018,50(8):103.
[20] LIU T,ZHANG L,JOO D,et al.NF-κB signaling in inflammation[J].Signal Transduction and Targeted Therapy,2017,2:17023.
[21] ANDREWS C,MCLEAN M H,DURUM S K.Cytokine tuning of intestinal epithelial function[J].Frontiers in Immunology,2018,9:1270.
[22] 张旭晖,曹银娣,孙智远,等.银杏-杜仲叶发酵物对脂多糖应激肉仔鸡的肠道功能和免疫反应的影响[J].动物营养学报,2023,35(4):2196-2208. ZHANG X H,CAO Y D,SUN Z Y,et al.Effects of fermented Ginkgo biloba-Eucommia leaves on intestinal function and immune response in stressed broilers challenged by lipopolysaccharide[J].Chinese Journal of Animal Nutrition,2023,35(4):2196-2208.(in Chinese)
[23] NEURATH M F.Cytokines in inflammatory bowel disease[J].Nature Reviews Immunology,2014,14(5):329-342.
[24] COCCIA M,HARRISON O J,SCHIERING C,et al.IL-1β mediates chronic intestinal inflammation by promoting the accumulation of IL-17A secreting innate lymphoid cells and CD4⁺ Th17 cells[J].Journal of Experimental Medicine,2012,209(9):1595-1609.
[25] ATREYA R,MUDTER J,FINOTTO S,et al.Blockade of interleukin 6 trans signaling suppresses T-cell resistance against apoptosis in chronic intestinal inflammation:Evidence in crohn disease and experimental colitis in vivo[J].Nature Medicine,2000,6(5):583-588.
[26] ASSEMAN C,MAUZE S,LEACH M W,et al.An essential role for interleukin 10 in the function of regulatory T cells that inhibit intestinal inflammation[J].Journal of Experimental Medicine,1999,190(7):995-1004.
[27] GüVENC M,CELLAT M,ÖZKAN H,et al.Protective effects of tyrosol against DSS-induced ulcerative colitis in rats[J].Inflammation,2019,42(5):1692.
[28] WEI W,MU S,HAN Y,et al.Gpr174 knockout alleviates DSS-induced colitis via regulating the immune function of dendritic cells[J].Frontiers in Immunology,2022,13:841254.
[29] DONG N,LI X,XUE C,et al.Astragalus polysaccharides alleviates LPS-induced inflammation via the NF-κB/MAPK signaling pathway[J].Journal of Cellular Physiology,2020,235(7-8):5525-5540.
[30] 余应嘉,叶淑芳,邓燕芳,等.大黄素甲醚对脂多糖诱导肠上皮细胞损伤自噬与缝隙连接蛋白的作用影响[J].中国医院药学杂志,2023,43(18):2032-2036. YU Y J,YE S F,DENG Y F,et al.Effect of physcion on autophagy and gap junction protein in LPS-induced intestinal epithelial cell injury model[J].Chinese Journal of Hospital Pharmacy,2023,43(18):2032-2036.(in Chinese)
[31] WANG X,YUAN Z,ZHU L,et al.Protective effects of betulinic acid on intestinal mucosal injury induced by cyclophosphamide in mice[J].Pharmacological Reports,2019,71(5):929-939.
[32] GAO W,ZHANG Y.Depression of lncRNA MINCR antagonizes LPS-evoked acute injury and inflammatory response via miR-146b-5p and the TRAF6-NFκB signaling[J].Molecular Medicine (Cambridge,Mass),2021,27(1):124.
[33] 李露,付王威,吴睿婷,等.基于UPLC-Q-TOF/MS的黑灵芝多糖对大鼠急性肠道炎症作用机制研究[J].食品工业科技,2021,42(6):310-317. LI L,FU W W,WU R T,et al.Effect of Ganoderma lucidum polysaccharides on intestinal inflammation in rats based on UPLC-Q-TOF/MS[J].Science and Technology of Food Industry,2021,42(6):310-317.(in Chinese)
[34] CORWIN E J.Understanding cytokines.PartⅠ:Physiology and mechanism of action[J].Biological Research for Nursing,2000,2(1):30-40.