Effect Of Streptococcus Agalactiae On Expression Of Leptin And Extracellular Matrix In Bovine Mammary Fibroblasts

Streptococcus agalactiae(S.agalactiae)is one of the major pathogens that can cause bovine mastitis,witch in turn leads to fibrosis of mammary gland.The bovine mammary fibroblasts(BMFB)are the major mesenchymal cells in mammary gland.The aim of this study was to study the effect of S.agalactiae on the expression of Leptin and its functional receptor(OB-R b)and extracellular matrix(ECM)in BMFB cultured in vitro,in order to explore the relationship between Leptin and bovine mammary fibrosis caused by S.agalactiae.In this study,different concentrations of heat-inactivated S.agalactiae(OCFU/mL,105CFU/mL,106CFU/mL and 108CFU/mL)were used to culture BMFB in vitro at different time(6h,12h,24h,48h and 72h),and the total RNA and protein were extracted from the cells,for examing the expression of Leptin and its functional receptors and extracellular matrix ?-smooth muscle actin(?-SMA)and type ? collagen(COL ? ?1)with qPCR and Western Blot.The expression of extracellular matrix hyaluronic acid(HA)in supernatant of cell culture was detected by ELISA.The research result indicates as fellow:(1)Different concentrations of S.agalactiae could promote the expression of Leptin and OB-R b mRNA and protein of BMFB in various degrees after different time.the expression of Leptin mRNA and protein were almost the same,and the expression of Leptin mRNA and protein with 105CFU/mL was significantly higher than that of the control group at 6h and 12h(P<0.01),and increase with the increase of the concentration at 24h,48h and 72h.The expression level of OB-R b mRNA and protein was consistent,and increase with the increase of the concentration of S.agalactiae,the expression of mRNA was the highest at 48h with 108 CFU/mL,and the expression of protein was the highest at 12h with 108CFU/mL.(2)Different concentrations of S.agalactiae could promote the expression of mRNA and protein of a-SMA and COL I a 1 in various degrees.The expression of a-SMA mRNA and protein was almost the same,and significantly higher than that of the control group with 105CFU/mL(P<0.01),the expression was the highest at 12 h;the expressionof mRNA and protein of COL I a 1 was inconsistent,and the expression of mRNA decreased first and then increased with the prolongation of time,the expression of mRNA was decreased at 24h and 48h,then increased at 72h,and the expression was the highest at 6h with 108CFU/mL,the expression of protein with 105CFU/mL and 106CFU/mL were significantly higher than those in the control group(P<0.01),and the expression with 108CFU/mL was lower than that of the control group.(3)The expression of HA in the supernatant of the cells was increased in various degrees,and the expression of HA was the highest at 24h with 106CFU/mL after different concentrations of S.agalactiae.In conclusion,heat-inactivated S.agalactiae can promote the expression of Leptin and OB-R b mRNA and protein of BMFB culture in vitro,which is related to the time and the concentration of S.agalactiae.After the action of S.agalactiae on BMFB,the expression of extracellular matrix(a-SMA,COL I a 1 and HA)increased.It's suggested that Leptin have a certain role in bovine mammary fibrosis caused by S.agalactiae.