| There is a big gap in the development of dairy industry between our country and developed countries, especially the milk fat and protein content are lower than the developed countries, such as United States, Canada. Feed resource is the important factor to limit the milk fat and milk protein synthesis. The current report said that in the same energy, straw and alfalfa as roughage respectively can cause significant difference of amino acids in the blood, and we know that amino acids can regulate the secretion of cytokines. Previous research in our lab found that cows feeding straw, TGF-β1 and IFN-γ levels in milk and blood were much higher than feeding alfalfa, TGF-β1 and milk protein rate present negative correlation. Therefore, we try to discuss the relationship between amino acids and high levels TGF-β1 and IFN-γ in blood, further to explore the the effect of TGF-β1 on fibrosis and anti-infection, which contribute to the scientific use of straw and prevented the infection of mammary gland reasonable, improve the breeding benefit. Therefore, we conducted some work as follows:First, tail vein blood was collected from health cows and peripheral blood cells(PBMCs) were separated by using the density gradient centrifugation, then we analysed the effect of amino acids on the secretion of TGF-β1 and IFN-γ in peripheral blood. TGF-β1 positive cells were sorted out by flow cytometry and the cell types which secreation TGF-β1 were identicated by PCR. Results showed that lysine, arginine, glycine, methionine could significantly promote the secretion of TGF-β1(p<0.05). Leucine, arginine and methionine could significantly promote the ecretion of IFN-γ in PBMCs(p<0.05). But other amino acids had no effect(p>0.05). Then we sorted TGF-β1 positive cells(24.98%)in PBMCs after the stimulation of arginine by the flow cytometry. And PCR results showed that the major cell types were monocytes, CD4 T cells and WC1 γδ T cells.Second, TGF-β1 is one of important fibrosis factors, so we try to explore the effects of TGF-β1 on the bovine mammary fibroblasts and the mice mammary gland tissue. The result showed that TGF-β1 through ERK1/2 signalling pathway to promote the expression of extracellular matrix α-SMA and I Collagen in bovine mammary gland(p<0.05), which were inhibited after added PD98059. Further, similar results were obtained in mice mammary gland tissue, TGF-β1 significantly promoted the expression of α-SMA and I Collagen in mammary(p<0.05), and PD98059 could restrain the effect of TGF-β1(p<0.05).Third, in order to determine the role of TGF-β1 with higher levels in mastitis, we studied the effect of TGF-β1 on the infection of Staphylococcus aureus in bovine mammary fibroblasts. The results showed that the adhesion and invasion of Staphylococcus aureus were significantly promoted after treating TGF-β1 12 h, which caused fibroblasts apoptosis(p<0.05).Above all: amino acid was related to high levels of TGF-β1 and IFN-γ closely. The monocytes, CD4 T cells and γδ T cells were involved in the synthesis and secretion of TGF-β1. In addation, with the long-term of high levels of TGF-β1 in blood could promote the occurrence of mammary gland fibrosis and promote the infections of Staphylococcus aureus, which led to the fibroblasts and apoptosis. Our studies provide new insights into the nutrition how causes undesirable changes in mammary gland structure and lay the foundation for the further exploration on mastitis pathogenesis. |
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