食淀粉乳杆菌对猪A群轮状病毒NSP4在小鼠肠组织表达的影响
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摘要
为探究食淀粉乳杆菌对猪轮状病毒(PRV)非结构蛋白NSP4表达的影响,对3日龄清洁级BALB/c小鼠分别在感染PRV前后灌服食淀粉乳杆菌,应用实时荧光定量PCR检测感染病毒后空肠组织中PRVNSP4的m RNA相对表达量,采用比色法测定空肠组织内钙离子含量变化,并通过ELISA定量检测空肠组织中整联蛋白α2β1、磷脂酶C和Dicer的含量。结果显示,饲喂食淀粉乳杆菌第5天和第7天时,预防组和辅疗组中NSP4的m RNA相对表达量显著低于病毒组(P<0.05);除预防组第11天外,感染病毒后各检测时间点预防组和辅疗组的钙离子含量显著低于病毒组(P<0.05);饲喂食淀粉乳杆菌第7天、第9天和第11天时,预防组和辅疗组中整联蛋白α2β1的含量显著低于病毒组(P<0.05);感染病毒后各检测时间点预防组和辅疗组中磷脂酶C和Dicer的含量均低于病毒组。以上结果表明:在小鼠感染轮状病毒前后灌服食淀粉乳杆菌均能显著抑制PRV NSP4的m RNA表达和整联蛋白α2β1的合成,并降低空肠组织中钙离子,磷脂酶C和Dicer的含量,这为研究益生菌抗轮状病毒机制提供参考。
To investigate the effect of Lactobacillus amylovorus on the m RNA expression of non-structural protein NSP4 in mice infected by porcine rotavirus(PRV),three-day-old clean grade BALB/c mice were fed with L.amylovorus before or after PRV infection.The relative m RNA expression levels of NSP4 in the jejunal tissues of mice infected with PRV were detected by real-time fluorescence quantitative PCR.The calcium ion content in the jejunal tissues were determined by colorimetry,and the content of integrinα2β1,phospholipase C and Dicer in the jejunal tissues were quantitatively detected by ELISA.The results showed that the relative m RNA expression levels of NSP4 in prevention and auxiliary treatment groups were significantly lower than that in rotavirus group on the fifth and seventh day after exposed to L.amylovorus(P<0.05).Except for the prevention group on the eleventh day,the calcium ion content in the prevention and auxiliary treatment groups were significantly lower than that in the rotavirus group at each detection time points with infected PRV(P<0.05).All of the content of integrin α2β1 in prevention and auxiliary treatment groups at seven,nine and eleven days post-exposed to L.amylovorus were significantly lower than that in the rotavirus group(P<0.05).The content of phospholipase C and Dicer in prevention and auxiliary treatment groups at each detection time point with infected PRV were lower than that in the rotavirus group.The results indicated that L.amylovorus can significantly inhibit the m RNA expression of PRV NSP4 and the synthesis of integrin α2β1,and can reduce the content of calcium ion,phospholipase C and Dicer,which will provide reference for studying the mechanism of probiotics against rotavirus.
To investigate the effect of Lactobacillus amylovorus on the m RNA expression of non-structural protein NSP4 in mice infected by porcine rotavirus(PRV),three-day-old clean grade BALB/c mice were fed with L.amylovorus before or after PRV infection.The relative m RNA expression levels of NSP4 in the jejunal tissues of mice infected with PRV were detected by real-time fluorescence quantitative PCR.The calcium ion content in the jejunal tissues were determined by colorimetry,and the content of integrinα2β1,phospholipase C and Dicer in the jejunal tissues were quantitatively detected by ELISA.The results showed that the relative m RNA expression levels of NSP4 in prevention and auxiliary treatment groups were significantly lower than that in rotavirus group on the fifth and seventh day after exposed to L.amylovorus(P<0.05).Except for the prevention group on the eleventh day,the calcium ion content in the prevention and auxiliary treatment groups were significantly lower than that in the rotavirus group at each detection time points with infected PRV(P<0.05).All of the content of integrin α2β1 in prevention and auxiliary treatment groups at seven,nine and eleven days post-exposed to L.amylovorus were significantly lower than that in the rotavirus group(P<0.05).The content of phospholipase C and Dicer in prevention and auxiliary treatment groups at each detection time point with infected PRV were lower than that in the rotavirus group.The results indicated that L.amylovorus can significantly inhibit the m RNA expression of PRV NSP4 and the synthesis of integrin α2β1,and can reduce the content of calcium ion,phospholipase C and Dicer,which will provide reference for studying the mechanism of probiotics against rotavirus.
引文
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[27]贾慧琼,安敏,徐熙,等.植物乳杆菌STⅢ对大鼠小肠上皮细胞钙离子吸收的影响[J].中国微生态学杂志,2016(01):6-9.JIA Hui-qiong,AN Min,XU Xi,et al.Experimental study on the effect of Lactobocillus plantarum STⅢon calcium absorption in rat intestinal epithelial cells[J].Chinese Journal of Microecology,2016(01):6-9.(in Chinese)
[2]任大勇.益生乳酸杆菌的黏附及免疫调节作用研究[D].长春:吉林大学,2013.Ren Da-yong.Research on adhesion and immunoregulation of probiotic lactobacillus strains[D].Changchun:Jilin University,2013.(in Chinese)
[3]LIN C K,TSAI C,LIN P P,et al.Lactobacillus acidophilus LAP5 able to inhibit the Salmonella choleraesuis invasion to the human Caco-2 epithelial cell[J].Anaerobe,2008,14(5):251-255.
[4]GAO X,HUANG L,ZHU L,et al.Inhibition of H9N2 virus invasion into dendritic cells by the S-layer protein from L.acidophilus ATCC 4356[J].Frontiers in Cellular and Infection Microbiology,2016,16:137.
[5]赵毅博,田宗民,王莹莹,等.食淀粉乳杆菌代谢产物抑菌与抗病毒的比较研究[J].中国畜牧兽医,2014,41(8):242-246.ZhAO Yi-bo,TIAN Zong-min,WANG Ying-ying,et al.Comparative study on effect of bacteriostasis and antivirus of Lactobacillus amylovorus metabolites[J].China Animal Husbandry&Veterinary Medicine,2014,41(8):242-246.(in Chinese)
[6]MIRAZIMI A,MAGNUSSON KE,SVENSSOR L.A cytoplasmic region of the NSP4 enterotoxin of rotavirus is involved in retention in the endoplasmic reticulum[J].Journal of General Virology,2003,84(Pt 4):875-883.
[7]OLAYA G,ULLOA R,VELEZ R,et al.In vitro antiviral activity of Lactobacillus casei and Bifidobacterium adolescentis against rotavirus infection monitored by NSP4 protein production[J].Journal of Applied Microbiology,2016,120(4):1041-1051.
[8]OUSINGSAWAT J,MIRZA M,TIAN Y,et al.Rotavirus toxin NSP4 induces diarrhea by activation of TMEM16A and inhibition of Na+absorption[J].Pflugers ArchivEuropean Journal of Physiology,2011,461(5):579-589.
[9]赵建勇,独军正,高闪电,等.整联蛋白受体介导的病毒感染作用[J].中国人兽共患病学报,2009,25(4):379-382.ZHAO Jian-yong,DU Jun-zheng,GAO Shan-dian,et al.Integrin receptor-mediated viral infection[J].Chinese Journal of Zoonoses,2009,25(4):379-382.(in Chinese)
[10]潘丽.大豆凝集素对整联蛋白介导的猪肠上皮细胞增殖与凋亡机制的研究[D].长春:吉林农业大学,2017.PAN Li.Study on the mechanism of soybean lectin on integrin-mediated proliferation and apoptosis of porcine intestinal epithelial cells[D].Changchun:Jilin Agricultural University,2017.(in Chinese)
[11]RUTNAM ZJ,WIGHT TN,YANG BB.miRNAs regulate expression and function of extracellular matrix molecules[J].Matrix Biology,2013,32(2):74-85.
[12]BERNSTEIN E,CAUDY AA,HAMMOND SM,et al.Role for a bidentate ribonuclease in the initiation step of RNA interference[J].Nature,2001,409(6818):363-366.
[13]张睿.益生菌增加细胞表面半乳糖在抑制RV感染中的作用[D].哈尔滨:东北农业大学,2017.ZHANG Rui.Effect of Probiotic Increased Galactose on the Inhibition of RV Infection[D].Harbin:Northeast Agricultural University,2014.(in Chinese)
[14]ZHANG M,ZENG CQ,MORRIS AP,et al.A functional NSP4enterotoxin peptide secreted from rotavirus-infected cells[J].Journal of Virology,2000,74(24):11663-11670.
[15]MAJEROWICZ S,KUBELKA CF,STEPHENS P,et al.Ultrastructural study on experimental infection of rotavirus in a murine heterologous model[J].Mem Inst Oswaldo Cruz Riode Janeiro Vology,1994,89(3):395-402.
[16]林海军.轮状病毒小鼠动物模型的建立与初步应用[D].厦门:厦门大学,2014.LIN Hai-jun.Establishment and Preliminary Applications of the Rotavirus Sucking Mouse Model[D].Xiamen:Xiamen University,2014.(in Chinese)
[17]ESTES MK,KANG G,ZENG CQ,et al.Pathogenesis of rotavirus gastroenteritis[J].Novartis Found Symposium,2001,238:82-96,96-100.
[18]陈军华,刘作义.轮状病毒感染发病机制研究进展[J].国外医学(儿科学分册),2004,31(5):237-239.CHEN Jun-hua,LIU Zuo-yi.Research progress in the pathogenesis of rotavirus infection[J].Foreign Medical Sciences of Pediatrics,2004,31(5):237-239.(in Chinese)
[19]HU L,CRAWFORD SE,HYSER JM,et al.Rotavirus non-structural proteins:structure and function[J].Current Opinion Virology,2012,2(4):380-388.
[20]LIBRI V,MIESEN P,VAN RIJ RP,et al.Regulation of microRNA biogenesis and turnover by animals and their viruses[J].Cellular and Molecular Life Sciences,2013,70(19):3525-3544.
[21]KANELLOPOULOU C,MULJO SA,KUNG AL,et al.Dicer-deficient mouse embryonic stem cells are defective in differentiation and centromeric silencing[J].Genes&Development,2005,19(4):489-501.
[22]DONG Y,ZENG CQ,BALL JM,et al.The rotavirus enterotoxin NSP4 mobilizes intracellular calcium in human intestinal cells by stimulating phospholipase C-mediated inositol1,4,5-trisphosphate production[J].Proceedings of the National Academy of Sciences of the United States of America,1997,94(8):3960-3965.
[23]SAAD N,DELATTRE C,URDACI M,et al.An overview of the lastadvances in probiotic and prebiotic field[J].LWT-Food Science and Technology,2013,50:1-16.
[24]MARTENS E C,CHIANG H C,GORDON J I.Mucosal glycan foragingenhances fitness and transmission of a saccharolytic human gut bacterial symbiont[J].Cell Host&Microbe,2008,4(5):447-457.
[25]FREITAS M,AXELSSON L G,CAYUELA C,et al.Indigenous micr-obes and their soluble factors differentially modulate intestinal glycosylation steps in vivo.Use of a“lectinassay”to survey in vivo glycosylation changes[J].Histochemistry and Cell Biology,2005,124(5):423-433.
[26]CHEN M F,WENG K F,HUANG S Y,et al.Pretreatment with a heat-killed probiotic modulates monocyte chemoat-tractant protein-1 and reduces the pathogenicity of influenza and enterovirus 71 infections[J].Mucosal Immunology,2017,10(1):215-227.
[27]贾慧琼,安敏,徐熙,等.植物乳杆菌STⅢ对大鼠小肠上皮细胞钙离子吸收的影响[J].中国微生态学杂志,2016(01):6-9.JIA Hui-qiong,AN Min,XU Xi,et al.Experimental study on the effect of Lactobocillus plantarum STⅢon calcium absorption in rat intestinal epithelial cells[J].Chinese Journal of Microecology,2016(01):6-9.(in Chinese)