沙门菌感染小鼠氧化应激模型的建立及其分子机制研究

doi:10.16431/j.cnki.1671-7236.2021.10.036

摘要/Abstract

摘要： 试验旨在建立鼠伤寒沙门菌诱导的昆明小鼠氧化应激模型并探讨其分子机制。将18只小鼠随机分为3组，每组6只，分别灌胃生理盐水（对照组）、低剂量沙门菌菌液（试验组1）和高剂量沙门菌菌液（试验组2）。灌胃24 h后解剖小鼠并采集样品，检测血清氧化应激指标，观察肝脏、十二指肠、空肠和回肠切片并评分，测定肝脏和十二指肠抗氧化酶、抗氧化信号通路关键蛋白和紧密连接蛋白的mRNA表达量。结果表明，与对照组相比，试验组2小鼠血清中超氧化物歧化酶（SOD）和谷胱甘肽过氧化物酶（GPx1）活力显著或极显著降低（P<0.05；P<0.01），而丙二醛（MDA）含量极显著上升（P<0.01）；试验组1和2小鼠十二指肠表现为绒毛断裂脱落和肠道固有层溃疡坏死，肝脏出现炎性细胞浸润、肝索紊乱和点状坏死；试验组2小鼠十二指肠和肝脏中锰超氧化物歧化酶（SOD2）、GPx1、血红素加氧酶（HO-1）、自噬相关蛋白（p62）、核因子E2相关因子2（Nrf2） mRNA表达量显著或极显著降低（P<0.05；P<0.01），十二指肠闭锁小带蛋白1（ZO-1）和密封蛋白（OCLN）以及肝脏ZO-1和紧密连接蛋白-8（CLDN8） mRNA表达量极显著降低（P<0.01）。综合上述试验结果，使用高剂量沙门菌成功建立了小鼠氧化应激模型，并发现高剂量沙门菌可能通过降低p62和Nrf2的转录来抑制十二指肠和肝脏中抗氧化酶的表达，造成十二指肠和肝脏细胞屏障功能损伤。

关键词: 沙门菌; 氧化应激; 紧密连接

Abstract: The study was aimed to establish the oxidative stress model of Kunming mouse induced by Salmonella Typhimurium, and explore its molecular mechanism. 18 mice were randomly divided into 3 groups with 6 mice in each group. Normal saline (control group), low dose Salmonella solution (group 1) and high dose Salmonella solution (group 2) were given by gavage respectively. Mice were dissected and samples were collected after gavage for 24 h. Serum oxidative stress indexes were detected. HE sections of liver, duodenum, jejunum and ileum were observed and scored. The mRNA expression of liver and duodenal antioxidant enzymes, key proteins in the antioxidant signal pathway, and tight junction proteins were detected. The results showed that:Compared with the control group, superoxide dismutase (SOD) and glutathione peroxidase (GPx1) activity in the serum of the group 2 decreased significantly or extremely significantly (P<0.05;P<0.1), while malondialdehyde (MDA) increased extremely significantly (P<0.01). The duodenal villi were broken and dropped off, and ulceronecrotic of the intestinal lamina propria, and the liver mainly showed inflammatory cell infiltration, hepatic cord disorders and punctate necrosis in groups 1 and 2. Compared with the control group, the expression of manganese superoxide dismutase (SOD2), GPx1, heme oxygenase-1 (HO-1), sequestosome 1 (p62) and nuclear factor E2-related factor 2 (Nrf2) mRNA decreased significantly or extremely significantly in duodenum and liver (P<0.05;P<0.01), while the expression of ZO-1 and OCLN mRNA in duodenal, and the expression of ZO-1 and claudin-8 (CLDN8) mRNA in liver decreased extremely significantly (P<0.01) in group 2. The above results showed that the oxidative stress model of mice was successfully established by high dose Salmonella solution, and it was found that high dose Salmonella solution might inhibit the expression of antioxidant enzymes in duodenum and liver by reducing the transcription of p62 and Nrf2, resulting in the damage of cell barrier function in duodenum and liver.

Key words: Salmonella; oxidative stress; tight junctions

中图分类号:

S852.61

刘雪姣, 李海花, 王怡梦, 尚智援, 吴悦, 乔家运. 沙门菌感染小鼠氧化应激模型的建立及其分子机制研究[J]. 中国畜牧兽医, 2021, 48(10): 3834-3844.

LIU Xuejiao, LI Haihua, WANG Yimeng, SHANG Zhiyuan, WU Yue, QIAO Jiayun. Establishment of Oxidative Stress Model in Mice Infected with Salmonella and Its Molecular Mechanism[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(10): 3834-3844.

参考文献

[1] CHAKRABORTY S, LIU L, FITZSIMMONS L, et al.Glycolytic reprograming in Salmonella counters NOX2-mediated dissipation of ΔpH[J]. Nature Communications, 2020, 11(1):1783.
[2] AKAIKE T.Host defense and oxidative stress signaling in bacterial infection[J]. Nihon Saikingaku Zasshi, 2015, 70(3):339-349.
[3] WIDLANSKY M E, GUTTERMAN D D.Regulation of endothelial function by mitochondrial reactive oxygen species[J]. Antioxidants & Redox Signaling, 2011, 15(6):1517-1530.
[4] IRRAZABAL T, THAKUR B K, KANG M, et al.Limiting oxidative DNA damage reduces microbe-induced colitis-associated colorectal cancer[J]. Nature Communications, 2020, 11(1):1802.
[5] 卢德勋.动物营养学科发展在战略方向上的重大突破:构建动物健康营养理论和技术体系及其实际应用[J]. 动物营养学报, 2021, 33(1):1-12. LU D X.A major breakthrough in development of animal nutrition in strategic direction:Building an animal health and nutrition theory and technology system and its application[J]. Chinese Journal of Animal Nutrition, 2021, 33(1):1-12.(in Chinese)
[6] CHEN Q, WANG Y, JIAO F, et al.Betaine inhibits Toll-like receptor 4 responses and restores intestinal microbiota in acute liver failure mice[J]. Scientific Reports, 2020, 10(1):21850.
[7] DE OLIVEIRA A M, DE FREITAS A F S, DE SOUZA COSTA M D, et al.Pilosocereus gounellei (Cactaceae) stem extract decreases insulin resistance, inflammation, oxidative stress, and cardio-metabolic risk in diet-induced obese mice[J]. Journal of Ethnopharmacology, 2021, 265:113327.
[8] LI C, TAN F, YANG J, et al.Antioxidant effects of Apocynum venetum tea extracts on D-galactose-induced aging model in mice[J]. Antioxidants (Basel), 2019, 8(9):381.
[9] MAZKOUR S, SHEKARFOROUSH S S, BASIRI S, et al.Effects of two probiotic spores of Bacillus species on hematological, biochemical, and inflammatory parameters in Salmonella Typhimurium infected rats[J]. Scientific Reports, 2020, 10:8035.
[10] 罗波文, 邹田德, 陈丽玲, 等.嗜酸乳杆菌对氧化应激仔猪小肠上皮细胞抗氧化能力和紧密连接蛋白表达的影响[J]. 动物营养学报, 2020, 32(5):2108-2115. LUO B W, ZOU T D, CHEN L L, et al.Effects of Lactobacillus acidophilus on antioxidative ability and expression of tight junction protein in intestinal epithelial cells of piglets under oxidative stress[J]. Chinese Journal of Animal Nutrition, 2020, 32(5):2108-2115.(in Chinese)
[11] ZHANG L J, TU R Q, WANG Y, et al.Early-life exposure to lead induces cognitive impairment in elder mice targeting SIRT1 phosphorylation and oxidative alterations[J]. Frontiers in Physiology, 2017, 8:446.
[12] QIAN Y, ZHANG J, ZHOU X K, et al.Lactobacillus plantarum CQPC11 isolated from Sichuan pickled cabbages antagonizes D-galactose-induced oxidation and aging in mice[J]. Molecules, 2018, 23(11):3026.
[13] 宫秀燕, 韦明, 蒋秋斐, 等.凝结芽孢杆菌对肠炎沙门氏菌感染肉鸡生产性能和抗氧化功能的影响[J]. 中国畜牧杂志, 2015, 51(17):74-79. GONG X Y, WEI M, JIANG Q F, et al.Effect of Bacillus coagulans on growth performance and antioxidant function of broilers challenged with Salmonella Enteritis[J]. Chinese Journal of Animal Science, 2015, 51(17):74-79.(in Chinese)
[14] 窦彩霞, 王倩, 安建勇, 等.Keap1-Nrf2/ARE信号通路及其在畜禽抗氧化中的研究进展[J]. 中国畜牧兽医, 2019, 46(9):2567-2574. DOU C X, WANG Q, AN J Y, et al.The research progress of Keap1-Nrf2/ARE signaling pathway and its role in the antioxidation of livestock and poultry[J]. China Animal Husbandry & Veterinary Medicine, 2019, 46(9):2567-2574.(in Chinese)
[15] HOS N J, GANESAN R, GUTIÉRREZ S, et al.Type Ⅰ interferon enhances necroptosis of Salmonella Typhimurium-infected macrophages by impairing antioxidative stress responses[J]. Journal of Cell Biology, 2017, 216(12):4107-4121.
[16] 詹晖, 田林林, 钟常城, 等.植物乳杆菌WLPL01艾草发酵液干预鼠伤寒沙门氏菌感染小鼠的作用[J]. 南昌大学学报(理科版), 2019, 43(4):351-358. ZHAN H, TIAN L L, ZHONG C C, et al.Effect of Artemisia argyi fermentation liquid by Lactobacillus plantarum WLPL01 on infection of Salmonella Typhimurium in mice[J]. Journal of Nanchang University(Natural Science), 2019, 43(4):351-358.(in Chinese)
[17] ZHANG L, GUI S Q, WANG J, et al.Oral administration of green tea polyphenols (TP) improves ileal injury and intestinal flora disorder in mice with Salmonella Typhimurium infection via resisting inflammation, enhancing antioxidant action and preserving tight junction[J]. Journal of Functional Foods, 2020, 64:103654.
[18] 马东霞.Kupffer细胞中microRNA-155的缺失减轻小鼠肝脏部分热缺血再灌注损伤[D].武汉:华中科技大学, 2016. MA D X.MicroRNA-155 deficiency in Kupffer cells ameliorates liver partial hot ischemia-reperfusion injury in mice[D].Wuhan:Huazhong University of Science and Technology, 2016.(in Chinese)
[19] CHIU C J, MCARDLE A H, BROWN R, et al.Intestinal mucosal lesion in low-flow states:Ⅰ.A morphological, hemodynamic, and metabolic reappraisal[J]. Archives of Surgery, 1970, 101(4):478-483.
[20] KAMEL H M H, HUME S P, CARR K E, et al.Development of villous damage in mouse small intestine after local hyperthermia or irradiation[J]. Journal of Submicroscopic Cytology and Pathology, 1988, 20(1):185-193.
[21] CHEN X, XI Z, LIANG H, et al.Melatonin prevents mice cortical astrocytes from hemin-induced toxicity through activating PKCα/Nrf2/HO-1 signaling in vitro[J]. Frontiers in Neuroscience, 2019, 13:760.
[22] 盛琳琳, 曹秀琴, 甄庆洁, 等.嗜肺军团菌激活Nrf2-Keap1通路抑制小鼠RAW264.7巨噬细胞自噬[J]. 细胞与分子免疫学杂志, 2019, 35(10):878-885. SHENG L L, CAO X Q, ZHEN Q J, et al.Legionella pneumophila activates Nrf2-Keap1 pathway to inhibit autophagy of mouse RAW264.7 macrophages[J]. Chinese Journal of Cellular and Molecular Immunology, 2019, 35(10):878-885.(in Chinese)
[23] RUAN Y C, WANG Y, DA Silva N, et al.CFTR interacts with ZO-1 to regulate tight junction assembly and epithelial differentiation through the ZONAB pathway[J]. Journal of Cell Science, 2014, 127(20):4396-4408.
[24] SASSI A, WANG Y, CHASSOT A, et al.Interaction between epithelial sodium channel γ-subunit and Claudin-8 modulates paracellular sodium permeability in renal collecting duct[J]. Journal of the American Society of Nephrology, 2020, 31(5):1009-1023.
[25] LI X F, ZHANG X J, ZHANG C, et al.Ulinastatin protects brain against cerebral ischemia/reperfusion injury through inhibiting MMP-9 and alleviating loss of ZO-1 and occludin proteins in mice[J]. Experimental Neurology, 2018, 302:68-74.
[26] 王菲, 黄毅, 李延森, 等.Keap1/Nrf2/ARE通路相关基因在热诱导的氧化应激小鼠肝脏中的表达[J]. 中国生物化学与分子生物学报, 2014, 30(3):284-290. WANG F, HUANG Y, LI Y S, et al.Expression of Keap1/Nrf2/ARE pathway-related genes in protective response to heat-induced oxidative stress in the liver of mice[J]. Chinese Journal of Biochemistry and Molecular Biology, 2014, 30(3):284-290.(in Chinese)
[27] YANG S H, YU L H, LI L, et al.Protective mechanism of sulforaphane on cadmium-induced sertoli cell injury in mice testis via Nrf2/ARE signaling pathway[J]. Molecules, 2018, 23(7):1774.
[28] 张斌, 夏作理, 赵晓民, 等.氧化应激模型的建立及其评价[J]. 中国临床康复, 2006, 44:112-114. ZHANG B, XIA Z L, ZHAO X M, et al.Establishment and evaluation of oxidative stress models[J]. Chinese Journal of Clinical Rehabilitation, 2006, 44:112-114.(in Chinese)
[29] 李驰, 闫峻, 穆淑琴, 等.一次性腹腔注射Diquat诱导构建肉仔鸡慢性氧化应激模型[J]. 西北农业学报, 2017, 26(4):509-513. LI C, YAN J, MU S Q, et al.The construction model of chronic oxidative stress in broilers induced by intraperitoneal injection of Diquat[J]. Acta Agriculturae Boreali-occidentalis Sinica, 2017, 26(4):509-513.(in Chinese)
[30] 王慧瑞, 贺海旺, 于佳彤, 等.3-硝基丙酸诱导小鼠睾丸氧化应激模型的建立[J]. 延边大学农学学报, 2020, 42(4):62-66. WANG H R, HE H W, YU J T, et al.Establishment of mouse testicular oxidative stress model induced by 3-nitropropionic acid[J]. Journal of Agricultural Science Yanbian University, 2020, 42(4):62-66.(in Chinese)
[31] 强文军, 王莹, 路茜, 等.β-1, 3-葡聚糖对肠炎沙门氏菌感染鸡肠道氧化还原状态的影响[J]. 中国畜牧杂志, 2010, 46(23):51-55. QIANG W J, WANG Y, LU Q, et al.The effect of β-1, 3-glucan on the redox status of the intestinal tract in chickens infected with Salmonella Enteritidis[J]. Chinese Journal of Animal Science, 2010, 46(23):51-55.(in Chinese)
[32] ZAKI M H, FUJII S, OKAMOTO T, et al.Cytoprotective function of heme oxygenase 1 induced by a nitrated cyclic nucleotide formed during murine salmonellosis[J]. Journal of Immunology, 2009, 182(6):3746-3756.
[33] CORR S C, PALSSON-MCDERMOTT E M, GRISHINA I, et al.MyD88 adaptor-like (Mal) functions in the epithelial barrier and contributes to intestinal integrity via protein kinase C[J]. Mucosal Immunology, 2014, 7(1):57-67.
[34] YU H B, CROXEN M A, MARCHIANDO A M, et al.Autophagy facilitates Salmonella replication in HeLa cells[J]. mBio, 2014, 5(2):e00865.
[35] BAREGAMIAN N, SONG J, BAILEY C E, et al.Tumor necrosis factor-alpha and apoptosis signal-regulating kinase 1 control reactive oxygen species release, mitochondrial autophagy, and c-Jun N-terminal kinase/p38 phosphorylation during necrotizing enterocolitis[J]. Oxidative Medicine Cellular Longevity, 2009, 2(5):297-306.
[36] KÖHLER H, SAKAGUCHI T, HURLEY B P, et al.Salmonella enterica serovar Typhimurium regulates intercellular junction proteins and facilitates transepithelial neutrophil and bacterial passage[J]. American Journal of Physiology Gastrointestinal & Liver Physiology, 2007, 293(1):178-187.
[37] 池肇春.肠道微生物与自身免疫性肝病研究进展与评价[J]. 世界华人消化杂志, 2019, 27(1):50-62. CHI Z C.Intestinal microbiome and autoimmune liver disease[J]. World Chinese Journal of Digestology, 2019, 27(1):50-62.(in Chinese)
[38] 王改, 王志华, 段红年, 等.乌司他丁对过氧化氢诱导的肠上皮屏障损伤的保护作用[J]. 胃肠病学, 2017, 22(4):224-228. WANG G, WANG Z H, DUAN H N, et al.Protective effect of ulinastatin on hydrogen peroxide-induced intestinal epithelial barrier disruption[J]. Chinese Journal of Gastroenterology, 2017, 22(4):224-228.(in Chinese)
[39] 陈景杰, 陈新瑶, 董星, 等.猴头菇多糖对氧化应激状态下猪小肠上皮细胞紧密连接相关基因表达的影响[J]. 中国兽医科学, 2017, 47(1):128-134. CHEN J J, CHEN X Y, DONG X, et al.Effects of Hericium erinaceus polysaccharide on gene expression of tight junction in intestinal porcine enterocyte cells induced by oxidative stress[J]. Veterinary Science in China, 2017, 47(1):128-134.(in Chinese)
[40] 吴姚平, 武晓丽, 徐锋, 等.鼠伤寒沙门氏菌对肠道屏障的破坏作用[J]. 南昌大学学报:理科版, 2017, 41(3):265-269. WU Y P, WU X L, XU F, et al.Salmonella Typhimurium breaks intestinal barrier of mouse[J]. Journal of Nanchang University(Natural Science), 2017, 41(3):265-269.(in Chinese)
[41] WANG L H, LI L, LV Y, et al.Lactobacillus plantarum restores intestinal permeability disrupted by Salmonella infection in newly-hatched chicks[J]. Scientific Reports, 2018, 8(1):2229.
[42] SPLICHAL I, DONOVAN S M, SPLICHALOVA Z, et al.Colonization of Germ-free piglets with commensal Lactobacillus amylovorus, Lactobacillus mucosae, and probiotic E.coli Nissle 1917 and their interference with Salmonella Typhimurium[J]. Microorganisms, 2019, 7(8):273.
[43] ZHANG X L, CHENG X P, YU L, et al.MCOLN1 is a ROS sensor in lysosomes that regulates autophagy[J]. Nature Communications, 2016, 7(1):12109.
[44] ISHIMURA R, TANAKA K, KOMATSU M.Dissection of the role of p62/Sqstm1 in activation of Nrf2 during xenophagy[J]. Febs Letters, 2014, 588(5):822-828.
[45] 赖华毅.鼠伤寒沙门氏菌spvB基因对宿主细胞自噬的影响及其分子机制的研究[D].南昌:南昌大学, 2018. LAI H Y.Effects of spvB gene in Salmonella Typhimurium on antophagy of host cells and its molecular mechanism[D].Nanchang:Nanchang University, 2018.(in Chinese)