| Bovine mastitis is one of the most prevalent diseases in dairy industry,and it is characterized by a mammary gland inflammatory response caused by metabolic and physiologic changes,traumaor,more frequently,contagious or environment pathogenic microorganisms.A large amount of literatures have confirmed that epithelial cells,macrophages,neutrophils,lymphocytes and other stromal cells constitute the mammary gland defensive system,which participates in cellular immune responses in the process of bovine mastitis.Fibroblasts are the largest number of stromal cells in the mammary glands,but their roles and functions in bovine mastitis and bovine mastitis protracted course of fibrosis is unclear.In this study,we demonstrate that inflammation-associated fibroblasts(INFs)extracted from bovine mammary glands with clinical mastitis had different expression pattern regarding to several extracellular matrix(ECM)proteins,chemokines and cytokines compared to normal fibroblasts(NFs)from dairy cows during lactation to investigate whether stromal fibroblasts participate in the process of bovine mastitis.Then the effects of stromal cell derived factor 1(SDF-1)which was obviously up-regulated in INFs on the cell biology function and mechanism of bovine mammary gland epithelial cells were examined to explore the role fibroblasts in mastitis and in the process of mammary gland fibrosis development.This study may have vital significance on prevention and control of bovine mastitis and fibrosis.First,this study applied a real-time quantitative PCR(qPCR)and protein imprinting technology(Westernblot)to test the expression of ECM protein on gene mRNA and protein levels,such as Collagen-?,Vimentin and matrix metalloproteinase 1(MMP1);Then we detected the expression of cytokines and chemokines on mRNA gene and protein levels by qPCR and enzyme-linked immunosorbent assay(ELISA)respectively,including IL-8,CXCL2 and SDF-1.Through the cell scratch experiment we tested the migration ability of two kinds of fibroblast;We usedthe virulence composition of escherichia coli lipopolysaccharide(LPS)and staphylococcus aureus lipidteichoic acid(LTA)to establish a vitro induced fibroblasts cell model in order to test the expression of ECM related protein,cytokines and chemokines.Through comparing the biological characteristics of INFs and NFs from different fields we confirmed whether fibroblasts participate in the process of bovine mammary gland inflammation.Then,through establishing co-cultured model of fibroblasts and mammary gland epithelial cells in vitro to study the changes of INFs on mammary epithelial cell morphological and the expression of EMT Maker such as ?-SMA and cytokeratin.Additionally,we used a series of molecular biology methods to study the mechanism of SDF-1 which is high expression in INFs on mammary epithelial cells.Finally,we established a mouse model induced by SDF-1 to verify the effect on promoting inflammatory.The results showed the expression and secretion levels of ECM proteins Collagen-1,Vimentin,MMP-1,IL-8,CXCL2 and SDF-1 inflammatory related factor were significantly higher in INFs cell model induced by LPS and LTA compared with NFs,indicating that fibroblasts participate in the process of bovine mammary gland inflammation response and play a specific role in function.The results of co-cultured model in vitro showed that INFs can induce mammary gland epithelial cells to change from the paving stone samples into spindle shape,and promote the expression of EMT marker ?-SMA and cytokeratin,indicating that under the condition of co-culture,INFs can induce epithelial cells to occur EMT.Furthermore,we found the secretion/expression of SDF-1 was higher in cell model induced by LPS and LTA and mastitis tissue than NFs and normal control group.Exogenous SDF-1 activates the NF-?B pathway to promote mammary gland epithelial cells to occur EMT,and induce inflammatory reaction as well as inhibit the epithelial cell proliferation.In addition,SDF-1 was able to induce mastitis and slight fibrosis of mouse mammary gland,which was attenuated by a specific inhibitor of the receptor of SDF-1.Results showed that SDF-1 was secreted by INFs to mediate the mammary gland epithelial EMT and induce inflammatory process.To sum up,our findings indicate that stromal fibroblasts within mammary glands with mastitis contribute to EMT and inflammatory responses of epithelial cells through the secretion of SDF-1.Then the stromal fibroblasts participate in bovine mastitis and fibrosis protracted by bovine mastitis.The findings illuminate the role of stromal fibroblasts on epithelial cells in the inflammatory process from a new view preliminarily,in order to explore the mechanism of mastitis and mammary gland fibrosis. |
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