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

Effects of Bacillus amyloliquefaciens SC06 and Escherichia coli K88 on Expression of Genes Related to Transporters,Tight Junctions,Apoptosis and Immunity in IPEC-1 Cells

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(hereinafter 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 10^8 CFU/mL SC06 for 6 h,and then cells in group K88 and group SC06+K88 were treated with DMEM/F12 medium containing 10^8 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 factorα(TNF-α),interleukin-6(IL-6),interleukin-8(IL-8)and transforming growth factorβ(TGF-β)genes,and significantly upregulated the expression of nuclear factorκB-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-βand 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.