Study Of Effect On MMPs/TIMPs And UPA System Expression And The Signaling Pathway In Bovine Mammary Fibrosis Induced By Staphylococcus Aureus

Bovine mammary fibrosis is a disease which characterized by ECM overdeposition and myofibroblast proliferation.S.aureus is one of the main pathogens causing bovine mastitis.The sustained injury causes breast structure disorder,development of fibrosis and even breast sclerosis.Our previous studies were not involved in regulating metabolism of ECM.Therefore,this study aimed at MMPs/TIMPs and uP A system,and its related pathway in the process of bovine mammary fibrosis induced by S.aureus in vivo and in vitro.The BMFBs and BMECs were stimulated with 0,105,106,108 CFU/mL heat-inactivated S.aureus for 6,12,24 and 48 h.The expression levels of MMP-1,-2,-3,-9,-13,TIMP-1,-2,uPA,uPAR,PAI-1 and COL I gene and protein were detected by qPCR and Western blot,and the activity of MMP-2 and-9 in cell supernatant was detected by gelatinase assay.106 CFU/mL heat-inactivated S.aureus induced BMFBs then transcriptome sequencing was performed.Differentially expressed genes and signaling pathways were analyzed by bioinformatics.ERK1/2,P38,JNK and phosphorylation levels expression were verified by qPCR and Western blot.Using MAPKs specific inhibitors,the activity of MMP-2 and-9 in cell supernatant was detected by gelatinase assay.MMP-2,TIMP-1,-2,uPA and PAI-1 protein expression were detected by Western blot.S.aureus-induced mice breast fibrosis model observed each group at different infection stages by Masson staining.MMP-1,-3,-13,TIMP-1,uPA and PAI-1 mRNA expression were detected by qPCR,the distribution of MMP-1,-3,-13,TIMP-1,uPA and PAI-1 were detected by immunohistochemistry.The results show:(1)Different concentrations heat-inactivated S.aureus upregulated MMP-1,-2,-3,-9,-13,TIMP-1,-2,uPA,uPAR,PAI-1 and COL I mRNA expression in BMFBs,but steadily at later period.Also,protein expression levels were upregulated.The activity of MMP-2 was very obvious than MMP-9,but its activity significantly higher in 24 h and 48 h.MMP-1,-2,-3,-9,-13,TIMP-1,-25 uPA,uPAR,PAI-1 and COL I mRNA expression in BMECs,significant expression in 6 h and 48 h.while the uPA,PAI-1 mRNA expression levels were downregulated with a time-dependent manner.While MMP-1,-2,-3,-9,-13,TIMP-1,-2,uPA,uPAR,PAI-1 and COL I protein expression were upregulated with a time-dependent manner.The activity of MMP-2 and MMP-9 was similar,and its activity significantly higher in 24 h and 48 h.The expression trend of MMP-1,-2,-3,-9,-13,TIMP-1,-2,uPA,uPAR,PAI-1 and COL I was showed in BMFBs and BMECs by the changes of the time:MMP-1,-2,-9,-13 and uPA showed a certain descent trend,which was contrary to the expression trend of COL I.The expression trend of MMP-3 and COL I was consistent,showing an upregulation.TIMP-1,-2 and PAI-1 showed a certain upward trend,showing inhibition.The expression trend of MMP-2,-3,-9,uPA,TIMP-1,-2 was consistent with COL I in BMECs,and reached the peak at 12 h,then gradually slowed downregulation.The expression of MMP-1,-13 and PAI-1 was opposite to COL I,showing a more obvious downward trend.Transcriptome sequencing revealed MMPs,TIMPs,some collagen and uPA system,involving the PI3K-AKT,Toll-like receptor,MAPKs,TNF and IL-17 signaling pathways by S.aureus induced BMFBs.S.aureus promoted ERK1/2,P38 and JNK mRNA and protein expression in BMFBs,MAPKs(ERK1/2,P38 and JNK)specific inhibitor can inhibit MMP-2 activity,and inhibition effect was significant in 24 h.MAPKs specific inhibitors reduced S.aureus-induced MMP-2,TIMP-1,-2,uPA and PAI-1 expression,and inhibition effect was significant in 24 h,and the existence of inhibitors combined effect.S.aureus induced mammary fibrosis in mice,promoting the expression of MMP-1,-3,-13,TIMP-1,uPA and PAI-1 in breast tissue.Moreover,MMP-1,-3,-13,TIMP-1,uPA and PAI-1 were mainly expressed in macrophage,epithelial cells and hyperplasia fibroblasts.Conclusions:The results of this study indicate that different concentration of heat-inactivated S.aureus induced BMFBs and BMECs MMP-1,-2,-3,-9,-13 and TIMP-1,-2 expression,and activating the uPA system involved in regulation of MMPs expression.Activated MAPKs signaling pathways involved in the regulation of the main MMP-2,TIMP-1,-2 and uPA/PAI-1 expression.In addition,the study also identified S.aureus induced mice mammary gland fibrosis,upregulated MMP-1,-3,-13,TIMP-1,uPA and PAI-1 expression,and they were mainly expressed in macrophage,epithelial cells and hyperplasia fibroblasts.