The Protective Effects Of Astragalus Polysaccharides On Bovine Mammary Epithelial Cells Apoptosis Induced By LPS In Vitro

In this study, the bovine mammary epithelial cells cultured in vitro was used as experimental materials, to built the bovine mammary epithelial cell apoptosis model by the Escherichia coli Lipopolysaccharide (LPS). Based on this, the protevtive effect of Astragalus polysaccharides on bovine mammary epithelial cell apoptosis induced by LPS was studied; And discussing the cintent change rules of a number of cytokines and enzymes in inflammation cells proteced by Astragalus polysaccharides.The results should be helpful to provide renferences for the prevention and controling animal mastitis by Chinese herbal medicine like Astragalus polysaccharides as immune physiological regulator.1. The apoptosis model of bovine mammary epithelial cell built in vitroUsing tissue culture method, combined usage of enzyme digestion method and repeated adherent method to establish the bovine mammary epithelial cell culture system in vitro, And meta-pragmatic LPS (0,10,20,50,100 μg/mL) direct effects on the second generation of the bovine mammary epithelial cells, seting up multiple time points (3,8,16and 24 h), respectively the CCK-8 flow cytometry instrument Agarose gel electrophoresis and transmission electron microscopy (sem) method, experimental technologies such as histologic changes in activity of bovine mammary epithelial cells And the change of the biochemical characteristics, in order to appear specificity mark of apoptosis as the guidelines to determine the role of apoptosis induced effect time and concentration by LPS.The test results showed that the 1 00μg/mL LPS effect of 8 h was the ideal criteria for the bovine mammary epithelial cell apoptosis model builing.2. The protective effect of Astragalus polysaccharide on the bovine mammary epithelial cells apoptosis induced by LPSTo study the protective effects of Astragalus polysaccharides on the bovine mammary epithelial cells apoptosis induced by LPS, according to first experiment results mammary epithelial cells in good growth sate were divided into 5 groups:the blank control group,LPS model group Astragalus polysaccharides[1,3 and 5 mg/mL)+LPS model groups.Adopt CCK-8, Flow cytometry, Agarose gel electrophoresis and transmission electron microscopy (sem), experimental technology methods to determine the indicators, The results showed that:compared with the control group, a significant reduction in the cell activity of LPS group, compared with LPS group, Astragalus polysaccharide cell activity of each drug group significantly enhanced, and a dose-response relationship.The Astragalus polysaccharide can significantly block the apoptosis of cells in S phase, thus facilitating the increase of cell DNA synthesis. The results suggested that Astragalus polysaccharide has the role of the protecting bovine mammary eptithelial cells from apoptosis induce by LPS.3. Modulation and mechanism of Astragalus polysaccharide in LPS-induced inflammatory in bovine mammary epithelial cellsIn order to further explore the Astragalus polysaccharide regulates the inflammatory response mechanism of primary cultured bovine mammary epithelial cells, this experiment adopts radioimmunoassay method measures inflammation cells and the change rule of cytokine levels the secretion of TNF-a, IL-1β,IL-6, IL-2;And using ELISA to detect the change rule of all kinds of related enzymes NAGase AKP MPO activity of iNOS by Astragalus polysaccharides protection, The test results showed that 1,3,5 mg/mL of Astragalus polysaccharides will not produce toxic effects to the bovine mammary epithelial cells, Ding Yueyun doctoral thesis,finding 20 mg/mL of Astragalus polysaccharides significantly promote bovine mammary epithelial cells in vitro proliferation is consistent; Astragalus polysaccharides pretreatment cell can obviously decrease content of the TNF-α、IL-1β、IL-6 and the activity of NAGase、AKP、MPO、 iNOSAll these results showed that Astragalus polysaccharide could protect bovine mammary epithelial cells from apoptosis induced by LPS, preliminary confirmed that the mechanism of anti-inflammatory effect of Astragalus polysaccharide is to inhibiting the secretion of various cytokines, reduce the activity of related enzymes, thus reducing the neutrophil infiltration in the mammary, reduce the mammary tissue inflammatory damage.