Effects Of AvBD2,Bacillus Subtilis And Lactoferrin On Intestinal Barrier Function Of Piglets And Its Mechanism

Research and development of safe,environmentally friendly and efficient new feed additives to replace the traditional feed antibiotics is one of the hot spots in animal nutrition research.In this paper,AvBD2,Bacillus subtilis and lactoferrin were used to study the intestinal epithelial permeability,tight junction integrity and expression of closely related genes in weaned piglets by in vitro cell experiments.The protective effect and mechanism of AvBD2,Bacillus subtilis and lactoferrin on the intestinal epithelial mechanical barrier of weaned piglets were revealed from the molecular level.AvBD2,Bacillus and lactoferrin results of IPEC-J2 appreciation rate and toxic e ffects of show that,AvBD2-A / B had a stronger effect on the LDH release rate of IPEC-J2,and the LDH release rate was the lowest at concentrations of 3.125 ?g / mL and 6.25 ? g / mL and the release rates were 4.76% and 4.22%.AvBD2-C did not significantly promote the LDH release rate of IPEC-J2 at a concentration of 6.25 ?g / mL,the LDH release rate was 13.61%,the LDH release rate was the lowest at a concentration of 6.25 ? g / mL.Bacillus subtilis and lactoferrin promoted the release of LDH in IPEC-J2,and the LDH release rate was the lowest at concentrations of 200?g / mL and 300?g / mL and the release rates were 9.35% and 12.23%.After 21 days of continuous incubation of IPEC-J2 cells in porcine jejunum epithelial cell line,the transmembrane resistance of epithelial cells reached about 3500 ? after 18 days and then stabilized,indicating that IPEC-J2 cells had differentiated.The permeability of the cells showed that the permeability of FD4 in the Transwell bottom medium was maintained at about 2.15% at 6 h,indicating that the permeability of epithelial cells was little.Under the stimulation of LPS,the penetration rate of FD4 reached 22%,pre-treated with AvBD2,Bacillus subtilis and lactoferrin,FD4 permeability was less than 8%.The results showed that AvBD2,Bacillus subtilis and lactoferrin could effectively reduce the permeability of LPS to epithelial cells.In the LPS stimulation,a short period of time a sharp decline in resistance,LPS group resistance value is reduced to 53%,then the resistance has increased,but the last resistance continues to decline.Pretreatment with AvBD2,Bacillus subtilis and lactoferrin in the experimental group,each time the resistance values are higher than the LPS group,Indicating that AvBD2,Bacillus subtilis and lactoferrin can alleviate the damage effect of LPS on IPEC-J2 barrier integrity.Cultured to IPEC-J2 for 21 days,it was clear from the observation of transmission electron microscopy that AvBD2,Bacillus subtilis and lactoferrin had no destructive effect on the tight junctions between cells.LPS can destroy the tightly linked structure of IPEC-J2.AvBD2,Bacillus subtilis and lactoferrin play a protective or mitigating role in the destruction of IPEC-J2 tight junction structure under LPS stimulation.RT-PCR analysis of tight junction protein gene expression levels showed that,AvBD2,Bacillus subtilis and lactoferrin showed a certain time effect on the expression of tight junction protein and mucin-related genes.AvBD2-A treatment for 12 h,the target gene ZO1 transcription level increased,the transcription level of target genes ZO2,C laudin1,MUC1,MUC4 and MUC20 was increased at 24 h after treatment with AvBD2-A,the transcription level of target genes ZO1,ZO2,MUC1,MUC4 and MUC20 was increased at 48 h after treatment with AvBD2-A,and the difference was significant.AvBD2-B treatment for 12 h,the target gene ZO1 transcription level increased,AvBD2-B treatment 24 h,the target gene Claudin1 transcription level increased;significant difference.After 24 h of AvBD2-C treatment,the transcription level of target gene MUC4 was increased;the difference was significant.Bacillus subtilis treatment for 12 h and 24 h,the target gene ZO1,ZO2,Claudin1,MUC1,MUC4 and MUC20 transcription level increased,Bacillus subtilis was treated for 24 h and 48 h,and the transcription level of the target gene Occludin was increased;the difference was significant.Lactoferrin treatment for 24 h,the target gene Claudin1,MUC4 and MUC20 transcription level increased;significant difference.In addition to the above,the target gene transcription level difference was not significant.The experimental results of MEK1 / 2,p38,PKC and PI3 K were detected by AvBD2,Bacillus subtilis and lactoferrin.MEK1 / 2 signal transduction pathway.After the addition of inhibitor U0126,the expression of all the target genes decreased,the difference was significant.Compared with the inhibitor group.After treatment with AvBD2-A,the expression levels of Occludin,MUC4 and MUC20 were significantly increased,and the differences were significant.After treatment with AvBD2-B,the expression of target gene ZO1,ZO2 and Claudin1 increased,and the expression of MUC20 decreased,and the difference was significant.After treatment with AvBD2-C,the expression levels of target genes ZO1,ZO2 and Occludin were increased and the difference was significant.The expression of ZO1,ZO2,Claudin1,Occludin,MUC1 and MUC4 were increased after Bacillus subtilis treatment,and the difference was significant.After treatment with lactoferrin,the expression levels of target genes MUC1,MUC4 and MUC20 were increased,and the difference was significant.P38 signal transduction pathway.The expression of all the target genes was decreased and the difference was significant.Compared with the inhibitor group.The expression levels of target genes such as Claudin1,Occludin and MUC1 were significantly increased after AvBD2-A treatment.The expression of ZO1,ZO2,Claudin1 and Occludin increased significantly after AvBD2-B treatment,and the difference was significant.The expression levels of RO2,Claudin1,Occludin,MUC1 and MUC4 were increased after treatment with AvBD2-C,and the expression of MUC20 was decreased and the difference was significant.The expression of ZO1,ZO2,C laudin1,Occludin,MUC1 and MUC20 were increased after Bacillus subtilis treatment,and the difference was significant.After treatment with lactoferrin,the expression level of target gene MUC20 was increased and the difference was significant.PKC signal transduction pathway.The expression of ZO1,ZO2,Claudin1,Occludin,MUC1 and MUC20 decreased with the addition of inhibitor Staurosporine,and the difference was significant.Compared with the inhibitor group.After treatment with AvBD2-A,the expression level of all the target genes was improved and the difference was significant.After expression of AvBD2-B,the expression levels of target genes ZO1,ZO2,Occludin,MUC1 and MUC4 were increased,and the expression of MUC20 was decreased,and the difference was significant.After expression of AvBD2-C,the expression levels of target genes ZO1,ZO2,Occludin,MUC1 and MUC4 were increased,and the expression of MUC20 was decreased and the difference was significant.After expression of Bacillus subtilis,the expression levels of target genes ZO1,ZO2,Occludin,MUC1 and MUC4 were increased and the expression of MUC20 was decreased and the difference was significant.After treatment with lactoferrin,the expression levels of Occludin,MUC1 and MUC4 were increased,and the expressio n of gene ZO2 decreased and the difference was significant.PI3 K signal transduction pathway.The expression of ZO1,ZO2,C laudin1,Occludin and MUC1 decreased,and the expression of MUC20 was increased and the difference was significant.Compared with the inhibitor group.After treatment with AvBD2-A,the expression levels of Occludin,MUC1,MUC4 and MUC20 were increased,and the difference was significant.The expression levels of target genes ZO1,ZO2,Claudin1,MUC1 and MUC4 were increased after treatment with AvBD2-B.The expression of MUC20 was decreased and the difference was significant.The expression of target genes ZO1,MUC1 and MUC4 increased after treatment with AvBD2-C,and the difference was significant.The expression of ZO1,ZO2,MUC1,MUC4 and MUC20 were increased and the difference was significant after treatment with Bacillus subtilis.After the treatment of lactoferrin,the expression levels of MUC1 and MUC4 were increased,and the expression of gene ZO1 and ZO2 decreased,and the difference was significant.In summary.AvBD2-A and Bacillus subtilis can increase the expression level of target gene through MEK1 / 2 and p38 signaling pathway,PKC signal pathway and PI3 K signal pathway.AvBD2-B can increase the expression of target gene by p38 signaling pathway.AvBD2-C can increase the expression level of target gene through MEK1 / 2 signal pathway and PI3 K signal pathway.Lactoferrin can increase the expression of target gene through MEK1 / 2 and p38 signaling pathway,protect intestinal barrier and maintain the stability of intestinal environment.