| Bovine mastitis is one of the most harmful bovine diseases worldwide,and results in serious economic losses due to a potential long-term negative effect on udder health and milk production.The most common udder pathogens isolated from cases of clinical mastitis in dairy herds are a combination of Gram-negative and Gram-positive opportunistic pathogens.Gram-negative bacteria are responsible for about 40%of all clinical cases of bovine mastitis.Among them,Escherichia coli is the most common cause of bovine clinical mastitis.This mastitis can be induced by the intramammary administration of the E.coli endotoxin lipopolysaccharide(LPS),which exhibits a dose-dependent inflammatory response in the quarters of cow.Methionine-methionine(Met-Met)is a functional dipeptide.Although the role of dipeptide in milk protein synthesis is clearly established,whether Met-Met has anti-inflammatory effect and protective mechanism in bovine mammary epithelial(MAC-T)cells inflammation remains unknown.The specific objectives of the current study were to investigate the potential protective effects of Met-Met on LPS-induced MAC-T cell inflammation and to elucidate the related mechanisms using transcriptomics,metabolomics and RNA interference technology.The details are as follows:1.The effect of Met-Met on LPS-induced inflammation in bovine mammary epithelial cellsThis chapter aims to study the effect of Met-Met on LPS-induced inflammation in MAC-T cells.In our experiment,different concentrations of Met-Met and LPS were used to stimulate MAC-T cells,and the cell viability,inflammatory cytokines and inflammatory mediator expression were detected at different time points.The results showed that 2 mM Met-Met significantly promoted cell viability at 3 and 6 h,while 5,10 mM Met-Met showed cytotoxicity at 24 h,so the best subsequent option was 2 mM Met-Met with 6 h treatment.Compared with the control group,LPS could quickly induce the expression of inflammatory cytokines TNF-α,IL-8,and IL-1β to increase at 1 h,and reach the peak in 3 hours(Ptreatment<0.05),and pretreatment with 2 mM Met-Met significantly down-regulated the expression of TNF-α(Ptreatment<0.05),IL-8(Ptreatment<0.01)and IL-1β(Ptreatment<0.05).Compared with the LPS group,2 mM Met-Met could significantly inhibit the expression of the inflammatory mediator AP-1(P<0.05)at different time points,and different concentrations of Met-Met showed a concentration-dependent inhibition of TNF-α and IL-1β.Pretreated with 2 mM Met-Met could also obviously reduce the extracellular secretion of TNF-α(P<0.05)and IL-8(P<0.01)in MAC-T cells.Based on the concentration of TNF-α secreted from cells,three gradients of TNF-α were further set up to stimulate MAC-T cells to emulate the cascade inflammatory response caused by cytokines,and further the expression of inflammation-related cytokines were detected.Compared with the TNF-α group,pretreated with the 2 mM Met-Met exhibited inhibition against low,medium and high concentrations of TNF-α(13.3 pg/ml,P<0.01;40 pg/ml,P<0.05;120 pg/ml,P<0.05)and the expression of IL-8 was significantly suppressed(13.3 pg/ml,P<0.01;40 pg/ml,P<0.01;120 pg/ml,P<0.01).These results indicated that Met-Met could relieve mammary epithelial cells inflammation caused by LPS,and could inhibit the TNF-α-induced cascade inflammatory response.2.Metabolic mechanism of Met-Met in relieving LPS-induced inflammation in bovine mammary epithelial cellsBased on metabolomics analysis,this chapter focused on the metabolic mechanism of Met-Met in relieving LPS-induced bovine mammary epithelial cells inflammation.The experiments were divided into CON,LPS,Met-Met+LPS and Met-Met groups,each group had 3 biological replicates,and the cells were collected for metabolomics analysis.The overall metabolic profile of each treatment group changed significantly compared with the control group.The number of down-regulated DMs in the LPS vs.CON group was more than the up-regulated DMs(P<0.01);Metabolite-metabolite interaction network analysis found that DMs were mainly adenosine monophosphate,adenine,leucine,and phosphorylcholine;KEGG enrichment analysis indicated that DMs were mainly enriched in purine metabolism,synthesis and degradation of valine,leucine,isoleucine,and glycerophospholipid metabolism pathways.There were 17 overlapping DMs in the LPS vs.CON&Met-Met+LPS vs.LPS intersection group,of which 10 were up-regulated in the LPS vs.CON group and down-regulated in the Met-Met+LPS vs.LPS group,and 7 were down-regulation in the LPS vs.CON group and up-regulated in the Met-Met+LPS vs.LPS group;Metabolite-metabolite interaction network analysis showed that DMs were mainly tryptophan,valine,isoleucine,and adenosine monophosphate,adenosine triphosphate,adenine,embeic acid,palmitic acid;KEGG pathway analysis indicated that overlapping DMs were mainly enriched in the synthesis and degradation of valine,leucine and isoleucine,and the synthesis of aminoacyl-tRNA,purine metabolism,synthesis of non-unsaturated fatty acids and other pathways.The most correlated DMs in the Met-Met vs.CON group were L-methionine,S-methylthio-5’-adenosine,phenylacetic acid,adenine,adenosine monophosphate,uridine 5’-monophosphate and uridine 5’-Diphosphate;KEGG enrichment analysis showed that DMs were significantly enriched in cysteine,methionine metabolism,pyrimidine metabolism,purine metabolism,niacin and nicotinamide metabolism,aminoacyl-tRNA synthesis pathway.These results suggested that bovine mastitis caused numerous metabolites alteration,and the metabolic pathways of amino acids,purines and fatty acids might involve in the anti-inflammatory process of Met-Met in alleviating bovine mammary epithelial cells inflammation.3.The molecular mechanism of Met-Met alleviating LPS-induced inflammation in bovine mammary epithelial cellsBased on transcriptomics analysis,the mechanism of Met-Met alleviating LPS-induced inflammation in bovine mammary epithelial cells was studied.The experiment design was same as that in the 2 part,each group was processed accordingly and the cells were collected for RNA-seq and qPCR analysis.Compared with the control group,the overall transcription profile of each treatment group changed significantly.The number of up-regulated genes in the LPS vs.CON group was more than the number of down-regulated genes(P<0.01);Protein-protein interaction(PPI)network analysis revealed that the core genes(hub genes)are mainly related to cytokines,such as CXCL2,CXCL8,TNF,CSF2,NFKBIA,etc.;GO enrichment analysis found that differential genes(DEGs)are majorly involved in inflammatory response,cytokine regulation biological processes;KEGG enrichment analysis indicated that DEGs were mainly enriched in NFκB,MAPK,TNF signaling pathways.There were 46 overlapping genes in the LPS vs.CON&Met-Met+LPS vs.LPS intersection group,of which 40 were up-regulated in the LPS vs.CON group and then down-regulated in the Met-Met+LPS vs.LPS group,and six genes were down-regulated in the LPS vs.CON group and then were up-regulated in the Met-Met+LPS vs.LPS group;GO enrichment analysis found that overlapping genes were mainly involved in lipopolysaccharide response and inflammatory response;KEGG pathway analysis showed that overlapping genes were mainly enriched in the NFκB,MAPK and IL-17 signaling pathways.The number of down-regulated genes in the Met-Met vs.CON group was more than the number of up-regulated genes(P<0.01);PPI network analysis found that hub genes such as SIPR1,CCR7,HCAR1,CXCL2,IL-1A were mainly involved in inflammatory and histone ubiquitination processes;GO enrichment analysis found that DEGs were mainly involved in biological processes such as amino acid catabolism and kinase activity regulation;KEGG enrichment analysis showed that DEGs were significantly enriched in JAK-STAT signaling pathway,glycine,serine,and threonine metabolic pathways.These results suggested that bovine mastitis contributed to gene transcriptome changes in bovine mammary epithelial cells.NFκB,MAPK and JAK2-STAT5 signaling pathways might participated in the process of Met-Met alleviating mammary epithelial cells inflammation.4.The regulatory mechanism of JAK2-STAT5 and NFκB/MAPK pathway in the anti-inflammatory process of Met-MetThis chapter aims to study the regulatory mechanism of JAK2-STAT5 and NFκB/MAPK pathways in the process of Met-Met alleviating the inflammation of bovine mammary epithelial cells.The experiments were conducted in the LPS and Met-Met+LPS groups,and the protein levels of JAK2-STAT5 and NFκB/MAPK were detected at different time points(0,15,30,45,60,and 90 min).The phosphorylation and non-phosphorylation levels of JAK2-STAT5,NFκB,and MAPK proteins were detected under the condition of JAK2 gene silencing.The results showed that Met-Met could quickly activate p-JAK2/JAK2(P<0.01),p-STAT5/STAT5(P<0.05)and significantly inhibit the level of p-IκB/IκB,p-NFκB/NFκB(P<0.05)and p-P38/P38,p-JNK/JNK(P<0.01),whereas,contribute to the level of p-ERK1/2/ERK1/2(P<0.05);Interfering JAK2 significantly reduced the phosphorylation and non-phosphorylation levels of JAK2 and STAT5(P<0.01),and obviously reversed the Met-Met-mediated inhibition of p-IκB,NFκB and JNK under the inflammatory state(P<0.05).These results indicated that the activation of JAK2 was a necessary condition for Met-Met to alleviate the bovine mammary epithelial cells inflammation.Met-Met exerted anti-inflammatory effects by activating JAK2-STAT5 and then inhibited the NFκB and partial MAPK signaling pathways.In summary,MAC-T cells inflammation accompanied with decreased anti-inflammatory amino acids,increased pro-inflammatory fatty acids,and disordered purine metabolism.Met-Met activated the JAK2-STAT5 pathway and then inhibited the NFκB and MAPK signaling pathways to alleviate the inflammation of bovine mammary epithelial cells,and improve metabolic disorders.This discovery provides a theoretical basis for the development of a functional oligopeptide Met-Met to improve animal immunity. |
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