| Probiotics plays the multiple roles,which includes regulation of intestinal microenvironment;inhibition or kill of intestinal pathogens;improvement of intestinal micro-ecological balance;regulation of intestinal mucosal immunity and maintenance of intestinal barrier function.Reconstruction of intestinal flora may affects the neuro-endocrine-immune signaling pathway of the microbe-brain-gut axis.Vasoactive intestinal peptide(VIP)is an important neurotransmitter that mediates the brain-gut axis.And mast cells(MCs)are the key effector cells which were closely related to brain-gut axis.However,the exact mechanism of regulatory effect of VIP/MCs-mediated Lactobacillus casei ATCC 393 on intestinal barrier dysfunction remains unclear.This study was conducted to investigate the effect of Lactobacillus casei ATCC 393 on intestinal barrier dysfunction and its possible relationship with VIP and MCs.For this,we carried out the following experiments:establishment of an model of enterotoxin-producing Escherichia coli(ETEC K88)-induced intestinal barrier dysfunction models in vitro and in vivo for evaluation the role of Lactobacillus casei ATCC 393(L.casei ATCC 393)on repair of intestinal barrier injury by;L.casei ATCC 393/VIP/MCs co-culture cell model was established to evaluate the effect of L.casei ATCC 393 and VIP on the activation of Porcine Mucosal Mast Cells(PMMCs);Moreover,establishment of VIP receptor antagonists and MCs blocking-mediated C57BL/6 mouse models were established to investigate the role of VIP and MCs in regulatory effect of L.casei ATCC 393 on ETEC K88-induced intestinal barrier dysfunction.The obtained main results are as follows:1.Study on the effect of L.casei ATCC 393 on intestinal barrier dysfunction caused by ETEC K88a.L.casei ATCC 393 alleviated ETEC K88-induced intestinal barrier dysfunction in IPEC-J2 cells.Compared with control,ETEC K88 infection decreased the viability of IPEC-J2 cells and significantly destroyed its morphological structure.L.casei ATCC 393 pretreatment can effectively alleviate the toxic effect of ETEC K88 on IPEC-J2 cells;L.casei ATCC 393 pretreatment significantly inhibits the decrease of TEER value and increase of FITC-Dextran flux in IPEC-J2 cells challenged by ETEC K88 infection;L.casei ATCC 393 was able to competitively adhere to GFP-ETEC K88 on IPEC-J2 cells;L.casei ATCC 393 can effectively alleviate the decrease of Occludin and ZO-1 proteins and NOD1,NOD2,TLR2,TLR4,TLR5 and TLR9 mRNA expression level caused by ETEC K88 infection.b.L.casei ATCC 393 alleviated ETEC K88-induced intestinal barrier dysfunction in C57BL/6 mice.ETEC K88 infection leads to an increased level of inflammatory cytokines in serum;L.casei ATCC 393 can alleviate duodenal structure damage caused by ETEC K88 infection and inhibit the release of inflammatory cytokines in serum;L.casei ATCC 393 can regulate intestinal microecological disorders caused by ETEC K88 infection,maintain intestinal microecological balance,and inhibit decrease of ZO-1 and Occludin proteins and NOD1,NOD2,TLR2,TLR4,TLR5 and TLR9 mRNA expression level and increase in FITC-Dextran flux which caused by ETEC K88 infection.The above results demonstrate that L.casei ATCC 393 can effectively alleviate intestinal barrier dysfunction caused by ETEC K88 infection2.Study on the effect of MCs on the regulation of L.casei ATCC 393-mediated intestinal barrier dysfunction.a.L.casei ATCC 393 effectively alleviates the increase of intestinal permeability in IPEC-J2 cells by regulating the activation of PMMCs.L.casei ATCC 393 effectively inhibits the activation of PMMCs and release of increase of β-hex,histamine,tryptase,IL-6,IL-8,GM-CSF,IFN-γ,and TNF-α,meanwhile,alleviate the decrease of TEER value and increase of FITC-Dextran flux in IPEC-J2 cells;b.L.casei ATCC 393 can effectively alleviate intestinal barrier dysfunction in C57BL/6 mice by regulating mast cells activation.Compared with ETEC K88 infection group,Cromolyn Sodium pretreatment significantly attenuated the injury of duodenal and jejunal structure caused by ETEC K88 infection,increased inflammatory cytokines level in serum,significantly inhibited the increase of degranulated MCs in ileum and decrease in the protein expression level of Occludin,Claudin-1,TLR2 and TLR4.Moreover,it also maintained the microecological homeostasis of cecal in experimental mice.Compared with the MCs blocker treatment group,L.casei ATCC 393 has the similarly biological effect to Cromolyn Sodium that effectively alleviates intestinal barrier dysfunction caused by ETEC K88 infection in C57BL/6 mice.The above results demonstrate that L.casei ATCC 393,as potential MCs stabilizer,may attenuate intestinal barrier dysfunction caused by ETEC K88 infection through TLRs-mediated MCs signaling pathway3.Study on the effect of VIP-mediated MCs in protection of intestinal barrier dysfunction by L.casei ATCC 393.a.VIP can regulate the activation of MCs.Compared with ETEC K88 and LPS model groups,VIP pretreatment significantly inhibits activation of MCs and release of β-hex,histamine and tryptase induced by ETEC K88 and LPS;b.VIP and L.casei ATCC 393 can synergistically inhibit the decrease of TEER value and increase of FITC-Dextran flux induced by ETEC K88 infection in IPEC-J2 cells.c.VIP6-28 pretreatment inhibited the protective effect of L.casei ATCC 393 on intestinal barrier dysfunction caused by ETEC K88 infection in C57BL/6 mice.Moreover,with the blocking of VIP signal transduction,ETEC K88 infection caused a significant increase of inflammatory cytokines in serum,while it can be alleviated by administration with L.casei ATCC 393.And VIP can mediate the regulatory effect of L.casei ATCC 393 on intestinal microecology in mice;Compared with ETEC K88 infective group,L.casei ATCC 393 could not exert inhibitory effect on the increase of epithelial permeability caused by ETEC K88 infection in the presence of VIP6-28.The above results demonstrate that VIP may mediate the protective effect of L.casei ATCC 393 on intestinal barrier dysfunction via MCs. |
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