Mechanism Of Spermidine Regulating Cell Proliferation,Migration,and Inflammatory Response In Porcine IPEC-J2 Cells

Polyamines,including putrescine,spermidine and spermine,are a class of active small molecules and necessary for animal cell growth.Putrescine is a precursor for the synthesis of spermidine and spermine.These three polyamines can be converted to each other by the enzymes of polyamine synthase,polyamine oxidase and spermidine/spermine acetyltransferase.Our prescious study found that putrescine has a good effect on promoting cell perliferation,migration and anti-inflammatory of pig intestinal epithelial cells(IPEC-J2).However,it is not clear whether these function are performed by putrescine itself or by its metabolite spermidine or spermine.In present study,we took IPEC-J2 cells as the model,and used the polyamine metabolism pathway inhibitors DEGBG and DFMO to block the mutual transformation between polyamines,to reveal the mechanism of polyamines promoting cell perliferation,migration and anti-inflammatory.IPEC-J2 cells were seeded in 96-well plates at0.5×10~5cells/mL,and different concentrations of spermidine(1?mol/L,2?mol/L,4?mol/L,8?mol/L,16?mol/L,32?mol/Land 64?mol/L)were usedto study the proliferation effect of spermidine on IPEC-J2 cells and screened out the optimal amount of spermidine.Different concentrations of DEGBG(0.25 mmol/L,0.5 mmol/L,1 mmol/L and 2 mmol/L)were added to the medium to determine the approptiate level of DEGBG to inhibit intracellular spermidine production.The effects on cell proliferation,migration and inflammatory response were detected by adding DEGBG to the IPEC-J2cell culture medium,and LPS stimulation was used as a model of cell damage.Then added spermidine or putrescine to study its repairing effect on cell damage caused by DEGBG or LPS.Cellular RNA and protein were extracted for detection of gene expression of key genes involoved in related signaling pathways.The study found that adding 1?mol/L,2?mol/L,4?mol/L,8?mol/L and 16?mol/L spermidine to the culture medium can promote the proliferation of IPEC-J2 cells,of which 8?mol/L spermidine has the best effect on promoting proliferation.Adding 1mmol/L of DEGBG to the culture medium for 24 h significantly reduced the intracellular level of spermidine and inhibited the proliferation of IPEC-J2.Supplementing putrescine in the medium containing DEGBG did not restore the proliferation rate of IPEC-J2.However,the proliferation of IPEC-J2 was restored by adding spermidine.The addition of DEGBG had no effect on the expression of total ERK protein,but significantly reduced the expression of phosphorylated ERK protein.Exgenous addition of spermidine eliminated the inhibitory effect on phosphorylated ERK protein expression.Adding DEGBG to the medium also significantly inhibited cell migration,while exogenous addition of spermidine restored the cell migration.Spermidine inhibited the increase in the expression of TNF-?,a cellular inflammaoty factor stimulated by LPS.The addition of DEGBG to the culture medium caused the increase of the expression of IL-8,IL-6,TNF-?,COX-2,IL-1?gene while the addition of exogenous spermidine could inhibit the increase of IL-8,IL-6,TNF-?gene expression.The addition of DEGBG to the medium had no effect on the total protein expression of NF-?B,but increased the expression of phosphorylated NF-?B protein.Exogenous addition of spermidine reduced the expression of phosphorylated NF-?B.The results of this study indicated that putrescine promotes the proliferation and migration of IPEC-J2 cells through its metabolizer spermidine.Under normal circumstances,spermidine has no effect on the expression of inflammatory factors in cells.However,the lack of spermidine in the cell will increase the inflammatory response of the cell,and the supplementation of spermidine can reduce the inflammatory response.Our results have laid a theoretical foundation for the application of polyamines in piglets and the development of polyamines as new feed additives.