The Molecular Mechanism Of PEPT1 Regulating The Inflammatory Response In Bovine Rumen Epithelial Cells

In dairy cow production,in order to increase milk production,high-concentrate diets are added to the diet.However,long-term feeding of high-concentrates will induce a large number of gram-negative-bacterial death in the rumen.Some antigen peptides are removed from the surface-of bacteria.A large amount of lipopolysaccharide(LPS)and bacterial dipeptides and bacterial tripeptides(collectively referred to as small bacterial peptides)is produced in the rumen,which causes some nutritional and metabolic diseases and systemic pro-inflammatory reactions,such as rumenitis,liver abscess,laminitis,intermittent diarrhea,etc.,and-leads to a decrease in milk production.Previous studies reported that it mainly focused on the effects of LPS and-short chain fatty acids(Short chain fatty acid,SCFA)on rumen epithelial inflammation and demonstrated that PEPT1 is responsible for the transport and absorption of bacterial small-peptides into small intestinal cells,which subsequently-causes a rapid inflammatory response in the small intestine and severe colitis.However,it has not been investigated whether PEPT1 can mediate bacterial small peptides regulating the inflammatory response of neogastric epithelial cells(Bovine rumen epithelial-cells,BRECs)in-dairy cows.Therefore,the present study aims to deeply explore the molecular mechanism of PEPT1 regulating BRECs-inflammatory response through quantitative PCR,gene knockdown,transcriptome analysis,and Western blots,immunofluorescence,and provide a theoretical basis for studying BRECs inflammatory response and immune-response.Chapter 2:Effects.of high concentrate-rumen fluid on inflammatory response in bovine rumen epithelial cellsThe purpose of this-experiment is to study the effect of high-concentrate rumen fluid on the inflammatory response of bovine rumen epithelial cells.The experiment was divided into two groups:the medium containing 10%normal rumen fluid(as Control)and the medium containing 10%high-concentrate rumen fluid(HCRL)were-each cell cultured for 6 hours.,with 6 replicates in-each group.After 6 hours of culture,the cells were collected to extract total cell RNA and total cell protein.The results showed that compared with control group,high-concentrate diet rumen fluid significantly increased(P<0.05)the expression of BRECs pro-inflammatory factors IL-1β,IL-6 and TNF-α.In addition,HCRL can-significantly promote(P<0.05)the expression of CCL2,CCL20,CXCL2,CXCL3,CXCL8 and CXCL9 chemokines in BRECs.The HCRL significantly reduced(P<0.05)the expression of TLR2 and TLR4,but did not change the expression of TLR4 adaptor proteins CD14,MD2 and MyD88 and IRAK1 and TRAF6 in downstream signaling pathways.Notably,the HCRL can significantly-enhance(P<0.05)the expression of PEPT1 in BRECs.It is suggested that PEPT1 may-transport the small bacterial peptides into the cells,causing an inflammatory response.The HCRL significantly promoted(P<0.05)the up-regulation of MDA and H2O2 in BRECs.However,the contents of SOD,GSH-Px and T-AOC were significantly reduced(P<0.05).The results show that the HCRL can promote the inflammatory response of rumen epithelial cells and strengthen the inflammation response.In addition,the HCRL has a damaging effect on the rumen epithelium of dairy cows.Chapter 3:Transcriptome analysis of mRNA differential genes in wild-type and PEPT1 knockout bovine rumen epithelial cellsThe purpose-of this experiment is to study the molecular mechanism of PEPT1 regulating the inflammatory response of BRECs.The experiment was divided into 2 groups:wild-type BRECs and-PEPT1 knockout BRE Cs.BRECs knocked out PEPT1 were obtained through the CRISPR/Cas9 system.Through the whole transcriptome analysis of the differential gene expression of wild-type BRECs and PEPT1 knockout BRECs transcription level.The results showed that after TA cloning and sequencing,it was found that PEPT1 knockout resulted in 3 parents,of which two parents were effective knockouts.Compared with the wild type,the PEPT1 is almost not expressed in BRECs knocked out PEPT1 by Western blots.The results showed that compared with wild-type BRECs,the-expression of IL-6 was signifi cantly down-regulated(P<0.001)in BRECs knocked out PEPTI.The CCL2 and CCL5 chemokines were extremely significantly down-regulated(P<0.001)in BRECs knocked out PEPT1.It is worth noting that expression of most CXCL chemokines has been down-regulated in BRECs knocked out PEPT1.Compared with wild-type BRECs,the expression of CXCL2,CXCL3,CXCL5,CXCL9,CXCL12 and CXCL 16 chemokines were extremely significantly down-regulated-(P<0.001)in BRECs knocked out PEPT1 The above results suggest that PEPT1 can regulate the inflammation response in of BRECs.The downstream signaling molecules IRAK4 kinase(P<0.05)and IRF1 transcription factor(P<0.01)were significantly down-regulated in BRECs knocked out PEPT1.In addition,NOD2 involving pattern recognition receptors was significantly down-regulated(P<0.05),but the expression of NOD1 receptors was not changed.Inaddition,the expression of RIPK2,a key kinase downstream of the NOD receptor signaling pathway,was also significantly down-regulated(P<0.001)in BRECs knocked out PEPT1,but did not change the expression of RIPK1 and RIPK3 kinases.The BRECs-knocked out PEPT1 has no profound change in the expression of ERK2,ERK3,ERK4,and ERK5 kinase in BREC s,however,the expression of ERK1 kinase and NF-κB1 transcription factor were extremely significantly down-regulated(P<0.01).Importantly,the PEPT1 is positively correlated with ERK1 kinase and NF-κB1 transcription factor.These results indicate that PEPT1 regulating the inflammatory response of BRECs may be mediated by MAPK signaling pathway ERK1 kinase and NF-κB1 transcription factor.Chapter 4:PEPT1 mediated the high-concentration rumen fluid to regulate the inflammatory response in bovine rumen epithelial cellsThe purpose of this experiment is to study PEPT1 mediated HCRL to regulate the inflammatory response in BRECs.The experiment is divided into 4 groups:wild-type BRECs,knockout PEPT1 BRECs,wild-type BRECs with 10%HCRL and knockout PEPT1 BRECs with 10%HCRL.The results showed that compared with wild-type BRECs with 10%HCRL,IL-1β and TNF-α expreesion were extremely significantly down-regulated(P<0.01)in knockout PEPT1 BRECs with 10%HCRL.Compared with wild-type BRECs,the-expression of CXCL2 and CXCL3 chemokines were extremely significantly enhanced(P<0.01)in wild-type BRECs with 10%HCRL.Compared with wild-type BRECs with 10%HCRL,the expression of CCL2,CXCL3 and CXCL9 chemokines were significantly down-regulated(P<0.05)in knockout PEPT1 BRECs with 10%HCRL.In addition,compared with wild-type BRECs with 10%HCRL,the expression of RIPK2 kinase was extremely significantly down-regulated(P<0.001)in knockout PEPT1 BRECs with 10%HCRL.Using indirect immunofluorescence analysis,the red fluorescence of p-ERK1/2 was reduced in knockout PEPT1 BREC s compared with wild-type BRECs.The red fluorescence of p-ERK1/2 was significantly enhanced in BRECs with 10%HCRL.However,the red fluorescence of p-ERK1/2 was reduced in knockout PEPT1 BRECs with 10%HCRL.The results of Western blots showed that,compared with wild-type BRECs,phosphorylated p-ERK1/2 was reduced in knockout PEPT1 BRECs.Compared with the BRECs with 10%HCRL,the expression of phosphorylated p-ERK1/2 was down-regulated in knockout PEPT1 BRECs with 10%HCRL.The above results demonstrated that PEPT1 mediates ERK1/2 to regulate the inflammatory response of BRECs.Chapter 5:PEPT1 mediating bacterial small peptides to regulate the inflammatory response in bovine rumen epithelial cellsThe purpose of this experiment is to study PEPT1 mediating bacterial dipeptide MDP and bacterial tripeptide Tri-DAP to regulate the inflammatory response in BRECs.The experiment is divided into 5 groups:wild-type BRECs,wild-type BRECs with MDP,knockout PEPT1 BRECs with MDP,wild-type BRECs with Tri-DAP,and knockout PEPT1 BRECs with Tri-DAP.These results show that,compared with the control group,the bacterial dipeptide MDP and the bacterial tripeptide Tri-DAP can significantly promote the expression of BRECs IL-6 and TNF-α.Compared with the wild-type BRECs with MDP and Tri-DAP,the expression of IL-6 and TNF-α were significantly down-regulated in knockout PEPT1 BRECs with MDP and Tri-DAP,and the expression of CCL2,CXCL2,CXCL3 and CXCL9 chemokines were down-regulated.Compared to the control group,the expression of NOD2 and RIPK2 were significantly enhanced in the wild-type BRECs with MDP and Tri-DAP,but,significantly reduced in knockout PEPT1 BRECs with MDP and Tri-DAP.Using indirect immunofluorescence analysis,compared with the control group,the red fluorescence intensity of p-ERK 1/2 were significantly enhanced in the wild-type BRECs with MDP.Compared with the BRECs with MDP,the red fluorescence intensity of p-ERK1/2 were significantly decreased in knockout PEPT1 BRECs with MDP.These results demonstrated that PEPT1 can mediate bacterial small peptides to regulate the NOD2/RIPK2 immune signaling pathway,-thereby regulating the expression of pro-inflammatory factors and chemokines.In conclusion,this study demonstrated that PEPT1 regulates p-ErK 1/2 phosphorylation and NF-κB1 transcriptional levels through NOD2/RIPK2 signaling pathway,thus regulating BRECs inflammation.