The Effect Of Escherichia Coli On TGF-β1, BFGF And PDGF MRNA Expression And Protein Secretion In Bovine Mammary Fibroblast Cultured

Escherichia coli is an important pathogen that causes bovine mastitis, can lead to various degree of mammary fibrosis. During the process of mammary gland fibrosis, the milk yield will decrease, the dairy cow will be eliminated as a result of mammary gland sclerosis in seriously cases.Bovine mammary fibroblasts (BMFBs) are important stromal cells of the mammary gland. Research on E. coli culture bovine mammary fibroblasts several factor expression in vitro has an important meaning of prevention mammary fibrosis.To better understand the effect of heat-inactivated Escherichia coli on the expression of several factors, TLR2, TLR4 and activation of nuclear transcription NF-κB and AP-1 in the bovine mammary fibroblasts (BMFB), BMFBs were stimulated with different concentrations of heat-inactivated E. coli (0 CFU/mL,105 CFU/mL,106 CFU/mL, and 108 CFU/mL) to analyze the mRNA expression of TLR2 and TLR4 with RT-PCR and protein production of TLR2, TLR4, NF-κB and AP-1 with Western-blot at different time points (6 h,12 h,24 h, and 48 h). The mRNA expression and protein production of TGF-β1,bFGF,PDGF-BB, TβRI, FGFR2, and PDGFRp with RT-PCR and Western-blot.The data demonstrated that:(1) the BMFBs stimulated with different concentrations of E. coli also released more TGF-β1, bFGF, PDGF-BB, TβRI, FGFR2, and PDGFRβ mRNA than the controls at each time point, with a time-dependent manner, peaking during stimulation, followed by a gradual decrease. The protein secretion was also in line with the mRNA expression. (2) The data also exhibited that TLR2 and TLR4 mRNA expression and protein secretion in BMFBs stimulated with 105,106, or 108 CFU/mL heat-inactivated E. coli gradually increased were in a dose- and time-dependent manner. The TLR2 mRNA expression and protein production in BMFBs were in a time-dependent manner, and the TLR4 mRNA expression and protein production in BMFBs were in a time-dependent manner. The expression and protein expression of TLR2 was in time and dose-dependent manner, the mRNA and the protein peaked at 48h (p<0.05). The expression and protein expression of TLR4 was in time and dose-dependent manner, the mRNA peaked at 24 h and the protein peaked at 12 h (p<0.05). (3) Treatment of cells with heat-inactivated E. coli resulted in an increase of p-NF-κB-p65 and p-c-jun expression, compared to the control groups. The p-p65 protein expression was in a dose- and time-dependent manner, while p-c-jun secretion was only in a time-dependent manner. The expression of p-NF-icB-p65 was in dose-dependent manner and then dropped after peaking at 12h (p<0.05). The expression of AP-1 was in dose-dependent manner and then dropped after peaking at 24h (p<0.05).Taken together, the results suggested that heat-inactivated Escherichia coli up-regulated the TGF-βi, bFGF, PDGF-BB, TGFβRI, FGFR2, PDGFRβ mRNA and protein expression. The increase in them expression was in time and dose-dependent manner. The results suggested that heat-inactivated Escherichia coli up-regulated the TLR2, TLR4 expression, activated NF-κB, AP-1 and induced the expression of pro-fibrotic cytokines in BMFB.