Study On The Mechanism Of Vitamin E Alleviating Oxidative Stress Damage Induced By H₂O₂ In MAC-T Cells

This thesis aims to use cell culture,proteomics and other technologies to reveal the mechanism of vitamin E alleviating the oxidative stress damage of dairy cow mammary epithelial cells at the molecular level.It is divided into the following four experiments.Test 1:Use H₂O₂ to treat cow mammary cells,screen out the appropriate concentration and time of H₂O₂ treatment,and construct an oxidative stress damage model.The experiment set 10 treatment concentrations（0,200,300,400,500,600,700,800,900 and 1000μM）and 5 treatment times（0,2,4,6 and 8h）.The first choice is to use the MTT method to measure the cell survival rate,and then use the colorimetric method to measure the superoxide dismutase（SOD）and catalase（CAT）activities,malondialdehyde（MDA）and reactive oxygen species（ROS）content in the cell culture medium.The results showed that treatment of cells with 600μmol/L H₂O₂for 6 hours would cause cell oxidative damage,which can be used as a suitable condition for constructing an oxidative stress model.Test 2:Based on the establishment of the oxidative damage model of dairy cow mammary epithelial cells,different concentrations of vitamin E were added,and the appropriate dosage of vitamin E was screened according to the changes in cell survival rate and antioxidant indexes.The experiment set 4 vitamin E treatment concentrations（0,10,20,and 40μM）and 5 treatment times（0,2,4,6 and 8h）,and preliminarily screened them through the MTT method and the determination of antioxidant indexes The appropriate concentration of vitamin E（20μM）and treatment time（6h）.Test 3:The effect of vitamin E on H₂O₂-induced membrane permeability of cow mammary epithelial cells and the expression of tight junction-related genes m RNA.The test was divided into control group,H₂O₂ group（600μM H₂O₂,6h treatment time）,H₂O₂+VE group（20μM VE,6h treatment time）.The transepithelial resistance（TEER）and culture supernatant of the cells were measured.The alkaline phosphatase（ALP）activity and the expression of tight junction genes ZO-1,occludin and claudin-1 were used to evaluate the effect of vitamin E on cell membrane permeability.The results showed that compared with the control group,the ALP activity in the cell culture medium of the H₂O₂ group increased significantly（P<0.01）,and the TEER value and the relative expression of tight junction genes were significantly reduced（P<0.01）;compared with the H₂O₂ group Compared with the H₂O₂+VE group,the ALP activity in the cell culture medium was significantly reduced（P<0.05）,and the TEER value and the relative expression of tight junction genes were significantly increased（P<0.01）.This shows that H₂O₂ extremely significantly inhibits the expression of tight junction genes and increases the permeability of the cow’s mammary gland epithelial membrane,while vitamin E significantly alleviates the effect of H₂O₂ on cells.Test 4:The effect of vitamin E on the protein expression of dairy cow mammary epithelial cells under oxidative stress.The cell samples of the control group,H₂O₂group and H₂O₂+VE group were analyzed by TMT-labeled proteomics.The protein difference analysis between the H₂O₂/Control group and H₂O₂-VE/H₂O₂ group of breast epithelial cells was carried out.A total of 44243 peptides and 6,696 proteins were identified.Through relative quantification and significance analysis,a total of793 differential proteins were identified,of which The H₂O₂/Control group up-regulated 479 differential proteins and down-regulated 77 differential proteins,and the H₂O₂-VE/H₂O₂ group up-regulated 60 differential proteins and down-regulated176 differential proteins.KEGG annotation and enrichment analysis found that most of the proteins are mainly related to the immune system and 8 signaling pathways such as signal transduction.In summary,20μM vitamin E treatment for 6h can not only effectively alleviate the oxidative damage of dairy cow mammary epithelial cells and the increase in cell membrane permeability induced by H₂O₂,but also can effectively alleviate the inhibition of H₂O₂ on the expression of tight junction-related genes to maintain dairy cows.The mechanical barrier function of breast epithelial cells.