Alleviating Effects And Mechanism Of Early Supplementation With Galacto-oligosaccharides On Intestinal Immune Stress In Suckling Piglets

In modern pig industry,the growth and survival rate of suckling piglets are the key indexes affecting pig production efficiency.However,the suckling piglets are vulnerable to the invasion of bacteria,viruses,and non-pathogens,which cause the immune stress in piglets because a piglet’s intestinal specific immune system is not fully developed.Immune stress can trigger intestinal oxidative stress of piglets.It has been proved that galacto-oligosaccharides（GOS）improve intestinal antioxidant function,improve intestinal integrity,and maintain intestinal immune homeostasis.However,whether GOS has protective effects on intestinal immune stress and oxidative stress of suckling piglets remains to be further studied.At present,lipopolysaccharides（LPS）treatment is a common strategy to construct an intestinal immune challenge model of piglets.Therefore,in vitro and in vivo experiments were used to elucidate the mechanism of GOS supplementation in alleviating LPS-induced inflammatory response,the oxidative stress and intestinal barrier damage in suckling piglets under immune chanllegen.The research is mainly divided into the following five parts:1.Effects of galacto-oligosaccharides on cytokines and antioxidant levels of IPEC-J2cells treated with lipopolysaccharidesIn this chapter,IPEC-J2 cell line was used to investigate the effects of GOS on LPS-induced change in cytokines,antioxidant levels and cell permeability.In experiment 1,IPEC-J2 cells were cultured with GOS at the concentration of 0,0.5,1,5,10 and 20 mg/m L for 24h.The optimal treatment doses of GOS were 0.5 and 1 mg/m L based on the cell survival rate.Meanwhile,IPEC-J2 cells were stimulated with LPS at the concentration of 0,0.5,1,5μg/m L,10 and 20μg/m L for 24 h,respectively.The optimal dose of LPS was 1μg/m L.Subsequently,IPEC-J2 cells were divided into 4 groups,namely the control group,LPS group,LPS+0.5mg/m L GOS group and LPS+1 mg/m L GOS group.The control group was cultured with DMEM/F12 medium,and the LPS group was cultured with DMEM/F12 medium containing1μg/m L LPS.The LPS+0.5 mg/m L GOS group was cultured with DMEM/F12 medium containing 1μg/m L LPS and 0.5 mg/m L GOS,and the LPS+1 mg/m L GOS group was cultured with DMEM/F12 medium containing 1μg/m L LPS and 1 mg/m L GOS.The results showed that LPS exposure increased cellular permeability as well as the production of pro-inflammatory cytokines and ROS（P<0.05）,and decreased the level of antioxidant enzymes（P<0.05）.In contrast,GOS prevented LPS-induced changes in IPEC-J2 cells（P<0.05）.We also found that GOS inhibited LPS-induced the increase of nuclear factor kappa-B（NF-κB）expression,which may be the main reason for the decrease of proinflammatory cytokines production.2.Effects of galacto-oligosaccharides on growth performance,intestinal morphology,digestion and absorption function of immune-challenged pigletsThe objective of this chapter was to investigate the effects of GOS on growth performance,plasma biochemical indexes,serum immunoglobulin and cortisol levels,intestinal morphology,disaccharidase and digestive enzyme activity,as well as m RNA expression of genes associated with digestion and absorption of immune-challenged piglets.In experiment 2,a total of 18 newborn piglets were selected from 2 litters（9 piglets per litter）.Each litter was divided into 3 groups,namely the control group（CON）,the LPS challenge group（LPS）and the GOS group challenge with LPS（LPS+GOS）,respectively.During the first 13 days after birth,piglets in LPS+GOS group were given 1 g/kg GOS solution every day,and piglets in CON and LPS groups were given the same volume of physiological saline every day.On the 14^th day of the experiment,piglets in LPS and LPS+GOS groups were intraperitoneally injected with 80μg/kg LPS solution,and piglets in the control group were intraperitoneally injected with the same volume of physiological saline.Piglets were slaughtered and sampled 2 h after LPS injection.The results showed that the early GOS supplementation increased average daily gain on day 7-14（P<0.05）,and increased small intestinal weight and small intestinal length（P<0.05）.Intraperitoneal injection of LPS significantly increased the levels of alanine aminotransferase,total bilirubin,triglyceride in plasma and cortisol in serum（P<0.05）,and decreased the levels of total cholesterol and high density lipoprotein cholesterol in plasma（P<0.05）.Moreover,intraperitoneal injection of LPS significantly decreased the activities of lactase and amylase in duodenum and jejunum,maltase and sucrase in ileum（P<0.05）,and significantly down-regulated the m RNA expression of genes associated with digestion and absorption in small intestine（P<0.05）.The early GOS supplementation could partly inhibit the changes of these indexes.3.Effects of galacto-oligosaccharides on intestinal antioxidant capacity and barrier function of immune-challenged pigletsThe objective of this chapter was to investigate the effects of early GOS supplementation on antioxidant level and barrier function in LPS-challenged piglets.The experiment design in this chapter is the same as experiment 2.The results showed that intraperitoneal injection of LPS also significantly increased the production of ROS and MDA in small intestine（P<0.05）,decreased the levels of antioxidant enzymes in jejunum and ileum（P<0.05）.In addition,LPS up-regulated the gene expression of pro-apoptotic factors in small intestine and the activity of diamine oxidase in plasma（P<0.05）,decreased the activity of diamine oxidase and the protein expresseions of tight junctions in small intestine（P<0.05）.The early GOS supplementation partly inhibited LPS-induced abnormal changes.Moreover,the early GOS supplementation inhibited the LPS induced decreased protein expression of Nrf2-related proteins（P<0.05）.4.Effects of galacto-oligosaccharides on intestinal microbial composition,microbial metabolites of immune-challenged pigletsThe objective of this chapter was to investigate the effects of early GOS supplementation on intestinal microbial composition and microbial metabolites of LPS-challenged piglets.The experiment design is the same as experiment 2.The analysis of microbial composition showed that LPS significantly down-regulated the relative abundance of Firmicutes and Lactobacillus in jejunal and ileal digesta of piglets（P<0.05）,and up-regulated the relative abundance of Actinobacteria in ileal digesta of piglets（P<0.05）.The early GOS supplementation significantly inhibited the LPS-induced increase in the relative abundance of Actinobacteria in ileal digesta（P<0.05）.In addition,the early GOS supplementation significantly increased the relative abundance of Streptococcus,Veillinella and unclassified Lactobacillales in ileal digesta compared with CON and LPS groups（P<0.05）.The analysis of short chain fatty acids showed that GOS supplementation significantly increased the concentrations of acetate,propionate and butyrate in jejunal and ileal digesta compared with the control group（P<0.05）.The results of bile acid composition showed that early GOS supplementation inhibited the LPS-induced decrease of chendoxychololic acid（CDCA）level.5.Effects of galacto-oligosaccharides on intestinal inflammatory response of immune-challenged pigletsThis chapter aims to investigate whether GOS alleviates the intestinal inflammation induced by LPS through the microbial-bile acids mechanism in piglets.The in vivo experiment design is the same as experiment 2.The in vitro experiment design is as followings:the intestinal lamina propria CD11b⁺cells of mice cultured in cell culture plate,after culturing for the night,the cells were divided into the following different groups:control group,30μM CDCA group,100 ng/m L LPS group,30μM CDCA+100 ng/m L LPS group,100μM SQ22536+30μM CDCA group and 100μM SQ22536+30μM CDCA+100 ng/m L LPS group.The result showed that the early GOS supplementation inhibited LPS-induced increase of the proinflammatory cytokines expression and the increased of NF-κB phosphorylation（P<0.05）.In addition,we found that the early GOS supplementation inhibited LPS-induced decreased m RNA expression of takeda G-protein receptor 5（TGR5）and the reduction of cyclic adenosine monophosphate（c AMP,TGR5 downstream factor）level（P<0.05）.We also found that differential bile acid（CDCA）could induce the m RNA expression of TGR5 and the production of c AMP in lamella propria derived CD11b⁺cells（P<0.05）.Adenylate cyclase（participating in the generation of c AMP）inhibitors eliminated the protective effect of CDCA on lamina propria devived CD11b⁺cells（P<0.05）.In summary,this study draws the following conclusions:（1）GOS alleviate LPS-induced intestinal epithelial inflammatory response and oxidative stress.（2）The early GOS supplementation promotes the growth of piglets and alleviates the digestive and absorption dysfunction caused by LPS stimulation.（3）The early GOS supplementation alleviates the intestinal oxidative stress response and intestinal barrier dysfunction induced by LPS stimulation.（4）The early GOS supplementation increases the abundance of short-chain fatty acid- producing bacteria and the concentration of short-chain fatty acid,and regulates the bile acids pool.（5）The early GOS supplementation contributes to the intestinal health of piglets by decreasing the production of pro-inflammatory cytokines and inhibiting the activation of NF-κB,which is through microbial-bile acids-dependent TGR5-c AMP signaling pathway.