解淀粉芽孢杆菌SC06与肠毒性大肠杆菌K88对IPEC-1细胞转运载体、紧密连接、凋亡和免疫相关基因表达的影响
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摘要
本试验以IPEC-1细胞为材料,探究解淀粉芽孢杆菌SC06(以下简称SC06)与肠毒性大肠杆菌K88(以下简称K88)对肠上皮屏障功能相关基因表达的影响。将IPEC-1细胞分为4个组并作不同处理:CK组为空白对照,不作处理; SC06组和SC06+K88组用含有108CFU/mL SC06的DM EM/F12培养基先预处理6 h,之后K88组和SC06+K88组加入含有108CFU/mL K88的DM EM/F12培养基处理3 h;乳酸脱氢酶(LDH)活性测定另设以含1%Trinton X-100的DM EM/F12培养基处理的Trinton组为阳性对照。各组细胞均培养9 h。结果表明:1)与CK组相比,K88和SC06单独处理均显著降低了葡萄糖转运载体2 (GLUT2)和小肽转运载体1(PepT1)基因的相对表达量(P<0.05),SC06单独处理显著增加了丙氨酸/丝氨酸/半胱氨酸/苏氨酸转运载体2(ASCT2)和兴奋性氨基酸转运载体1(EAAC1)基因的相对表达量(P<0.05);与K88单独处理相比,SC06预处理显著抑制了K88诱导的LDH活性和EAAC1基因相对表达量的增加(P<0.05)。2)与CK组相比,K88单独处理显著上调了闭锁小带蛋白-1(ZO-1)、闭合蛋白(occludin)基因的表达(P <0.05),显著下调了密封蛋白(claudin)-3、claudin-4和黏蛋白-1(MUC1)基因的表达(P <0.05); SC06单独处理显著上调了ZO-1、claudin-4基因的表达(P <0.05); K88和SC06单独处理均显著促进了天冬氨酸蛋白水解酶-3(caspase-3)、凋亡相关因子(Fas)及B淋巴细胞瘤-2(Bcl-2)基因的表达(P<0.05),显著抑制了天冬氨酸蛋白水解酶-9(caspase-9)与Bcl-2相关X蛋白(Bax)基因的表达(P<0.05),且K88单独处理还显著降低了天冬氨酸蛋白水解酶-8(caspase-8)基因的表达(P<0.05)。与K88单独处理相比,SC06预处理显著阻止了由K88诱导的ZO-1、caspase-3和Bcl-2基因相对表达量的增加(P<0.05)及claudin-3、claudin-4、MUC1、caspase-9和Bax基因相对表达量的降低(P <0.05)。3)与CK组相比,K88单独处理显著上调了肿瘤坏死因子α(TNF-α)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)及转化生长因子β(TGF-β)基因的表达(P<0.05),并显著上调了核转录因子-κB-p50(NF-κB-p50)、肿瘤坏死因子受体相关因子-6(TRAF-6)、髓样分化因子88(MyD88)、核苷酸结合寡聚域1(NOD-1)及Toll样受体-4(TLR4)基因的表达(P <0. 05); SC06单独处理显著降低了IL-6、NOD1与Toll样受体-6(TLR6)基因的表达(P<0.05),显著上调了TG F-β和MyD88基因的表达(P<0.05)。与K88单独处理相比,SC06预处理显著抑制了由K88导致的MyD88、NOD-1及TLR4基因相对表达量的增加(P<0.05)。综上所述,K88诱导IPEC-1细胞发生炎症反应,破坏肠上皮细胞的完整性,SC06在一定程度上有缓解作用。
This experiment was undertaken to evaluate the effects of Bacillus amyloliquefaciens SC06( herein-after referred to as SC06) and Escherichia coli K88( hereinafter referred to as K88) on the expression of genes related to intestinal epithelial barrier function by material of IPEC-1 cells. The IPEC-1 cells were divided into four groups with different treatments: cells in group CK were not treated and used as blank control,while cells in group SC06 and group SC06+K88 were pretreated with DMEM/F12 medium containing 108 CFU/mL SC06 for 6 h,and then cells in group K88 and group SC06+K88 were treated with DMEM/F12 medium containing108 CFU/mL K88 for 3 h. For the determination of dehydrogenase( LDH) activity,cells in group Trinton were treated with DMEM/F12 medium containing 1% Trinton X-100 and used as positive control.Cells in each group were cultured for 9 h. The results showed as follows: 1) compared with group CK,the relative expression levels of glucose transporter 2( GLUT2) and small peptide transporter 1( PepT1) genes were significantly decreased by K88 and SC06 alone treatment( P < 0.05). SC06 alone treatment significantly increased the relative expression levels of alanine/serine/cysteine/threonine transporter 2( ASCT2) and excitatory amino acid transporter 1( EAAC1) genes( P<0.05). Compared with group,SC06 pretreatment significantly suppressed the higher LDH activity as well as the relative expression level of EAAC1 gene induced by K88( P< 0. 05). 2) Compared with group CK,K88 alone treatment significantly upregulated the expression of zonula occludens protein-1( ZO-1) and occludin genes( P<0.05),and significantly downregulated the expression of claudin-3,claudin-4 and mucin 1( MUC1) genes( P<0.05). SC06 alone treatment significantly upregulated the expression of ZO-1 and claudin-4 genes( P<0.05). Both K88 and SC06 alone treatment significantly increased the expression of caspase-3,factor associated suicide( Fas) and B-cell lymphoma-2( Bcl-2)genes( P<0.05),and significantly inhibited the expression of caspase-9 and Bcl-2 associated X protein( Bax)genes( P<0.05). Moreover,K88 alone treatment also significantly decreased the expression of caspase-8 gene( P<0.05). Compared with group K88,the increase expression of ZO-1,caspase-3 and Bcl-2 genes and the decrease expression of claudin-3,claudin-4,MUC1,caspase-9 and Bax genes induced by ETEC K88 were significantly prevented by SC06 pretreatment( P<0.05). 3) Compared with group CK,K88 alone treatment significantly upregulated the expression of tumor necrosis factor α( TNF-α), interleukin-6( IL-6),interleukin-8( IL-8) and transforming growth factor β( TGF-β) genes,and significantly upregulated the expression of nuclear factor κB-p50( NF-κB-p50), tumor necrosis factor receptor-associated factor-6( TRAF-6),myeloid differentiation factor 88( MyD88),nucleotide binding oligomerization domain containing protein-1( NOD-1) and Toll like receptor 4( TLR4) genes( P<0.05). SC06 alone treatment significantly reduced the expression of IL-6,NOD-1 and Toll like receptor 6( TLR6) genes( P<0.05),and significantly upregulated the expression of TGF-β and MyD88( P<0.05). Compared with group K88,the increase expression of MyD88,NOD-1 and TLR4 induced by K88 were significantly prevented by SC06 pretreatment( P<0.05).In conclusion,K88 induces IPEC-1 cells inflammatory response and impairs integrity of epithelia,which is alleviated by SC06 to some extent.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2267-2277]
This experiment was undertaken to evaluate the effects of Bacillus amyloliquefaciens SC06( herein-after referred to as SC06) and Escherichia coli K88( hereinafter referred to as K88) on the expression of genes related to intestinal epithelial barrier function by material of IPEC-1 cells. The IPEC-1 cells were divided into four groups with different treatments: cells in group CK were not treated and used as blank control,while cells in group SC06 and group SC06+K88 were pretreated with DMEM/F12 medium containing 108 CFU/mL SC06 for 6 h,and then cells in group K88 and group SC06+K88 were treated with DMEM/F12 medium containing108 CFU/mL K88 for 3 h. For the determination of dehydrogenase( LDH) activity,cells in group Trinton were treated with DMEM/F12 medium containing 1% Trinton X-100 and used as positive control.Cells in each group were cultured for 9 h. The results showed as follows: 1) compared with group CK,the relative expression levels of glucose transporter 2( GLUT2) and small peptide transporter 1( PepT1) genes were significantly decreased by K88 and SC06 alone treatment( P < 0.05). SC06 alone treatment significantly increased the relative expression levels of alanine/serine/cysteine/threonine transporter 2( ASCT2) and excitatory amino acid transporter 1( EAAC1) genes( P<0.05). Compared with group,SC06 pretreatment significantly suppressed the higher LDH activity as well as the relative expression level of EAAC1 gene induced by K88( P< 0. 05). 2) Compared with group CK,K88 alone treatment significantly upregulated the expression of zonula occludens protein-1( ZO-1) and occludin genes( P<0.05),and significantly downregulated the expression of claudin-3,claudin-4 and mucin 1( MUC1) genes( P<0.05). SC06 alone treatment significantly upregulated the expression of ZO-1 and claudin-4 genes( P<0.05). Both K88 and SC06 alone treatment significantly increased the expression of caspase-3,factor associated suicide( Fas) and B-cell lymphoma-2( Bcl-2)genes( P<0.05),and significantly inhibited the expression of caspase-9 and Bcl-2 associated X protein( Bax)genes( P<0.05). Moreover,K88 alone treatment also significantly decreased the expression of caspase-8 gene( P<0.05). Compared with group K88,the increase expression of ZO-1,caspase-3 and Bcl-2 genes and the decrease expression of claudin-3,claudin-4,MUC1,caspase-9 and Bax genes induced by ETEC K88 were significantly prevented by SC06 pretreatment( P<0.05). 3) Compared with group CK,K88 alone treatment significantly upregulated the expression of tumor necrosis factor α( TNF-α), interleukin-6( IL-6),interleukin-8( IL-8) and transforming growth factor β( TGF-β) genes,and significantly upregulated the expression of nuclear factor κB-p50( NF-κB-p50), tumor necrosis factor receptor-associated factor-6( TRAF-6),myeloid differentiation factor 88( MyD88),nucleotide binding oligomerization domain containing protein-1( NOD-1) and Toll like receptor 4( TLR4) genes( P<0.05). SC06 alone treatment significantly reduced the expression of IL-6,NOD-1 and Toll like receptor 6( TLR6) genes( P<0.05),and significantly upregulated the expression of TGF-β and MyD88( P<0.05). Compared with group K88,the increase expression of MyD88,NOD-1 and TLR4 induced by K88 were significantly prevented by SC06 pretreatment( P<0.05).In conclusion,K88 induces IPEC-1 cells inflammatory response and impairs integrity of epithelia,which is alleviated by SC06 to some extent.[Chinese Journal of Animal Nutrition,2019,31( 5) : 2267-2277]
引文
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[4]朱红梅.产肠毒素性大肠杆菌致病特性及益生菌的筛选与疾病控制研究[D].硕士学位论文.厦门:厦门大学,2006.
[5]淳泽,何明清.芽孢杆菌对肠道致病菌体外生物拮抗作用的研究[J].四川农业大学学报,1994,12(增刊):627-634.
[6]周玲,王晓清,刘臻,等.营养素转运载体的研究进展[J].饲料研究,2013(4):18-23.
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[8] DONG X L,ZHANG N F,ZHOU M,et al.Effects of dietary probiotics on grow th performance,faecal microbiota and serum profiles in w eaned piglets[J].Animal Production Science,2014,54(5):616-621.
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[10] CUI Y J,LIU L,DOU X X,et al.Lactobacillus reuteri ZJ617 maintains intestinal integrity via regulating tight junction,autophagy and apoptosis in mice challenged w ith lipopolysaccharide[J].Oncotarget,2017,8(44):77489-77499.
[11] WANG Y,WU Y P,WANG Y B,et al. Bacillus amyloliquefaciens SC06 alleviates the oxidative stress of IPEC-1 via modulating Nrf2/Keap1 signaling pathway and decreasing ROS production[J].Applied Microbiology and Biotechnology,2017,101(7):3015-3026.
[12]杜威,黄琴,付爱坤,等.解淀粉芽孢杆菌SC06对环磷酰胺诱导的免疫抑制小鼠免疫功能的影响[J].中国畜牧杂志,2015,51(1):60-64.
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[16]计坚.解淀粉芽孢杆菌SC06介导猪肠道免疫机理的研究[D].博士学位论文.杭州:浙江大学,2013.
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[18] JAHROMI M F,ALTAHER Y W,SHOKRYAZDAN P,et al. Dietary supplementation of a mixture of Lactobacillus strains enhances performance of broiler chickens raised under heat stress conditions[J].International Journal of Biometeorology,2016,60(7):1099-1110.
[19] PAVLOVA I,MILANOVA A,DANOVA S,et al.Enrofloxacin and probiotic lactobacilli influence PepT1and LEAP-2 mRNA expression in poultry[J].Probiotics and Antimicrobial Proteins,2016,8(4):215-220.
[20]吴云鹏.植物乳杆菌调节猪肠上皮细胞屏障功能和转运载体的研究[D].博士学位论文.广州:华南农业大学,2016.
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