| Enterococcus faecium is a probiotic commonly used in piglet production,but its probiotic mechanism is unclear.In this study,we investigated the effects of Enterococcus faecium alive or heated on intestinal mucosal immunity and barrier function in piglets by in vivo piglet feeding trial and in vitro intestinal epithelial cell culture test.A total of 9 litters of piglets,with similar weight,parity and birth,were selected and randomly divided into 3 groups with 3 replicates(10-11 heads/replicate).The feeding trial was divided into three groups:Control group(10%sterilized skim milk solution),Enterococcus faecium group(alive,1×10~8 cfu/mL)and Enterococcus faecium group(heated,1×10~8 cfu/mL),the bacateria was feeding for six days.The trail period is 21 days,diarrhea and weight gain rate of continuous recording.In vitro intestinal epithelial cell culture test,IPEC-J2 cell line was treated by PBS,Enterococcus faecium(alive,1×10~8 cfu/mL),Enterococcus faecium(heated,1×10~8 cfu/mL),cell wall of Enterococcus faecium(100 ug/mL)and LPS(1ng/mL).The cell culture supernatant and cells were collected at 1,3,6,12 h,and the related indexes were measured.l.The results showed that the Enterococcus faecium alive or heated did not affect the body weight gain of suckling piglets,but they could reduce the diarrhea rate of piglets,and the spleen index of Enterococcus faecium alive group decreased significantly(p<0.05).The live bacteria improved the gene expression of ZO-1 of jejunum and ileum(p<0.05),had no effect on occludin gene expression;while the heated bacteria reduced the gene expression of ZO-1 and occludin of ileum(p<0.05)but these two proteins have no effect on the jejunum.Western blot showed that the expression of occludin and claudin protein in jejunum and ileum were increased by Enterococcus faecium alive and heated.The genes expression of ZO-1 and occludin of IPEC-J2 were up-regulated at 3h after treated by live Enterococcus faecium,heated Enterococcus faccium and cell wall of Enterococcus faecium(p<0.05).Enterococcus faecium(alive and heated)improved gene expression of claudin(p<0.05)at 1h.When the co-culture time was more than 6 hours,the expression of tight junction protein was down-regulated(p<0.05),The expression of these three tight junction proteins was significantly down-regulated in the LPS group(p<0.05),even during co-culture for 3 hours.Western blot results showed that,Within 3h,Enterococcus faecium can up-regulate the protein expression levels of occludin and claudin,The expression of claudin protein was up-regulated in the heat-activated bacteria and cell wall group at 1 h,and the difference was not significant at 3 h compared with the control group,at 6h,the expression of tight junction protein was decreased,however,the LPS group down-regulated the expression of occludin and claudin protein from 1 h.Protein expression was consistent with gene expression.2.Enterococcus faecium significantly up-regulated the mRNA expression of IL-1,IL-6,IL-8,TNF-? and TGF-?(p<0.05)in jejunum,while down-regulated the expression of IL-1,IL-6,IL-12 and TGF-? in ileal mucosa of piglets(p<0.05).In addition.,but it does not affect the expression of IL-12 in jejunum mucosa and IL-8,TNF-a in ileum mucosa,Enterococcus faecium heated group significantly down-regulated the expression of IL-1,IL-6,IL-8,IL-12?TNF-a in jejunum mucosa and IL-6,IL-8,IL-12 in ileum mucosa(p<0.05),the expression of TGF-? in jejunum mucosa and IL-1,TNF-?,TGF-? in ileum mucosa is unchanged.Effects of Enterococcus faecium alive and heated on the secretion of cytokines in small intestine of piglets were also different.Enterococcus faecium significantly promoted the secretion of TGF-? in the jejunum mucosa and ileum mucosa and IL-8 in the ileum mucosa(p<0.05),as well as,does not affect the concentrations of IL-8 in jejunum mucosa and IL-10,TNF-? in jejunum and ileum mucosa.But the levels of IL-10 and TGF-? in the jejunum mucosa of the heat-activated group were significantly increased(p<0.05),the production of IL-8,TNF-a and TGF-? in ileum mucosa also decreased significantly(p<0.05),the levels of IL-8?TNF-a in jejunum mucosa and IL-10 in ileum mucosa were not affected.And co-incubated with IPEC-J2 for 1 h,Enterococcus faeciu,m alive group significantly up-regulated the expression of IL-1,IL-8,TNF-a and TGF-?(p<0.05),but the expression levels of IL-6 and IL-12 were significantly inhibited(p<0.05);Enterococcus faecium heated group only significantly up-regulated the expression of IL-6,IL-8 and TGF-?(p<0.05),While IL-1,IL-12 and TNF-? are not affected;Cell wall group only significantly up-regulated the expression of IL-8 and TNF-a(p<0.05),IL-1,IL-6,IL-12 and TGF-? did not affect.When co-cultured for 3 h,Enterococcus faecium alive group the expression of IL-1,IL-6,IL-8 and TNF-a was significantly up-regulated(p<0.05),heated bacteria group significantly promoted the expression of IL-1,IL-6,IL-8 and IL-12(p<0.05),Cell wall group the expression of IL-6 and IL-12 was significantly increased(p<0.05),but significantly inhibited the expression of TNF-a(p<0.05).No significant incubation of 1h or 3h,Enterococcus faecium alive significantly stimulated IPEC-J2 secretion of TGF-?,but significantly inhibited the production of IL-8 and IL-10,did not affect the concentration of TNF-a.While the heated bacteria were co-incubated with IPEC-J2,only significantly increased 1h TGF-? secretion,but no effect on 3h,and significantly reduced IL-8 and IL-10 production for 1 h and 3 h co-incubation,and had no effect on TNF-a.3.The live bacteria improved the genes expression of TLR-2 ? TLR-4 of jejunum(p<0.05)and TLR-9 of ileum(p<0.05),however,TLR-9 and TLR-2 and TLR-4 genes in ileum mucosa were not affected;while the heated bacteria reduced the gene expression of TLR-4 of jejunum and TLR-9 of ileum(p<0.05),but did not affect the expression of TLR-2 in the jejunum and ileal mucosa and the TLR-9 gene in the jejunum mucosa..When co-cultured with IPEC-J2 for 3 h,The gene expression of TLR-2 in the Enterococcus faecium and cell wall group was significantly increased(p<0.05),but the expression of TLR-9 was not affected;However,both heat-activated gyroup and LPS group significantly reduced the gene expression of TLR-9(p<0.05),but did not affect the expression of TLR-2;TLR-4 gene was up-regulated of IP EC-J 2 at 3h in group cof Enterococcus faecium alive group of Enterococcus faecium heated and group LPS(p<0.05),while Enterococcus faecium cell wall reduced the TLR-4 gene expression(p<0.05).In conclusion:The live and heated bacteria of Enterococcus faecium have similar beneficial effects(reduce pig diarrhea),but they are not exactly the same on the regulation of small intestine mucosal immunity and barrier function in piglets:Enterococcus faecium enhance piglets intestinal mucosa(including pig Intestinal epithelial cells)tight junction proteins expression and activation of TLR-2,4,9,and induce tolerance-based immune response;while heated bacteria can only up-regulate the expression of tight junction proteins and intestine proteins and TLR-4 in porcine intestinal epithelial cells,but also can induce tolerance-based immune response of intestinal mucosa. |
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