Influence Of Porcine Beta Defensin 2 On The Gene Ex-pression Of NF-κB And MAPK Signaling Pathways In E.coli Infection IPEC-J2 Cell

The mammalian defensins are widely concerned because of their strong antibacterial ability,wide antibacterial effect,and not easy to cause drug resistance.Therefore,defensins are considered to be a new type of antimicrobial agents to replace traditional antibiotics.Porcine defensin 2 showed strong salt tolerance and thermal stability in vitro,and a lower concentration of defensins could kill Escherichia coli and Staphylococcus aureus.p BD2 may also play a role in inflammation,but its mechanism is not clear.TLRs mediated IκB-α/NF-κB and MAPK play a key role in inflammation.In order to investigate whether p BD2 plays an important role in the inflammatory response of intestinal epithelial cells by TLRs mediated signaling pathway,Escherichia coli was used to infect IPEC-J2 cells in this study.The expression of TLR1-9 was detected by fluorescence quantitative method,and the optimal concentration and time of E.coli infection of IPEC-J2 cells were explored.Different concentrations(0μg/m L、20μg/m L、40μg/m L、80μg/m L)of p BD2 were added into cell culture medium,and cells were collected at 2h,4h,6h and 8h respectively.The expression of TLRs(Toll-like receptors)and pathway protein gene was detected by fluorescent quantitative and Western blot method in order to obtain the effect of defensins on intestinal epithelial cells.The results are as follows:(1)In Escherichia coli infected IPEC-J2 cells,the optimal concentration and time of infection were 107 and 1h,respectively.Microscopic observation showed that after infection with 2h,a small number of cells died.After infection with 4h and 8h,there was an increase in the death rate.After the infection of 8h,a group of dead cells appeared,and the cell morphology changed.The expression of ZO-1 and ZO-2 gene in intestinal tract was significantly decreased,which indicated that the infection of Escherichia coli resulted in the damage of cell barrier.(2)Only TLR4、TLR5 and TLR7 increased significantly in TLRs in the cells infected with Escherichia coli.IκB-α/NF-κB and TAK1/JNK signaling pathway related gene expression was significantly increased,the expression of inflammatory factor IL8 was also significantly increased,indicating that TLR4 、 IκB-α/NF-κB and TAK1/JNK signaling pathway may affect the inflammatory response.(3)After the addition of p BD2 into E.coli infected IPEC-J2 cells,it was found that the change of IκB-α/NF-κB and p38 MAPK was not obvious,but the TAK1/JNK was significantly decreased,which indicated that the defensins may attenuate the inflammatoryresponse through TAK1/JNK signaling pathway.(4)The endogenous p BD2 was significantly decrased after infection with 2h,but it was found that the p BD2 was increased when adding exogenous p BD2.