Effects Of Spermine Supplementation On Intestinal Epithelial Cell Restoration In Piglets

Spermine is a common functional substance in cells,which can improve the antioxidant status of animals and promote the body immunity.However,the effect of spermine on the restoration of intestinal epithelial injury in piglets is not clear.In this study,the effect of spermine on the restoration of intestinal epithelial cell injury was investigated in vitro and in vivo.Experiment 1:The effects of spermine supplementation on the restore of injured intestine in pigletsThe objective of this study was to investigate the effects of spermine administration on the restoration of intestinal epithelial cell of piglets and the m RNA levels of Ras-related C3botulinum toxin substrate 1（Rac1）/Phospho-lipase C-γ1（PLC-γ1）signaling pathway of the jejunum and ileum in piglets.Eighty piglets were weaned at 9-day of age.Before starting the experiment,piglets were fed with the milk-based diet for 2-day.Piglets were divided into spermine group which fed spermine in 0.4 mmol kg^-1 body weight and control group in7-hour,3-day,6-day and 9-day.The results showed that（1）Spermine reduced the inflammation gene expression of jejunum and ileum in piglets:spermine significantly reduced jejunal and ileal tumor necrosis factorα（TNF-α）,interleukin-1β（IL-1β）,interleukin-6（IL-6）,interleukin-8（IL-8）and interferonγ（IFN-γ）m RNA levels,significantly decreased ileal interleukin-2（IL-2）the m RNA levels,and significantly increased interleukin-10（IL-10）and transforming growth factorβ1（TGF-β1）m RNA levels in jejunum and ileum（P<0.05）.Extended spermine supplementation significantly decreased the m RNA levels of TNF-α,IFN-γand IL-8 in the jejunum（P<0.05）,significantly increased IL-10 and TGF-β1 m RNA levels in jejunum,significantly decreased the m RNA levels of TNF-α,IL-1,IL-2,IL-6 and IL-8 in ileum（P<0.05）.（2）spermine enhanced the antioxidant status of jejunum and ileum of piglets:spermine significantly increased the m RNA levels of copper/zinc superoxide dismutase（SOD1）,glutathione peroxidase 1（GPx1）,glutathione-S-transferase（GST）and nuclear factor erythroid 2-related factor 2（Nrf2）in jejunum and ileum（P<0.05）,and significantly increased the m RNA levels of catalase（CAT）and glutathione reductase（GR）in ileum（P<0.05）.Prolonged spermine treatment time significantly increased jejunal SOD1,CAT and Nrf2 m RNA levels,and ileal SOD1,GR,GPx1 and Nrf2 m RNA levels were also significantly increased（P<0.05）.（3）spermine enhanced jejunal and ileum barrier function in piglets:spermine significantly increased the m RNA levels of zonula occludens-1（ZO1）,zonula occludens-2（ZO2）,occludin,claudin-1,claudin-2,claudin-3,claudin-12,claudin-14,claudin-15,claudin-16 in jejunum and ileum（P<0.05）,significantly decreased the m RNA levels of myosin light-chain kinase（MLCK）in the jejunum（P<0.05）.Extended spermine treatment time significantly increased the m RNA level of ZO-1,claudin-3,occludin,claudin-2,claudin-1,claudin-16 and claudin-15 in the jejunum（P<0.05）,significantly increased ZO1,claudin-12,ZO2,occludin,claudin-15,claudin-1,claudin-2,claudin-3,claudin-14,claudin-16 gene expression in the ileum（P<0.05）and significantly decreased the MLCK gene expression in the jejunum（P<0.05）.（4）serum D-lactate content was significantly reduced by 26.02%after spermine treatment（P<0.05）.Serum D-lactate content was positively related to the m RNA levels of proinflammatory cytokines,m RNA levels of antioxidant enzymes in the jejunum and ileum.（5）spermine enhanced Rac1/PLC-γ1 signaling pathways related gene expression in jejunum and ileum:spermine treatment significantly improved the jejunum and ileum Rac1,PLC-γ1,ras homolog gene family member A（Rho A）,cell division control protein 42（CDC42）gene expression（P<0.05）,jejunum myosin regulatory light chain（MRLC）gene expression（P<0.05）,and significantly decreased the ileum rho-associated,coiled-coil-containing protein kinase 1（Rock1）gene expression（P<0.05）.Extended spermine treatment time significantly increased the m RNA levels of Rac1,PLC-γ1,Rho A,CDC42 and MRLC in jejunum（P<0.05）,significantly increased ileum PLC-γ1 and Rho A m RNA levels（P<0.05）and significantly decreased ileum Rock1 gene expression（P<0.05）.The results of the experiment showed that spermine could reduce intestinal inflammation of piglets,enhance intestinal antioxidant status,maintain the integrity of intestinal barrier and restore intestinal epithelial injury of piglets,which may be related to Rac1/PLC-γ1 signaling pathway.Experiment 2:The effects of spermine supplementation on the intestinal epithelial cell restoration in piglets.In this study,IPEC-J2 cells were used as experimental materials to investigate the effects of spermine on the restoration of intestinal epithelial cell damage by constructing cell scratch model and the TNF-αdamage model.It mainly includes the effects of spermine treatment on cell proliferation,the effects of spermine and Rac1/PLC-γ1 specific inhibitor treatment on cell migration and the expression of Rac1 and PLC-γ1 proteins,the effects of spermine and Rac1/PLC-γ1 specific inhibitors on the m RNA levels of Rac1/PLC-γ1 signaling pathway,the expression of Rac1 and PLC-γ1 proteins and and tight junction protein expression after TNF-αtreatment.Results showed that（1）spermine promoted cell proliferation:1μM-10μM and 100μM spermine treatment can significantly increase cell proliferation（P<0.05）,1000μM,1500μM and 2000μM spermine can reduce cell proliferation（P<0.05）.（2）spermine promoted cell migration:1μM spermine significantly increased cell migration area and migration distance（P<0.05）,4μM spermine and 8μM spermine significantly reduced cell migration area,and the migration area and migration distance gradually decreased as the concentration of spermine increased（P<0.05）.（3）NSC-23766 and U73122 inhibited cell migration:135μM NSC-23766 significantly reduced cell migration area and migration distance in 6 hours（P<0.05）.75μM,90μM,105μM,120μM NSC-23766 significantly reduced the migration area and migration distance in 12 hours,and 120μM has the best effect（P<0.05）.120μM NSC-23766 significantly reduced cell migration area and migration distance in 12 hours（P<0.05）.1μM U73122,2μM U73122,3μM U73122 significantly reduced the cell migration area and migration distance,and the cell migration area and migration distance decreased with the increase of U73122 concentration（P<0.05）.1μM U73122 and 3μM U73122 significantly reduced cell migration area and migration distance in12 hours（P<0.05）,and 3μM U73122 has the best effect.（4）Compared with the 1μM spermine group,120μM NSC-23766+1μM spermine and 3μM U73122+1μM spermine group significantly reduced cell migration area and migration distance（P<0.05）.（5）1μM spermine significantly increased the protein expression levels of Rac1 and PLC-γ1（P<0.05）.（6）120μM NSC-23766 significantly reduced the Rac1 protein expression level（P<0.05）,and3μM U73122 significantly decreased the Rac1 and PLC-γ1 protein expression levels（P<0.05）.（7）Compared with the 1μM spermine group,1μM spermine+120μM NSC-23766 and1μM spermine+3μM U73122 significantly reduced the protein expression levels of Rac1 and PLC-γ1（P<0.05）.（8）Compared with the control group,40ng/m L TNF-αsignificantly reduced Rac1,Rho A,CDC42 gene expression levels（P<0.05）.0.1μM spermine significantly increased the expression levels of Rac1,PLC-γ1,Rho A,CDC42 and Rock1 genes as well as GTP-rac1,occludin and claudin-1 proteins（P<0.05）.Compared with the 40ng/m L TNF-α,40ng/m L TNF-α+0.1μM spermine significantly increased the expression levels of Rac1,PLC-γ1,MRLC,Rho A,CDC42 and Rock1 genes（P<0.05）,and significantly increased the expression levels of GTP-rac1 and phosphorylated PLC-γ1 proteins as well as ZO-1,occludin and claudin-1 proteins（P<0.05）.Compared with the 40ng/m L TNF-α+0.1μM spermine group,the 40ng/m L TNF-α+0.1μM spermine+160μM NSC-23766 significantly decreased Rac1,MRLC,CDC42 and Rock1 gene expression levels（P<0.05）,and the 40ng/m L TNF-α+0.1μM spermine+3μM U73122 significantly decreased Rac1,PLC-γ1 and MRLC gene expression levels（P<0.05）.40ng/m L TNF-α+0.1μM spermine+160μM NSC-23766and 40ng/m L TNF-α+0.1μM spermine+3μM U73122 significantly decreased the expression of GTP-rac1 and phosphorylated PLC-γ1 proteins（P<0.05）,and significantly decreased the expression levels of ZO-1,occludin and claudin-1 proteins（P<0.05）.In conclusion,spermine promoted intestinal epithelial cell restoration through cell migration,cell proliferation and tight junction protein expression.Rac1/PLC-γ1 signaling pathways mediated this process for spermine repairing intestinal epithelial cell damage in piglets.