Regulation Of MAPK Signaling Pathway On Inflammatory Cytokines And Cell Metabolism Of HD11

The infection of pathogenic microorganisms such as Escherichia coli,Salmonella often causes excessive inflammation reaction in animals,resulting in frequent intestinal inflammation diseases in poultry industry,which has caused enormous economic losses to livestock industry worldwide.The research on the pathogenesis and prevention of intestinal inflammation in poultry has always been a hot topic.The development of inflammation requires the participation of many kinds of immune cells,among which macrophages are the most important immune cell in inflammation reaction.In this study,HD11 was taken as the research object,through transcriptomics and metabonomics sequencing seek the dynamic changes of cell gene transcription and metabolism during inflammation reaction.Through joint analysis explore the interaction network between differential genes and differential metabolites and the correlation between key signal pathways and cell metabolic process.On this basis,this study effectively blocked the above key pathways to explore the important regulatory effect of blocking this pathway on proinflammatory factors and metabolic process of HD11.1.In this study,lipopolysaccharide(LPS)was used to construct an inflammation model in vitro.The effects of different concentrations of LPS on the viability and IL-1β m RNA expression of HD11 were detected by CCK8 and q PCR,and the changes of IL-1β m RNA expression were detected at different time after LPS stimulation.The results showed that in HD11,1μg/ml LPS stimulated cells for 6 h could induce IL-1β expression increase significantly(P < 0.05),and had no obvious toxic effect,so it could be the best scheme to construct inflammation model.2.HD11 were divided into control group(C group)and LPS inflammation group(LPS group).Using RNA-Seq and LC-MS analyze transcription and metabonomics of cell samples.(1)The results of transcriptomics showed that in LPS group,1319 differentially expressed genes were significantly up-regulated(P < 0.05),and 744 differentially expressed genes were significantly down-regulated(P < 0.05).Enrichment of KEGG pathways showed that differentially expressed genes were significantly focused on MAPK and NF-κB signaling pathways.A total of 42 genes associated with MAPK signaling pathway were identified by database analysis.Among them,35 genes including FOS,JUN,MYC,NF-κB and so on were significantly up-regulated(P < 0.05),7 genes including CAT were significantly down-regulated(P < 0.05).q PCR was used to verify some genes.The results showed that: The expressions of c-JUN,c-FOS,c-MYC genes in the LPS group were significantly increased(P < 0.05),indicating that LPS stimulation increased the expressions of transcription factors such as c-JUN,c-FOS,and NF-κB in HD11,and activated MAPK signaling pathway,and it was speculated that the activation of this pathway mediate the occurrence of cellular inflammation reaction.(2)Metabonomics showed that 88 differentially metabolites were screened between the control group and LPS group,among which glutamate,glutathione and other metabolites were significantly down-regulated(P < 0.05).KEGG pathway analysis showed that the differentially metabolites were significantly enriched in glutamate metabolism,glutathione metabolism,TCA cycle and other processes,indicating that these pathways may be related to the occurrence of inflammation reaction.Using gene-metabolite interaction network in Metabo Analyst 5.0,the results showed that the down-regulation of metabolites such as glutamate,L-Malic acid and Arachidonic Acid was related to the differentially expressed genes c-JUN,c-FOS,NF-κB and i NOS in MAPK pathway,which suggested that MAPK signaling pathway could regulate the metabolism of HD11.3.In order to explore the regulation of MAPK signaling pathway on HD11 cytokines and cell metabolism,the MAPK signaling pathway inhibitor-Astragaloside IV was used to specifically block this signaling pathway.HD11 s were divided into control group(C),LPS inflammation group(LPS group,L),AST IV+ LPS group(AST IV group,A+L)and AST IV group(AST IV group,A).q PCR,Western blot and ELISA were used to detect the m RNA expression and protein level changes of MAPK pathway related factors and cytokines in HD11 in different groups,and the changes of mitochondrial membrane potential,ROS production and glutathione metabolism in HD11 were detected by flow cytometry,fluorescence probe and kit.(1)In HD11,the contents of MAP3K1,MAP2K1/2/4,ERK,JNK,c-JUN,c-FOS m RNA and their phosphorylated proteins in L group were significantly higher than those in C group(P < 0.05);The expression of MAP3K1,MAP2K1/2/4,ERK,JNK,c-JUN and c-FOS m RNA and its phosphorylated protein content in A+L group were significantly lower than those in L group(P <0.05),which indicated that MAPK signaling pathway was significantly activated when HD11 s had inflammation reaction.AST IV can inhibit the over-activation of MAPK pathway.In HD11,the expression levels of IL-1β,TNF-α,IL-6,NF-κB,i NOS in L group were significantly higher than those in C group(P < 0.05).The m RNA and protein expressions of pro-inflammatory factors in A+L group were significantly lower than those in L group(P < 0.05),which indicated that AST IV could inhibit inflammation by inhibiting the activation of MAPK and reducing the expression of cytokines such as IL-1β,TNF-α,IL-6,NF-κB and i NOS.(2)Compared with C group,the mitochondrial membrane potential in L group decreased significantly(P < 0.05)and the production of ROS increased significantly(P < 0.05).Compared with L group,the mitochondrial membrane potential in A+L group was significantly increased(P <0.05),and the ROS production was significantly inhibited(P < 0.05),suggesting that LPS stimulation destroyed the mitochondrial structure and function of HD11 s,and increased ROS production.AST IV can inhibit the activation of MAPK,protect the morphology and function of mitochondria,change the metabolic process of cells,reduce the ROS content,and thus intervene the inflammation reaction.(3)The expression levels of GCLM,GPX1 and GPX4 m RNA in L group were significantly lower than those in C group(P < 0.05).The expression of GCLM,GPX1 and GPX4 m RNA in A+L group were significantly higher than L group(P < 0.05).Compared with C group,the contents of glutamate,T-GSH and GSH in L group decreased significantly(P < 0.05),while the contents of GSSG increased significantly(P < 0.05).Compared with L group,the contents of glutamate,T-GSH and GSH in A+L group increased significantly(P < 0.05),while the content of GSSG decreased significantly(P < 0.05),which indicated that in inflammation model of HD11 induced by LPS,the metabolic process of glutamate-glutathione could be improved by inhibiting the over-activation of MAPK pathway,so that the contents of glutamate and glutathione could be increased,and the antioxidant defense function could be exerted to inhibit the inflammation reaction.In conclusion,1μg/ml LPS stimulating cells for 6h can be the best scheme to construct the inflammation model of HD11.LPS activated MAPK signaling pathway,transcription factors c-JUN and c-FOS of MAPK signaling pathway can regulate the contents of metabolites such as glutamate,L-Malic acid and Arachidonic Acid.After MAPK signaling pathway is effectively blocked,on the one hand,it inhibits the activation of transcription factors c-JUN and c-FOS,and reduces the production of pro-inflammatory factors;on the other hand,it can regulate the metabolic level of immune cells,maintain mitochondrial homeostasis,reduce ROS production,improve glutamate-glutathione metabolism,and then participate in the regulation of inflammation reaction and cell function,providing important theoretical and experimental basis for further exploring the pathogenesis of inflammatory diseases and finding new targeted drugs.