Studies On The Effects Of Bacillus Amyloliquefaciens SC06 On The Alleviation Of Porcine Intestinal Epithelial Injury And Wnt/Notch/BMP Signaling Pathway

Intestinal health is crucial to piglets’growth and development.The intestinal damage caused by environmental stimulation,weaning stress,or infection in common,lead to deep diarrhea and vomiting,which could cause animal death in severe cases.Plenty of studies have shown that probiotics have good effects on the alleviating of intestinal damage,however,the underlying mechanisms need to be further studied.This study explored the correlation between the alleviation of intestinal injury caused by Bacillus amyloliquefaciens SC06 and the Wnt/Notch/BMP signaling pathway.In vivo,a total of 60 piglets for 40-day-old were selected and divided randomly into two groups,every group had three replicates,each replicated had 10 piglets,and were fed with a basal diet(CK group)or a basal diet supplemented with 10~8CFU/Kg Bacillus amyloliquefaciens SC06(Ba group)for 28 days.IPEC-J2 cells were used in the in vitro experiment and divided into four groups,the control(CK)group,the Diquat(DQ)group(treated with 950μM DQ for 6 h),the Ba SC06(Ba)group(treated with 108CFU/m L Ba SC06 for 6 h),and the B+D group(treated with 10~8CFU/m L Ba SC06 for6 h before the 6 h treatment of 950μM DQ).Here are the main results:1 Ba SC06 relieved the cell damage and apoptosis and decreased the expression of tight junctions caused by DQ in intestinal epithelial cells of pigsOur results in cell damages index measurement showed that compared with the CK group,DQ treatment significantly increased IPEC-J2 cell damages,LDH release,and apoptosis,and decreased tight junction expression.The Ba SC06 pretreatment alleviated cell damages and reversed the increasing of caspase-3 activity and the expression of cleaved caspase-3 and caspase-8,and the decreasing of the expression of Occludin,Claudin-1,and ZO-1,respectively(m RNA and protein).2 Ba SC06 affects the cell cycle and promotes the proliferation of intestinal epithelial cells of pigsThe result of cell cycle analysis shows that both DQ and Ba SC06 can promote cells to enter the S phase and promote the proliferation of epithelial cells.Compared with the CK group,the S phase of IPEC-J2 cells in the DQ and Ba groups increased from 15.8%to 24.6%and 23.9%,respectively.At the same time,the G0/G1 phase was also significantly reduced,but the above change was suppressed in the B+D group.The above results indicated that Ba SC06 can promote the proliferation of intestinal epithelial cells.3 Ba SC06 alleviated the oxidative stress damage by activating the Wnt signaling pathway in intestinal epithelial cells of pigsOur results in IPEC-J2 cells showed that compared with the CK group,the expression levels of p-GSK-3β,Lgr5+,β-catenin,and Bmi-1 proteins in the DQ and Ba groups were significantly increased,while those in the B+D group showed the opposite trend.At the same time,Ba SC06 treatment can increase the m RNA expression ofβ-catenin,Bmi-1,Lgr5+,Wnt3a,and FZD7 genes,which are related to the activation of the Wnt signaling pathway.After IPEC-J2 cells co-cultivation with Wnt signaling inhibitor ICG001,the activation of Wnt signaling pathway-related proteins and target genes caused by Ba SC06 was inhibited,while the protective effect of Ba SC06 on DQ-induced apoptosis and the tight junction damage also was inhibited in the B+D+ICG001group.Compared with the B+D group,cells in the B+D+ICG001 group which co-cultured with the inhibitor,the expression levels of tight junction proteins and genes such as Occludin and Claudin-1 were significantly reduced,and the m RNA expression levels of apoptosis-related genes(caspase-3 and caspase-8)increased markedly.All this evidence shows that Ba SC06 relies on Wnt signaling pathway to mediate DQ-induced intestinal injury repair.In vivo,we found that compared with the CK group,the Ba group shows higher Bmi-1,β-catenin protein expression,and higherβ-catenin,Bmi-1,Wnt3a,Olfm4,Cyclin D1,FZD7 genes m RNA expression level.Meanwhile,the expression of DKK1,the inhibitor of the Wnt signal pathway,was also found to have a significant decrease in the Ba group,and the expression of the GSK-3βgene was also decreased apparently,which further confirmed that Ba SC06 can activate the Wnt signaling pathway in the intestinal mucosa of piglets.4 Ba SC06 activated the Notch signal pathway but inhibited the BMP signal pathway in intestinal epithelial cells of pigsThe m RNA expression of Notch,Hes-1,Atoh1,and DLL-4 genes,which are related to the Notch signaling pathway,were increased after DQ treatment in IPEC-J2cells.In the Ba group,the expression of Notch,Hes-1,Atoh1,and DLL-4 genes still increased significantly,and the Notch signaling pathway was activated,but this increase appeared to be alleviated in the B+D group.In the Ba group,the m RNA expression of BMP2,BMP4,BMPR1A,BMPR1B,BMPR2,SMAD1,and SMAD4 genes in the cells decreased significantly.This result means the inhibition of the BMP signaling pathway.The same inhibition can also be seen in the DQ group.However,in the B+D group,the expression levels of the above genes rebounded and basically returned to the level of the control group.In summary,Ba SC06 inhibited the BMP signaling pathway and activated the Wnt and Notch signaling pathways,promoted the proliferation of intestinal epithelial cells,regulated the cell cycle,reduced cell apoptosis,enhanced intestinal tight junctions,and promoted DQ-induced intestinal epithelial cell damage repair.