| Staphylococcus aureus is a common Gram-positive pathogen that causes bovine mastitis, a persistent infection of the bovine mammary gland and leads to fibrosis of the mammary gland, mastoscirrhus, and even culling of the animal. Ligand from S. aureus with its receptor TLRs induced cytokine secretion by target cells in a myeloid differentiation factor-88 (MyD88) and TIR domain-containing adaptor protein (TIRAP) signaling pathway-dependent manner. Upon ligand binding, the activation of several signaling molecules leads to the release of transcription factors, such as activator protein-1 (AP-1) and nuclear factor-kappa B (NF-κB), which plays crucial roles in immune defense, cell differentiation, and apoptosis.To better understand the aetiopathogenesis of fibrosis during S. aureus-associated mastitis, BMECs and BMFBs were stimulated with different concentrations of heat-inactivated S. aureus (0 CFU/mL,105 CFU/mL,106 CFU/mL, and 108 CFU/mL) to analyze the expression and production of toll-like receptor 2 (TLR2), toll-like receptor 4 (TLR4), activator protein-1 (AP-1), nuclear factor-kappa B (NF-κB), transforming growth factor beta-1 (TGF-β1), plate-derived growth factor-BB (PDGF-BB) and basic fibroblast growth factor (bFGF) with qPCR and Western-blot at different time points (6 h,12 h,24 h, and 48 h). Specific NF-κB and AP-1 inhibitors were also used to investigate their effects on the regulation of TGF-β1, bFGF, and PDGF-BB expression.The data indicated that the BMECs stimulated with different concentrations of S. aureus released more TGF-β1, bFGF, PDGF-BB, TβRI, FGFR2, and PDGFRβ mRNA than the controls at each time point, with expression time-dependent manner. Protein secretion increased quickly, but stimulation decreased steadily at later period. And the BMFBs stimulated with different concentrations of S. aureus also released more TGF-β1, bFGF, PDGF-BB, TβRI, FGFR2, and PDGFRP 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. The data also exhibited that TLR2 and TLR4 mRNA expression and protein secretion in BMECs stimulated with 105, 106, or 108 CFU/mL heat-inactivated S. aureus 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 was in a time-deendent manner, however, the protein secretion was in a negative time dependent manner. Treatment of BMECs and BMFBs with heat-inactivated S. aureus resulted in an increase of p-NF-κB-p65 and p-c-jun expression, compared to the controls. The p-p65 protein pruduction was in a dose-and time-dependent manner, while p-c-jun secretion was only in a time-dependent manner. Having demonstrated that the greatest induction of TGF-β1, bFGF, and PDGF-BB was observed in cells induced with 105 CFU/mL S. aureus, the pathway by which the signal was transduced to the nucleus was investigated further. Whether the AP-1 and NF-κB pathways are essential in the S. aureus-induced upregulation of TGF-β1, bFGF, and PDGF-BB expression was tested using the AP-1 inhibitor Sp-600125 and the NF-κB inhibitor NAC. TGF-β1, bFGF, and PDGF-BB mRNA expression and protein secretion were significantly reduced when S. aureus-induced BMECs and BMFBs were treated with these inhibitors, compared to untreated cells or S. aureus-induced BMECs and BMFBs not treated with an inhibitor.The results demonstrated that S. aureus increaed TGF-β1, bFGF, PDGF-BB, TβRI, FGFR2, PDGFRβ, TLR2 and TLR4 mRNA expression and secretion, and could also upregulate p-NF-KB and p-AP-1 production. The increase in TGF-β1, bFGF and PDGF-BB expression was shown significant correlation to TLRs AP-1-and NF-κB-signaling pathway. Taken together, this study preliminary inicated the mechanism of the fibrosis induced by S. aureus-associated mastitis. |
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