The Effect Of Escherichia Coli On Expression Of Leptin And Extracellular Matrix In Bovine Mammary Fibroblasts

E.coli is one of the main pathogens that induces mastitis in the bovine.The cows infected with the bacteria may first develop mastitis and eventually develop mammary fibrosis.Bovine mammary fibroblasts(BMFBs)are the major interstitial cells of the breast tissue.When the breast tissue is damaged,fibroblasts are transformed into myofibroblasts,which secrete a large amount of extracellular matrix(ECM).Excessive deposition of ECM may lead to fibrosis.The purpose of this study was to investigate the effect of E.coli on the expression of leptin(LP)and its receptor(OB-Rb)and ECM in BMFBs,in order to explore the relationship of E.coli with LP in bovine mammary fibrosis.The effect of ECM expression was explored by E.coli causing the relationship between LP in the bovine mammary fibrosis.In this study,BMFBs cultured in vitro were used as the research object.After 6 h,12 h,24 h and 48 h,the serum-free medium and different concentrations of 105CFU/mL,106CFU/mL and 108CFU/mL inactivate E.coli were used.qPCR and Western-blot methods were used to detect mRNAs of Lp,OB-Rb,type I collagen(COLIα1)and a-smooth muscle actin(a-SMA)in BMFBs.The expression of protein was analyzed,and the expression of extracellular matrix hyaluronic acid(HA)in cell culture supernatant was detected by ELISA.The results as follow:(1)Different concentrations of heat-inactivated E.coli can significantly promote the expression of LP and OB-Rb mRNA and protein in different degrees after different time.The expression of LP mRNA and protein was basically consistent at 12 h and 24 h.The 106CFU/mL treatment group was significantly higher than the control group(P<0.01),and reached the peak at 72 h(P<0.01).The expression of OB-Rb mRNA and protein was basically the same.With the increase of concentration,the expression of mRNA was the highest at 72 h with 108 CFU/mL,and the expression of protein at 48 h with 108 CFU/mL.(2)Different concentrations of heat-inactivated E.coli on BMFBs can promote the mRNA and protein expression of a-SMA and COLIα1 in different extents.The expression of a-SMA mRNA and protein was inconsistent.The concentration of 106 CFU/mL and 108 CFU/mL was significantly higher than the control group(P<0.01),and the expression of a-SMA mRNA was the highest at 12 h with 108 CFU/mL;The expression of a-SMA protein in treatment group was highest at 48 h with 106 CFU/mL;the expression of COLIα1 mRNA decreased first and then increased with time,the expression decreased at 12 h and 48 h,then increased at 72 h,and the highest expression at 6 h with 106CFU/mL.The protein expression of treatment was significantly higher than the control group at 12 h,48 h and 72 h with 106CFU/mL and 108CFU/mL(P<0.01).(3)After different concentrations of heat-inactivated E.coli in BMFBs,the expression of HA in cell culture supernatant was higher than the control group,except for 48 h,the other time points 106CFU/mL treatment group were significantly higher than the control group(P<0.01),reached the highest at 12h(P<0.01).(4)Correlation analysis showed that the correlation analysis between LP and OB-Rb mRNA showed that the two factors were significantly correlated at 12 h(P<0.05),and the correlation analysis of protein levels showed that the two factors were significantly correlated at 48h(P<0.01).The expression of LP and COLIα1 mRNA was significantly correlated at 6h(P<0.05),and the protein was significantly correlated at 24 h and 48 h(P<0.01).The correlation between LP and a-SMA mRNA was significant at 12 h and 72 h(P<0.01).OB-Rb and COLIα1 mRNA were significantly correlated at 24 h and 72 h(P<0.01),and there was a significant correlation between protein at 6h(P<0.05).The correlation between OB-Rb and a-SMA mRNA was significantly correlated at 6h(P<0.05),and the correlation was significant at 12 h and 48 h(P<0.01).The correlation between OB-Rb and a-SMA protein at 72 h was extremely significant(P<0.01).LP was significantly associated with HA at 12 h and 24 h(P<0.05).In summary,the different concentrations of heat-inactivated E.coli promote the expression of LP and OB-Rb and extracellular matrices(a-SMA,COLIα1 and HA)to culture BMFBs in vitro.It is suggested that LP has a certain promoting effect on E.coli-induced bovine mammary fibrosis.