Protective Mechanism Of Chitosan Oligosaccharide Attenuation Of LPS Induced Inflammatory Injury In IPEC-J2 Cells

This study was to explore the protective mechanism of COS pre-protection before LPS induce IPEC-J2 cells inflammatory injury,in order to provide some theoretical reference for the application of cos as a feed additive in pig production.The proliferative activity of IPEC-J2 cells was detected by CCK-8 assay after IPEC-J2 cells were treated with LPS(0,250,500,1000,5000,10000 ng/m L)or COS(0,25,50,100,150,200,250,300,400,600 μg/m L)for 6 h or 24 h.The experiment was divided into control group(without COS or LPS),COS group(50 μg/m L),LPS group(500 ng/m L)and COS pre-protection group(COS 50 μg/m L + LPS 500ng/m L).Fluorescent quantitative PCR was used to detect the expression of related genes of inflammatory cytokines,tight link proteins and mucin protein.Western blot was used to detect the protein expression of My D88,p38 MAPK,Pho-p38 MAPK,JNK,IKKβ and NF-κB signal transduction pathways.The results showed that the viability of IPEC-J2 cells was decreased after treatment with different concentrations of LPS,and the proliferation of IPEC-J2 cells was significantly inhibited the concentration of LPS reached 100 ng/m L.When the concentration of LPS of 500 ng/m L,could decrease the viability of IPEC-J2 cells significantly to 62.7%(P<0.01).When the concentration of COS was 50-250 μg/m L,it could promote the proliferation of IPEC-J2 cells(P<0.01),and when the concentration was 50 μg/m L could promot the cell viability could to 127.5%.Compared with the control group,LPS had no effect on the expression of ZO-1 mRNA(P>0.05),COS pre-protection could up-regulate the expression of ZO-1 mRNA(P<0.05).Compared with the control group,LPS significantly decreased the expression of mucin MUC2 mRNA(P<0.01),COS pre-protection could alleviate the down-regulated expression of MUC2 mRNA by LPS(P>0.05).Compared with the control group,LPS significantly increased the expression of TLR4 and TNF-α mRNA(P<0.01).Compared with LPS,COS pre-protection could alleviate the up-regulated expression of TLR4 and TNF-α mRNA(P<0.01).Compared with the control group,LPS significantly up-regulated the expression of My D88,p38 MAPK,Pho-p38 MAPK,JNK,IKKβ and NF-κB protein(P<0.01).COS preprotection significantly alleviated the up-regulated expression of NF-κB protein by LPS(P<0.01).It was found that COS pre-protection could alleviate the down-regulation of ZO-1 mRNA in physical barrier of IPEC-J2 cells induced by LPS,it could significantly alleviate the down-regulation of MUC2 mRNA expression in the chemical barrier,TLR4 and TNF-α mRNA expression in the immune barrier induced by LPS(P<0.01),and alleviate the up-regulation of My D88,p38 MAPK,Pho-p38 MAPK,JNK,IKKβ and NF-κB protein induced by LPS.It is suggested that the anti-inflammatory effect of COS preprotection is through TLR4,My D88,MAPK and IKKβ pathway,inhibit the activation of downstream NF-κB signal pathway,reduce the release of inflammatory cytokines,increase the expression of intestinal tight junction protein and mucin gene,and inhibit the inflammatory response induced by LPS.