Anti-inflammatory Effect Of Methionine On Pathogenic Lipopolysaccharide And Its Application Prospect

Methionine(Met)is first restrictive amino acid in poultry and widely used in poultry production,it plays an important role in synthesizing organism protein and regulating immune function.Lipopolysaccharide(LPS)is one of the pathogenic factors of many infectious pathogens in poultry,which can cause inflammation of the organism and seriously affect the immune function and production performance of poultry.However,whether methionine can effectively regulate LPs induced innate immune mechanism and its molecular mechanism is unclear.This study established LPS-induced inflammatory response model of macrophages,to investigate the effect of methionine on LPS-induced inflammatory response of macrophage RAW264.7,and to explore its molecular mechanism from MAPK signaling pathway and methylation reaction process.This study conducted the following tests:1.Effect of Met treatment on RAW264.7 activity: CCK-8 was used to detect the activity of RAW264.7 in different Met concentrations(0.1 μM,1 μM,10 μM,0.1 mM,1 mM,10 mM).The results showed that there was no significant change in the activity of RAW264.7 with Met concentrations(p>0.05).2.Selection of the effective concentration of Met on LPS-induced RAW264.7 inflammation: Using Q-PCR technique to detect the relative gene expression of LPS-induced RAW264.7 cytokine IL-6,TNF-α and IFN-β by different Met concentrations(0.1 μM,1 μM,10 μM,0.1 mM,1 mM,10 mM).The results showed that the Met concentration of 10 mM could significantly inhibit the relative gene expression of cytokine(IL-6 、 TNF-α and IFN-β)produced by LPS-induced RAW264.7(p<0.05),then the concentration of methionine treatment was 10 mM in subsequent experiments.3.Effect of Met on the expression of cytokine(IL-6、TNF-α and IFN-β)induced by LPS in RAW264.7: The experiment was divided into 4 groups,the normal cultured RAW264.7 cells were control group,and the other three groups were Met,LPS and Met+LPS.Q-PCR and ELISA were used to detect the expression of Met on LPS-induced IL-6,TNF-α and IFN-β in RAW264.7 from gene level and protein level.The results showed that Met could significantly inhibit the expression of LPS-induced IL-6、TNF-α and IFN-β at both gene level and protein level in RAW264.7(p<0.05).4.Effects of Met on MAPK signaling pathway in LPS-induced RAW264.7: The experiment was divided into 2 groups,LPS and Met+LPS.The p38,Erk and JNK phosphorylation of LPS-induced MAPK signaling pathway by Met were detected by Western blot.The results showed that met could significantly reduce the phosphorylation level of p38,Erk and JNK in MAPK signaling pathway in LPS-induced RAW264.7(p<0.05).5.Effect of S-adenosine methionine(SAM)on the expression of cytokine(IL-6、TNF-α and IFN-β)induced by LPS in RAW264.7: The experiment was divided into 4 groups,the normal cultured RAW264.7 were the control group,the remaining three groups were SAM,LPS and SAM+LPS.The results showed that Sam,as a Met methyl donor,also had met at both gene and protein levels to significantly inhibit LPS-induced RAW264.7 cytokine IL-6,TNF-α and IFN-β expression(p<0.05).6.Effects of SAM on MAPK signaling pathway in LPS-induced RAW264.7: The experiment was divided into 2 groups,LPS and Met+LPS.Determination of p38,Erk,and JNK phosphorylation levels in LPS-induced MAPK signaling pathway by SAM using Western blot.The results showed that SAM was consistent with the Met effect,and also significantly inhibited the phosphorylation of p38,Erk and JNK in MAPK signaling pathway induced by LPS(p<0.05).7.Effect of autophagy LC3 by Met on LPS-induced RAW264.7: The experiment was divided into 4 groups,the control group was the normal cultured RAW264.7,and the other three groups were Met,LPS and Met+LPS.The expression and LC3 II/I of autophagy protein LC3 in LPS-induced RAW264.7 were detected by Western blot.The results showed that Met could significantly reduce the expression of LPS-induced LC3 II/I in RAW264.7(p<0.05).8.Effects of Met and SAM on the level of total genomic methylation in LPS-induced RAW264.7 inflammation: The experiment was divided into 6 groups: Ctr,Met,SAM,LPS,Met+LPS,SAM+LPS.The MeDIP-Seq technique was used to analyzed the whole genome methylation level of macrophage and differential methylation genes,then analysis of differential methylation genes between groups through Kyoto Encyclopedia of genes and Genomes(KEGG).The results showed that both Met and SAM could increase the methylation level of cell gene.Compared with the Met+LPS and LPS,the decreased-methylation gene was more than the increased-methylation gene,and 6 differential methylation gene enrichment was found in MAPK signaling pathway through KEGG,which indicated that Met was involved in methylation regulation of MAPK signaling pathway.Compared with the SAM+LPS and LPS,the decreased-methylation gene was more than the increased-methylation gene,and 4 differential methylation gene enrichment was found in MAPK signaling pathway through KEGG,which indicated that SAM was involved in methylation regulation of MAPK signaling pathway.