Methionine Protects Against Hyperthermia-induced Cell Injury In Cultured Bovine Mammary Epithelial Cells

The present study was performed to investigate the effects of methionine on mammary epithelial cells after the heat treatment.It measured the cell proliferation,antioxidant activity,apoptosis,the expression levels of related genes(HSF-1,HSP70,Bax and Bcl-2)and the expression levels of protein(HSP70)in response to high temperature and investigated the protective of methionine for the heat stress cows and their breast from the cellular level.This thesis includes two sections.1.Effects of different concentrations of methionine to the bovine mammary epithelial cell after heat treatment in vitro for the proliferation,antioxidant properties.Cells were cultured in DMEM medium with different methionine concentrations(0,30,60 mg/L)for 2d.Put the cell into water bath at 42℃,0.5h then recovery 6h at a temperature of 37℃.Cell viability was detected by MTT assay.The content of malondialdehyde(MDA),nitric oxide(NO),nitric oxide synthase(NOS),lactate dehydrogenase(LDH),the activities of superoxide dismutase(SOD),catalase(CAT)and glutathione peroxidase(GSH-Px)in the cell culture supernatant were determined by a colorimetric method.The result showed that:compared with the 37℃ groups,the content of MDA,NO,NOS,LDH was increased and the cell viability and the activity of SOD,CAT,GSH-Px was decreased in 42℃ groups.Compared with the 0 mg/L methionine groups,cell viability of 60 mg/L methionine groups were increased(P<0.05),the activity of the LDH was reduced(P<0.05),the content of the MDA,NO,NOS was significant reduced especially(P<0.01),CAT levels could be increased when added 30 mg/L,60 mg/L methionine in the medium of the heat treatment of mammary epithelial cells(P<0.05),the activity of SOD(P<0.05)and GSH-Px(P<0.01)in 60mg/L methionine groups was the highest.It suggested that there was a heat stress upon heat treatment of 42℃,cells subjected to the oxidative stress,free radicals of oxygen content increased.Methionine can alleviated the heat stress damage effectively and reduced the oxidative stress.2.Effects of different concentrations of methionine to the bovine mammary epithelial cell after heat treatment in vitro for the apoptosis and related gene and protein expression.Cells were cultured in DMEM medium with different methionine concentrations(0,30,60 mg/L)for 2d.Put the cell into water bath at 42℃,0.5h then recovery 6h at a temperature of 37℃,determination of apoptosis,the expression level of the HSF-1mRNA,HSP70mRNA,Bax/Bcl-2mRNA,HSP70 protein and activity of caspase-3.Apoptotic cells were detected by Annexin VFITC/PI double labeled flow cytometry.The result showed that:compared with the 37℃ groups,when added 30,60 mg/L methionine could significantly reduce late apoptosis/necrosis rate(P<0.05),and a significant difference in total mortality(P<0.01).Bovine mammary epithelial cells also exhibited a certain amount of HSP70 reserve after methionine pretreatment for 24 hours,and the expression level of the HSP70 gene and protein further increased with incubation at 42℃ for 30 min(P<0.01).Compared to the control,the expression of HSF-1 mRNA increased,and there was a significantly reduced expression of Bax/Bcl-2 mRNA and a reduced activity of caspase-3 against heat stress(P<0.01).Thus,methionine has cytoprotective effects on hyperthermia-induced damage in BMECs.