Studies On The Effect Of Exogenous TNF-α, IFN-γ And Bovine Milk Containing Five Kinds Of Cytokines In The Bovine Mammary Fibrosis

Bovine mammary fibrosis is an important pathological process which wasmainly caused by bovine mastitis that is a major disease in dairy cow. During theprocess of mammary gland fibrosis, the milk yield will decrease, the dairy cow will beeliminated as a result of mammary gland sclerosis in seriously cases. It’s significantthat study the role of cytokines in the bovine mammary gland fibrosis for preventionand control of bovine mammary gland fibrosis.There were few reports on mechanism of bovine mammary fibrosis. In this study,the bovine mammary epithelial cells and fibroblast were plated in six-well plates andafter overnight incubation at37℃the cells were treated withTNF-α（1,5,10ng/mL）and IFN-γ(5,10,20ng/ml) and incubate the two kinds of cells with cow milkcontaining TNF-α, IFN-γ, TGF-β1, bFGF, PDGF selected by Cytokine immunedetection for12h,24h,48h and72h. Total RNA from each sample was extractedusing Trizol reagent following the manufacturer’s instructions. we detected the effectof TNF-α，IFN-γ and cow milk containing five cytokines to the mRNA expression ofthe three transdifferentiation indicators (CTGF, α-SMA and COL Ⅰα1) in the twokinds of cells by Real-Time quantitative PCR with β-actin as an internal referencegene. We extracted protein in12h，24h，48h and72h, then detected the effect ofdifferent concentrations of TNF-α（1,5,10ng/mL），IFN-γ(5,10,20ng/ml) to the proteinexpression lever of α-SMA by Western blot method. Meanwhile, we incubated thetwo kinds of cells cultured with associated cell supernatant of TNF-α, IFN-γ (1:1,1:2,1:5) in vitro. Then detected the expression of mRNA of CTGF, α-SMA and COL Ⅰα1by Real-Time quantitative PCR, and detected the protein expression of α-SMA byWestern blot in12h,24h,48h and72h.The study indicated that exogenous TNF-α increased the expression of α-SMA, CTGF, COLⅠ α1mRNA in the bovine mammary epithelial cells and fibroblasts, andthe protein expression of α-SMA in bovine mammary fibroblasts, while the proteinexpression of α-SMA wasn’t detected in the bovine mammary epithelial cells.Exogenous IFN-γ inhibited the expression of α-SMA, CTGF and COLⅠ α1mRNA inthe bovine mammary epithelial cells and fibroblasts. Exogenous IFN-γ inhibited theexpression of α-SMA, CTGF and COL Ⅰα1mRNA and the protein expression ofα-SMA in the bovine mammary epithelial cells and fibroblasts. The cow milk selectedcontaining higher levels of TNF-α promoted the expression of α-SMA, CTGF,COL Ⅰα1mRNA in the BMEC and BMFB than the cow milk of low TNF-α. Thecow milk selected containing higher concentration of IFN-γ promoted the expressionof CTGF, COLⅠ α1mRNA in the BMEC than the cow milk of lower IFN-γ. Butα-SMA mRNA expressed lower by cow milk with IFN-γ of higher concentration thanlow in the BMEC. The cow milk selected containing higher concentration of IFN-γdecreased the expression of CTGF, COL Ⅰα1mRNA in earlier stage (12h,24h) andincreased in later stage(48h,72h) in the fibroblasts, but the higher concentrations ofIFN-γ have a inhibited tendency on the expression of α-SMA mRNA in thefibroblasts. The cow milk selected containing higher concentration of TGF-β1promoted the expression of α-SMA, CTGF, COL Ⅰ α1mRNA in the BMEC andBMFB than the cow milk of low TGF-β1. The cow milk selected containing higherconcentration of bFGF promoted the expression of CTGF, COLⅠ α1mRNA in theBMEC and BMFB than the cow milk of low bFGF, but the higher concentrations ofbFGF have a inhibited tendency on the expression of α-SMA mRNA. The cow milkselected containing higher concentration of PDGF-BB mainly plays a positive role onthe expression of CTGF and COL Ⅰα1mRNA in the bovine mammary epithelial cells,but the mRNA expression of α-SMA was increased with lower concentration ofPDGF-BB. The cow milk selected containing higher concentration of PDGF-BBmainly played a positive role on the expression of α-SMA mRNA in the bovinemammary fibroblasts, but the expression of CTGF and COL Ⅰα1mRNA wereincreased with lower concentration of PDGF-BB. The associated cell supernatant ofdifferent concentrations of TNF-α, IFN-γ inhibited the expression of α-SMA, CTGF and COL Ⅰα1mRNA and the protein expression of α-SMA in the bovine mammaryepithelial cells, and showed typical dose and time effect relationship.The associatedcell supernatant of different concentrations of TNF-α, IFN-γ inhibited the expressionof α-SMA, CTGF mRNA and the protein expression of α-SMA in the bovinemammary fibroblasts, but promote the expression of COL Ⅰα1mRNA in the bovinemammary fibroblasts.The results suggested that the two kind of exogenous cytokines and the cow milkcontaining cytokines have some certain effect on the expression of the fibrosisindexes both the bovine mammary epithelial cells and fibroblasts, and laid afoundation for the further study of the mechanisms of bovine mammary fibrosis.