Effects Of Iron Deficiency On Proliferation And Apoptosis In IPEC-J2 And Iron Supplementation On Intestinal Function In Suckling Pigs

Iron is an essential element for human and animals to maintain their function and physiological activities.Although iron is one of the most abundant trace elements on earth,iron deficiency（ID）and iron deficiency anemia（IDA）is the most common and frequent micronutrient deficiency worldwide.Iron supplementation is necessary to avoid iron deficiency anemia in early life such as infants and nursy pigs.Intestinal development and barrier function play an important role in body health.In the current study,in vivo and in vitro methods were used,iron deficiency was induced in IPEC-J2 and the effects of iron deficiency on intestinal development were studied.Different iron soureces were supplied for suckling piglets,the mechanism of iron on intestinal development and epithelial barrier,and the differences of orally FeSO₄ and FebisGly on intestinal physiology were studied.Experiment 1:Deferoxamine（DFO）treatment was used to induce iron deficiency in IPEC-J2 cells.The results showed no significant difference in cell viability treated with DFO at 50μM or 100μM for 6,12and 24 h（P>0.05）.DFO treatment at 50μM for 36 and 48 h significantly reduced the cell viability（P<0.05）.DFO treatment at 50μM for 24 and 48 h significantly reduced the Ferritin-H m RNA expression（P<0.05）.DFO treatment at 50μM for 36h decreased the cell proliferation rate.DFO treatment at 50μM for 48h significantly increased apoptosis,with significantly increased Cas-3,Cas 9 and Bax m RNA expression（P<0.05）.Significantly reduced permeability by DFO treatment at at 50μM and 100μM were observed afer 18,24 and 30 h（P<0.05）.According to the current study,increased iron deficiency negatively affected epithelial development by inhibiting cell proliferation,inducing cell apoptosis and increasing the permeability of epithelial cell layer.Experiment 2:eighteen one-day newborn piglets were divided two groups given intramuscular injection iron dextran at 1 m L for 100 mg Fe or equivalent saline.Pigs were fed by lactating sow and sampleing on day21.Resluts demonstrated no significant difference in growth performance between two groups（P>0.05）.Serum iron,hemoglobin,mean corpuscular volume（MCV）,mean hemoglobin content（MCH）and mean corpuscular hemoglobin concentration（MCHC）values were signifcanltly increased in iron supplementation group（P<0.05）.The expression of iron storage protein（Ferritin）in the duodenum and ileum were also significantly increased（P<0.05）.Iron supplementation significantly increased villous height in the duodenum and ileum（P<0.05）,and crypt depth in the duodenum（P<0.05）.Analysis of intestinal transcriptomic revealed that upregulated differential genes in iron supplementation group were associated with cell cycle and DNA replication pathways.Iron supplemetation significantly increased expression of p-AKT and m TOR proterin in the duodenum of suckling piglets（P<0.05）.Furthermore,iron supplementation significantly increased expressions of Claudin 1 and Occludin protein in the ileum,and goblet cells,Muc2 m RNA expression,IL-4 and IL-10 protetin expression in colon（P<0.05）.According to the current study,intramuscular iron supplementation increased intestinal iron level and promoted intestinal development through Akt/m TOR pathways.In addition,iron supplementation significantly increased tight junction,mucus production and anti-inflammatory cytokines expression for suckling piglets.Experiment 3:twenty-four neonatal pigs were were divided three groups and orally given two different iron sources FeSO₄ and FebisGly at25 mg/d Fe on day 2,7,12 and 17 after birth or physiological saline.Pigs were sampleing on day 21,and effects of FeSO₄ and FebisGly on iron utilization and intestinal barrier function of were compared.Results indicated that FeSO₄ and FebisGly significantly improved iron status and m RNA expression of genes related iron absorption（P<0.05）.Compared to FeSO₄,FebisGly significantly increased CHOL and HDL-C values and significantly reduced TG/HDL-C and TG/CHOL values（P<0.05）.No significantly differences in hepatic MDA、GSH and SOD among three groups（P>0.05）while FebisGly group show lower MDA and greater GSH in the duodenum（P<0.05）.Transcriptome analysis revealed that significantly different genes between FeSO₄ and FebisGly groups were enriched in Mineral absorption,p53 pathway,Oxidative Phosphorylation,Cell cycle,forkhead box O（FOXO）signaling pathway and Hippo signaling pathway in the duodenum of piglets.Comparred to Fe SO4,expressions of AMPK phosphorylation and FOXO3 protein were significantly increased in the FebisGly groups（P<0.05）.Comparred to CON and FebisGly groups,Fe SO4 group showed lowerα-diversity（OTU,Chao1,Simpson and Shannon index）（P<0.05）.Furthermore,greater Fusobacteria abundances were observed in FeSO₄ groups for Beta diversity（P<0.05）.FebisGly significantly increased Firmicutes,reduced Bacteroidetes and proteobacteria abundances in the colon（P<0.05）,then notably increased Lithocholic acid（LCA）,Ursodeoxycholic acid（UDCA）,Glycolithocholic acid（GLCA）and Glycoursodeoxycholic acid（GUDCA）in the bile acid pool of the colon（P<0.05）.RDA analysis indicated that Fusobacterium was mostly positively correlated with TG and TG/HDL-C in Fe SO4 group while Christensenellaceae＿R-7＿group,Ruminococcaceae＿UCG-002 and Ruminococcaceae＿UCG-005 were positively correlated with UDCA and GLCA while Terrisporobacter,Lachnoclostridium and Clostridium＿sensu＿stricto＿1 were positively correlated with HDCA.According to the current study,FeSO₄ and FebisGly significantly improved the iron status and intestinal iron absorption for suckling piglets.Compared to FeSO₄,FebisGly reduced MDA level,increased AMPK/FOXO3 expression and GSH level,modulated intestinal microbial composition and secondary bile acid production.In conclusion,iron deficiency inhibited intestinal epithelial development while iron injection improved intestinal tight junction,mucus production and immune defense.Oral FeSO₄ and FebisGly significantly improved iron level and intestinal development.Compared with FeSO₄,FebisGly promoted AMPK/FOXO3 pathway signal transduction and intestinal antioxidant capacity,modulated intestinal microbial composition and secondary bile acid production,which was more beneficial to improving intestinal health for suckling piglets.