Mechanism Of Recombinant Bovine Lipopolysaccharide Binding Protein Against Inflammatory Responses Induced By Lipopolysaccharide In Bovine Mammary Epithelial Cells

In this paper,the rbLBP and rbM-LBP protein were restructured by the prokaryotic expression system.The LPS induced inflammation model was established in the base of mammary gland epithelial cell(BMEC)culture in vitro.Moreover,the differentially expressed genes of inflammation model were analyzed by Gene chip technology.The effects of rbLBP on inflammation model also were evaluated.Our research will provide some useful references for further research in LBP applications and prevention in cattle mastitis.1 LBP carrier preparation,recombinant protein expression and purificationFull length bovine LBP and M-LBP cDNA were cloned into pET22b vector.The expressive plasmid pET22b-LBP and pET22b-M-LBP which contained the cDNA of the LBP under a T7-promoter were transformed into competent Escherichia coli BL21(DE3)cells and then were induced with IPTG.The purified bovine recombinant LBP and RBM-LBP were obtained by purification,and were identified by SDS-PAGE.Western blotting and protein spectrum analysis were used to determine the recombinant protein.The recombinant bovine LBP and rbM-LBP were cloned and expressed in Escherichia coli.System,and the active recombinant proteins purified from the Escherichia coli.2 Optimized culture and inflammatory model establishment of BMECsIn this study,BMECs were cultured and the inflammatoin model was established.Primary culture of BMECs were cultured tissue block method,cells grown well in DMEM.Cells were identified by cytokeratin andβ-casein.Results shows that the cell purity was up to 95%,and had perfect biological function.Stimulation of BMEC with LPS for 12h elicited a significant increase in mRNA expression for TNF-a and IL-1β(P<0.05),the expression showed a dose-dependent manner from 1 to 100μg of LPS per mL,100μg LPS stimulation showed a slightly decrease in those genes expression due to the cell toxicity effect.Secretion of TNF-a and IL-1βwas also increased when induced by LPS,and with a maximal level when stimulated with 10μg of LPS per mL.In this study,we successfully established the inflammatoin models of primary culture bovine mammary epithelial cells.3 Microarray analysis of differentially expressed genes in the lipopolysaccharide-induced pro-inflammatory response in BMECsTo investigate the immune response in the mammary gland upon infection,BMECs were induced with E.coli-derived lipopolysaccharide(LPS)to mimic the inflammatory process.Microarray analysis identified 958 differentially expressed genes among>43,000 transcripts,of which 472 were up-regulated and 486 were down-regulated following BMEC exposure to LPS.Gene ontology and KEGG analysis demonstrated the enrichment of transcripts related to immune defense and inflammatory response,including the toll-like receptor pathway and chemokine response.Quantitative PCR validated the microarray results,demonstrating up-regulation of transcripts involved in the TLR4 pathway,including LBP,TLR4,MyD88,NFKB,IL-1β,IL-8,NOS2,and TNF-α.TLR4 and NF-κB protein expression were increased in LPS-stimulated BMEC.The results demonstrate that the TLR4/NF-κB signaling pathway is activated by LPS in BMEC,suggesting that this microbial product may contribute to the inflammatory injury in E.coli infection.4 rbLBP regulated inflammation response in LPS-challenged BMECsTo verify the anti-inflammatory properties of LBP on the inflammatory response of primary BMECs induced by lipopolysaccharide(LPS),and to determine the underlying mechanism.Various concentrations(10 and 20μg/mL)of rbLBP could weaken the inflammation injury of BMEC induced by LPS without cytotoxicity.Toll-like receptor 4(TLR4),nuclear factorκB(NF-κB),IL-1β,and tumor necrosis factor a(TNF-a)from BMEC were decreased(p<0.05).TLR4 and NF-κB P65 protein levels were down-regulated,and nuclear transcription factorκB activity was also weakened.Results indicated that the protective effects of high concentrations of rbLBP on LPS-induced inflammation injury in BMEC were at least partially achieved by the decreased production of pro-inflammatory cytokines.rbLBP was found to directly inhibit LPS/TLR4-mediated NF-κB activation.One possible anti-inflammatory mechanism can be attributed to the negative role of rbLBP in suppressing TLR4/NF-κB activation mediated by LPS.5 rbLBP and rbM-LBP regulated inflammation response in LPS-challenged BMECsIn this experiment,rbLBP and rbM-LBP were acting on LPS-induced inflammation model of BMEC to investigate the possible role mechanisms.Results show,20μg/mL of rbLBP,rbM-LBP were reduced LPS-induced mammary epithelial cells in IL-1βand TNF-αmRNA expression,inhibited secretion of mammary epithelial cells TNF-a and IL-1β;significantly reduced LPS induced TLR4,mRNA and protein expression of NF-κB(P65)(P<0.05),inhibition of NF-κB(P65)activation.20μg/mL of rbLBP and the rbM-LBP significantly reduced LPS-induced early apoptotic cells(P<0.05).20μg/mL of rbLBP with rbM-LBP compare differences related factors affect the results were not significant(P>0.05).The results show that,LBP in mammary epithelial cells inhibited LPS-induced inflammatory response mechanism may be related to neutralize LPS activity,down-regulation TLR4 expression and inhibit NF-κB activation signal,inhibiting the secretion of inflammatory cytokines and expression.The rbLBP and rbM-LBP were not significant influence-related factors.