| After heat stress,the p38MAPK pathway has important effects on maintaining the vital life process,because of regulating immune responses.Many researches proved that p38MAPK pathway activated could alleviate the hot-stressed effects on livestock.However,studies on cellular and molecular levels were limited,especially when using bMECs(bovine mammary epithelial cells)after heat stress.In order to explore it,we use a specific inhibitor(SB203580)blocking the p38MAPK pathway in hyperthermia-induced bMECs,analysis the cell viability,apoptosis and oxidative damage,and investigate the protein expressions by using iTRAQ(isobaric tags for relative and absolute quantitation)in the present study.The study falls into two parts and points are as follows:1 The effects on the cell viability,apoptosis and oxidative damage after blocking the p38MAPK pathway in hyperthermia-induced bMECs.Cells in the control group were cultured in DMEM medium in incubator at 37℃.Cells of the experiment group were cultured in DMEM medium with different levels of inhibitor SB203580(0、1、5、10、20、40μmol/L)for 30min at 37℃,were put into incubator at 41℃at 0.5h,and were recoveried 6h at 37℃.Cells viability was detected by MTT assay.Cell apoptosis was investigated by an Annexin V/PI double staining kit with flow cytometry.The activities of SOD,GSH-px and the content of MDA were determinate by SOD,GSH-px and MDA kits.The results showed that comparing with the 37℃ groups,the cell viability was decreased(P<0.05)and the ratio of cell apoptosis was increased(P<0.01)in 41℃ groups.Compared to 0μmol/L at 41℃,adding 5、10μmol/L SB203580 reduced the apoptosis rate(P<0.05)under hyperthermia,and the group of lOpmol/L was lower than the groups of 5μmol/L at the 41℃ groups.Compared with the37℃ groups,the activities of SOD and GSH-px were declined(P<0.01)and the contents of MDA were improved(P<0.05)in 41℃ groups.Under hyperthermia-treated,the contents of MDA(P<0.05)were significantly decreased in 1μmol/L、5 μmol/L、10 μmol/L SB203580 group compared to without inhibitor(P<0.05).However,the activities of SOD and GSH-px were just reverse(P<0.01).In order to make sure the accuracy of this experiment,we used 10μmol/L inhibitor to further study.The hyperthermia induced the cells oxidative damage,and suppressed p38MAPK could remiss the oxidative damage of bMECs.2 The protein expression changes of p38MAPK pathway in hyperthermia-induced bMECs by using iTRAQ.Filtrating different proteins between hyperthermia-induced p38MAPK and inhibited p38MAPK in bovine mammary epithelial cells in level of protein.The control group,cells were cultured in DMEM medium in incubator 37℃.Cells in the experiment group were cultured in DMEM medium with different concentrations of inhibitor SB203580(0、10μmol/L)for 30 min and then were put into incubator at 41℃ at 0.5 h,then recoveried 6h at 37℃.iTRAQ combined with 2D LC-MS/MSwas used to inditify different proteins.By analysing classification of COG,it indicates that proteins rich in general function prediction only,posttranslational modification,protein turnover,chaperopnes and translation,ribosomal structure and biogenesis.Comparing 0 μmol/L SB203580,41℃ group with the control group and comparing 10 μmol/L SB203580,41℃ group with 0 μmol/L SB203580,41℃ group get different protein.The number of significantly different proteins are 34.Comparing 0 μmol/L SB203580,41℃ group with the control group,17 proteins significantly decreased expression while compared 10 μmol/L SB203580,41℃ group with 0 μmol/L SB203580,41℃ those proteins significantly increased expression.compared 0μmol/L SB203580,41℃ group with the control group,protein of the number of 17 significantly increased expression while compared 10 μmol/L SB203580,41℃ group with 0μmol/L SB203580,41 ℃.Those proteins significantly decreased expression.it indicates that those reversion proteins rich in complement and coagulation cascades、leukocyte transendothelial migration、protein digestion and absorption、MAPK signaling pathway. |
