| In recent years,chinese pig industries have achieved vigorous development,but due to the rapid development of intensive pig industry,various breeding problems have also occurred in the pig raising process,such as piglet with high diarrhea rate,high mortality rate and slow growth.Suckling period is a critical period for the growth and development of piglets.However,due to the weak disease resistance and the imperfect intestinal development of newborn piglets,piglets are easily attacked by pathogenic bacteria and toxic substances.It has been reported that early nutritional interventions could promote intestinal development,maturation of intestinal function and a perfect barrier defense system.A perfect barrier defense system in ture further improves the piglets’ capacity preventing foreign substances from entering the body,thereby promoting the development of the gastrointestinal tract and the normal operation of the body metabolism.Breast milk is the best source of nutrition for suckling piglets,which directly affects the intestinal health and body metabolism of suckling piglets.Lactoferrin is a non-heme iron-binding protein secreted by breast epithelial cells in breast milk.Therefore,this project utilized lactoferrin as an early intervention substance and explored the effects of early life lactoferrin on intestinal function and health,and further explored the mechanism of alterations of intestinal function affecting liver metabolism.1 Effect of early life lactoferrin intervention on growth performance,small intestinal development,digestive and absorptive functionin in suckling pigletsThis chapter aims to investigate the impact of early life lactoferrin(LF)intervention in growth performance and intestinal digestion and absorption function in suckling piglets.Sixty suckling piglets(1.51 ± 0.05 kg)obtained from six sows(10 piglets per litter)were assigned to a control(CON)group and an LF group in each litter,which were sow-fed.Piglets in the LF group were orally administered 8-12 mL of LF solution(0.5 g/kg body weight per day)for a week,and piglets in the CON group were orally administered the same dose of physiological saline.All piglets were weaned on day 21.Six piglets(n=6)from each group were euthanised on days 8 and 21.Samples of duodenum,jejunum and ileum tissues and mucosa were collected for further analysis.The early life LF intervention significantly increased the weaning weight of suckling piglets on day 21 and the average daily weight gain of suckling piglets from day 1 to day 21(P<0.05),and decreased the diarrhea incidence of suckling piglets from day 1 to day 7(P<0.05).In addition,early lactoferrin intervention increased the jejunal villi height and villi crypt ratio of suckling piglets on day 8(P<0.05),while significantly increasing the jejunal villi height of suckling piglets on day 21(P<0.05).Moreover,early lactoferrin intervention enhanced the activity of maltase and lactase in the jejunum and ileum of suckling piglets on day 8(P<0.05),while increasing the activity of jejunum maltase of suckling piglets on day 21(P<0.05).Compared with the control group,early lactoferrin intervention statistically upregulated the jejunal gene expression levels of APN,APA,GLUT,PEPT1 and ileal gene expression levels APA of suckling piglets on day 8(P<0.05),while upregulating the jejunal gene expression levels of APN,APA,GLUT2 and EAAC1 of suckling piglets on day 21(P<0.05).The above results indicated that early lactoferrin intervention improved the growth performance and decreased the diarrhea incidence of suckling piglets by enhancing the small intestinal digestive and absorptive function.2 Effect of early life lactoferrin intervention on small intestinal barrier function in suckling pigletsThis chapter aims to explore the effects of early lactoferrin intervention on the intestinal barrier function and microbial composition in suckling piglets.The design of the experiment is the same as that in chapter 4.Samples of urine,plasma,jejunal and ileal mucosa and j ejunal and ileal digesta were collected for further analysis.The results showed that early life lactoferrin intervention significantly reduced urinary lactulose-mannitol ratios in the suckling piglets on days 8 and 21,while increasing the activity of diamine oxidase in the jejunal mucosa and reducing the level of diamine oxidase in the plasma of suckling piglets on day 8(P<0.05).Additionally,early life lactoferrin intervention up-regulated the relative protein and gene expression of Occludin in the jejunum and ileum of suckling piglets on day 8(P<0.05),and up-regulated the relative protein and gene expression of Occludin in the jejunum of suckling piglets on day 21(P<0,05).Early life lactoferrin intervention significantly reduced the concentrations of IL-1β and TNF-α in the jejunal mucosa(P<0.05)and increased the concentration of IL-10 in the ileum on day 8(P<0.05).At 21 days of age,early life lactoferrin intervention increased the concentrations of IL-10 and sIgA in jejunal mucosa of suckling piglets(P<0.05)and decreased the concentration of TNF-α in jejunal and ileal mucosa(P<0.05).For microbial composition,at genus levels,early-life lactoferrin intervention increased the relative abundance and number of Lactobacillus(P<0.05)and decreased the relative abundance of genus Veillonella,Intestinibacter,Escherichia-Shigella and Howardella(P<0.05)in jejunal digesta on day 8,and reduced the relative abundance of Actinobacillus,Streptococcus and Terrisporobacter and the number of EscherichiaShigella in the ileal digesta on day 8(P<0.05).Furthermore,early life lactoferrin intervention significantly reduced the relative abundance of Actinobacillus and Veillonella in jejunal digesta on day 21(P<0.05).For the metabolic products of microbial,early life lactoferrin intervention increased the content of acetic acid in the ileal digesta on days 8 and 21(P<0.05).In summary,early-life lactoferrin intervention could prevent inflammation response and maintain the integrity of the intestinal barrier by reducing the intestinal concentration of proinflammatory cytokines(TNF-α),increasing intestinal concentrations of anti-inflammatory cytokines(IL-10)and slgA as well as increasing abundance of beneficial bacteria and decreasing abundance of pathogenic bacteria.3 Effects of early life lactoferrin intervention on iron homeostasis,antioxidant ability and cytokine levels in suckling pigletsThis chapter aims to explore the effects of early lactoferrin intervention on iron homestasis,antioxidant ability and cytokine levels in suckling piglets.The design of the experiment is the same as that in chapter 4.Samples of duodenum,liver,spleen,plasma and ileal digesta were collected for further analysis.The results showed that early-life lactoferrin intervention upregulated the levels of plasma hemoglobin,plasma iron and kidney iron in suckling piglets on day 8(P<0.05),and reduced the iron content in the terminal ileal digesta(P<0.05);meanwhile,early-life lactoferrin intervention increased hepatic iron in suckling piglets and reduced iron content in the terminal ileal digesta on day 21(P<0.05).In addition,early lactoferrin intervention significantly upregulated the relative expression levels of the genes of the duodenal cytochrome b(Dcytb)and lactoferrin receptor(LFR)in 8-day-old suckling piglets(P<0.05).In addition,early lactoferrin intervention increased the protein expression of divalent metal transporter 1(DMT 1)(P<0.05).At the same time,early lactoferrin intervention upregulated the protein expression of iron transporter(FPN 1)in the duodenal mucosa of suckling piglets on days 8 and 21(P<0.05).Furthermore,early lactoferrin intervention significantly increased GSH-Px activity in the plasma of suckling piglets on day 8 and increased T-AOC activity in the plasma of suckling piglets on day 21;early lactoferrin intervention enhanced SOD and GSH-Px activities in the duodenal mucosa of suckling piglets on day 21(P<0.05).In addition,early lactoferrin intervention increased the plasma levels of IL-10 and decreased the plasma and dundenal levels of IL-1β in the suckling pigelts on day 8(P<0.05).And,early lactoferrin intervention reduced the level of TNF-α in the plasma of suckling piglets on day 21(P<0.05).The above results indicated that early lactoferrin intervention improved iron levels in suckling piglets by regulating the iron transport-related protein expression and relative gene expression of the lactoferrin receptor(LFR)in the duodenum,as well as enhanced the antioxidant capacity and modulated the cytokine levels in the duodenum and plasma of suckling piglet.4 Effects of lactoferrin on lipopolysaccharide-induced inflammation,barrier function and oxidative stress in IPEC-J2 cellsThis chapter aims to explore the effects of lactoferrin on lipopolysaccharide-induced inflammation,barrier function and oxidative balance in porcine intestinal epithelial cells(IPEC-J2 cells).The IPEC-J2 cells were treated with or without LPS and LF for 24 h and analysed with various assays.The results indicated that the LPS treatment induced the secretion of pro-inflammatory cytokines[interleukin(IL)-1β,IL-8,and TNF-α](P<0.05),increased cell permeability,and downregulated the GSH-Px activity(P<0.05).The LF treatment decreased the secretion and gene expression of IL-1(3 and downregulated the phosphorylation levels of NF-κB,IκB and ERK1/2 in LPS-challenged cells(P<0.05).Moreover,the LF treatment decreased cell permeability,enhanced the expression of claudin1 protein,and inhibited expression of the myosin light chain kinase(MLCK)protein(P<0.05)in LPS-challenged cells.It also reduced the reactive oxygen species(ROS)and MDA production(P<0.05)as well as upregulated the GSH-Px activity and the expression of nuclear factor erythroid 2-related factor 2(Nrf2)protein(P<0.05).Taken together,these results suggest that LF alleviated the LPS-induced cellular inflammation through attenuation of nuclear factor kappa B(NF-κB)/mitogen-activated protein kinases(MAPK)pathway,maintaining cellular barrier integrity,and mitigating the oxidative stress.5 Effect of early life lactoferrin intervention on protein synthesis,energy metabolism and mitochondrial antioxidant activity in the liver in suckling pigletsThis chapter aims to explore the effects of early life lactoferrin intervention on the liver metabolism in the suckling piglets.Plasma,jejunum and liver samples were collected on day 8 and 21.The LF piglets had a decreased level of plasma urea nitrogen on day 8 and an increased level of plasma albumin on day 21.Pathway analysis of metabolomic profiles showed that LF treatment affected amino acid metabolism and energy metabolism in the liver of piglets.LF treatment enhanced phosphorylation levels of mTOR and p70S6K on day 8 as well as the phosphorylation level of p70S6K on day 21(P<0.05)in the liver of piglets.Moreover,LF treatment upregulated the expression of β oxidation related gene(carnitine palmitoyl transferase-1,CPT 1)(P<0.05)and affected tricarboxylic acid(TCA)cycle on day 21 in the liver.Furthermore,LF piglets had a lower level of malondialdehyde(MDA)and higher levels of glutathione(GSH)and glutathione peroxidase(GSH-Px)in the liver mitochondrial(P<0.05).Overall,the early life LF intervention affected liver metabolism of suckling piglets by inducing synthesize proteins,modulating fatty acid β oxidation and TCA cycle as well as enhancing antioxidative enzymes synthesis.Based on aforementioned results,it can be concuded that:(1)Early life lactoferrin intervention improved the jejunal morphological development,enhanced the intestinal digestion and absorption function,promoted the intestinal expression of tight junction protein,maintained the immune balance of the small intestinal mucosa,changed the composition of the small intestinal microbial community,increased the iron levels by regulating the duodenal expression of iron transport-related proteins,which thereby contributed to reducing diarrhea incidence and promoting the growth performance in suckling piglets.(2)Lactoferrin effectively alleviated the cellular inflammatory response and barrier damage in LPS-stimulated IPEC-J2 cells by downregulating the cellular phosphorylation levels of NF-κB,IκB and ERK proteins,reducing the secretion of pro-inflammatory factors(IL-1β),and enhancing the cellular barrier protein(claudin-1)expression.(3)Early life lactoferrin intervention regulated liver amino acid metabolism and activated liver mTOR signaling pathway to stimulate liver protein synthesis by enhancing intestinal digestion and absorption capacity of suckling piglets,and promoted energy metabolism by stimulating the liver fatty acid β oxidation and the TCA cycle in suckling piglets.In addition,early life lactoferrin intervention improved the antioxidant capacity of liver mitochondria and maintained the normal progress of liver metabolism. |
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