The Study Of Influences Of Epidermal Growth Factor On Intestinal Barrier Function And Phosphorus Absorption Of Weaned Piglets

In addition to serving as a major organ for nutrient digestion and absorption,the single layer of intestinal epithelium lining the gut act as a selective barrier to prevent the passing of toxins,allergens,and pathogens from the luminal content into the circulation system and other tissues.Integrity of the intestinal barrier is important for the pig husbandry.In vivo and in vitro researches have demonstrated that EGF had a role in repairing intestinal barrier function.However,the mechanism of EGF on intestinal barrier regulation under stress condition is rarely reported.Meanwhile,it seemed that EGF inhibited the active absorption of phosphorus.But,in theory,during the process of the intestinal barrier function repairing,it must be accompanied by a large number of DNA,RNA and protein syntheses,which the premise is more phosphorus absorbed by intestine.However,how EGF regulating the absorption of phosphorus to meet the needs of the body during the process of intestinal barrier function repaired,has not reported yet.Therefore,this research used weaned piglets as experimental model,combine in vitro cell culture method with in vivo animal experiment to study the effect of EGF on intestinal barrier function and phosphorus absorption response to LPS stimulation,to illuminate the mechanism of EGF on piglets intestinal damage repair and the relationship with phosphorus absorption,as to provide a theoretical basis for the application of EGF in the piglet feed.The present study consists of two parts: animal experiment and cell experiment.Part 1: the 21 days old weaned piglets were used as the animal model to study the effects of EGF on growth performance,intestional barrier function,and phorsphorus absorption.Twenty-four 21 days old piglets with an average weight of 5.76±0.38 kg were randly divided into four treatments,1)Control group(basal diets),2)EGF group(basal diets + 2.0 mg/Kg EGF),3)LPS group(intraperitoneal injection of 100 ?g/Kg LPS),4)EGF+LPS group(basal diets + 2.0 mg/Kg EGF+ intraperitoneal injection of 100 ?g/Kg LPS).Results showed that:(1)compared with LPS group,the ADG of EGF+LPS group decreased significantly(P < 0.05);EGF can improve the expression of tight junction genes,ZO-1,Claudin-1 and Occludin in jejunum and ileum of piglets stimulated by LPS(P < 0.05);EGF can increase proinflammatory factor,IL-1?,IL-6,TNF-? mRNA expression(P < 0.05)in jejunum and ileum of piglets stimulated by LPS;EGF can promote the MUC2 mRNA expression(P < 0.05)in jejunum and ileum of piglets stimulated by LPS;EGF can inhibit the colonization of harmful bacteria(salmonella)in jejunum of piglets stimulated by LPS(P < 0.05);EGF through activating EGFR to inhibit TLR4/NF-?B pathway,leading to reduce TNF-? release,thus inhibiting the activation of the miRNA-122 a,promoting the expression of Occludin,to protect the intestinal barrier function.(2)EGF can inhibit the expression of NaPi-?b in jejunum and ileum of piglets,while,EGF promoted the NaPi-?b expression stimulated by LPS,which indicated that EGF can promote the active absorption of phosphorus under stress condition.Part 2: the porcine intestinal epithelial cells(IPEC-J2)were used as the cell models to study the effects of EGF on apoptosis,oxidative injury,tight junction as well as immune function.Firstly,different concentration of EGF(0,50,100,150,200,250 ng/mL)and LPS(0,0.1,1.0,10.0 mu g/mL),and different incubation time(6,12,24,36,48 h)were texted to chose the optimum doses of EGF,LPS and the suitable cultivation time.Accordingly,100 ng/mL EGF,1.0 ?g/mL LPS,and cell cultured for 24 h were chosen for subsequent experiments.Based on above,IPEC-J2 cells were divided into four grous,1)Control group(0 ng / mL EGF,0 ?g/mL LPS),2)EGF group(100 ng / mL EGF,0 ?g/mL LPS),3)LPS group(0 ng / mL EGF,1.0 ?g/mL LPS),4)EGF+LPS group(100 ng / mL EGF,1.0 ?g/mL LPS).Results showed that:(1)compared with LPS treatment,EGF+LPS treatment significantly increase(P < 0.05)cell growth and Bcl-2 expression,significantly reduce(P < 0.05)LDH activity,apoptosis rate,Fas,Bax,Cascase-3,Cascase-8,Cascase-9 gene expression,and P53,Fas,Bax,Caspase3 protein expression.These results suggested that EGF through inhibiting proapoptotic genes expression,and promoting antiapoptotic genes expression to against LPS-induced cell damage.(2)Compared with LPS treatment,EGF+LPS treatment significantly reduce(P < 0.05)MDA levels,significantly increase(P < 0.05)T-AOC,CAT,GSH-Px and SOD levels,significantly increase(P < 0.05)CAT,GSH-Px,SOD,Nrf2,HO-1,NQO1 mRNA expression,significantly up-regulated(P < 0.05)the protein expression of Nrf2,HO-1,NQO1.Collectively,the present data indicated that EGF would enhance Nrf2-mediated expressions of proteins,such as HO-1 and NQO1,and stimulate secretion of antioxidative enzymes,such as SOD,CAT and GSH-Px to reduce LPS induced oxidative injure in IPEC-J2 cells.(3)Compared with LPS treatment,EGF+LPS treatment significantly increase(P < 0.05)tight junction(ZO-1,Claudin-1,Occludin)mRNA and protein expression,significantly increase(P < 0.05)EGFR,MAPK,mTOR,PI3 K,p70S6K mRNA expression,significantly increase(P < 0.05)EGFR,MAPK,mTOR,PI3 K and p70S6 K protein expression,significantly increase(P < 0.05)the phosphorylation levels of MAPK,mTOR,PI3 K and p70S6 K protein.The present data indicated that EGF has a protective effect on tight junction induced by LPS,and EGF may be through the EGFR/MAPK and PI3K/mTOR signaling pathways regulating tight junction function of IPEC-J2 cells.(4)Compared with LPS treatment,the expression of iNOS and COX2,the production of NO,PGE2,the secretion of proinflammatory cytokines(IL-1?,IL-6,TNF-?)in EGF+LPS treatment significantly decreased(P < 0.05);the immunoglobulin(IgA,IgG,IgM)level in EGF+LPS treatment significantly increased(P < 0.05);the expression of CD14,TLR4,MYD88,NF-?B mRNA,the expression of TLR4,NF-?B protein and the phosphorylation levels of NF-?B protein in EGF+LPS treatment significantly decreased(P < 0.05);the expression of I?B? mRNA and protein in EGF+LPS treatment significantly increased(P < 0.05),and the phosphorylation levels of I?B? protein in EGF+LPS treatment significantly decreased(P < 0.05),these data indicated that EGF probably by inhibiting TLR4 / NF-?B signaling pathways,thus reducing the iNOS,COX-2,NO,PGE2,IL-1?,IL-6,TNF-? production to alleviate the immune stress induced by LPS.(5)EGF can inhibit the expression of NaPi-?b in IPEC-J2 cells,while,on the contrary,EGF can promote the NaPi-?b expression induced by LPS,which indicated that EGF can promote the active absorption of phosphorus under stress condition.In summary,according to the results of cell and animal experiments,EGF through reducing apoptosis,improving cell redox status,maintaining tight junction structure,alleviating immunological stress,promoting mucin secretion,reducing the colonization of harmful bacteria to protect the intestional barrier function when animals or calls under stress state.Also EGF can promote the NaPi-?b expression under stress condition,accelerating intestinal repair process.