Effect Of Selenomethionine On The Damage In Bovine Mammary Epithelial Cells Induced By Staphylococcus Aureus

Staphylococcus aureus(S.aureus)is one of the common pathogens in cow mastitis,which leads to chronic mastitis and huge economic losses.Selenium(Se)is one of the essential trace elements in mammals,which plays a role in regulation of the redox system and immune system by selenoproteins.Selenomethionine(SeMet)is often applied on the farms as supplementation.Compared with inorganic Se,SeMet is characteristic of low toxicity and high bioavailability.Therefore,this study aimed to determine the effect of SeMet on the antioxidant capacity of bovine mammary epithelial cells(BMECs),and confirm the regulation of SeMet on damage in BMECs induced by S.aureus,which may provide theoretical basis for prevention and treatment of mastitis.(1)To determine the effect of SeMet on the antioxidant capacity in BMECs,BMECs were incubated with 2,4,and 8 μmol/L SeMet for 24 h.Then,the activities of antioxidant enzymes,reactive oxygen species(ROS),gene expressions of selenoproteins,and Nrf2 signaling pathway were detected,respectively.Results showed that 2,4,and 8 μmol/L SeMet significantly enhanced the activities of glutathione peroxidase(GPx),superoxide dismutase(SOD),and total antioxidant capacity(T-AOC)in BMECs(p<0.01).SeMet at 4 and 8 μmol/L significantly enhanced the catalase(CAT)activity(p<0.01).2,4,and 8 μmol/L SeMet significantly upregulated the gene expressions of GPX1,GPX4,and SELENOP(p<0.01).Additionally,4 and 8μmol/L SeMet upregulated the gene expressions of TRXR1,NRF2,and HO-1 significantly,and downregulated KEAP1 gene expression extremely(p<0.05 or p<0.01).2μmol/L SeMet significantly upregulated NQO-1 gene expression(p<0.01).(2)To determine the effect on BMECs induced by S.aureus,BMECs was cultured with S.aureus(MOI=1:1).Then,the gene expressions of Nrf2 signaling pathway were examinated at 2,4,6,and 8 h after infection.The protein expressions of NF-κB signaling pathway were detected at 0.5,1,2,3,4,5,and 6 h after infection.Additionally,the gene expressions of related inflammatory factors were detected at 0.5,1,2,3,4,6,and 8 h after infection.The results showed that gene expression of NRF2 was significantly increased(p<0.01),and KEAP1 gene expression was significantly decreased(p<0.05 or p<0.01),at 2 and 4 h after infection.At 6 and 8 h after infection,NRF2 gene expression was decreased significantly(p<0.01),and KEAP1 gene expression was increased significantly(p<0.05)at 8 h after infection.Meanwhile,the phosphorylation level of p65 was significantly increased(p<0.05 or p<0.01)at 0.5,1,2,3,4,5,and 6 h after infection,and the phosphorylation level of IκBα was significantly increased(p<0.05 orp<0.01)at 1,2,3,and 4 h after infection.Additionally,at 1,2,3,4,6,and 8 h after infection,the gene expressions of TNF-α,IL-1β,and IL-8 were upregulated significantly(p<0.05 or p<0.01).(3)To investigate the effect of SeMet on the oxidative stress and inflammation caused by S.aureus in BMECs,BMECs were incubated with 4μmol/L SeMet and invaded by S.aureus.The activities of antioxidant enzymes,ROS production,mitochondrial membrane potential(MMP),and the gene expressions of selenoproteins and Nrf2 signaling pathway were detected at 8 h after infection.Simultaneously,immunofluorescence technique was used to observe the nuclear translocation of Nrf2 protein.At 3 h after infection,protein expressions of NF-κB signaling pathway and gene expressions of related inflammatory factors were detected.Results showed that the ROS production was significantly decreased,and MMP was significantly increased in SeMet intervention group(p<0.05),compared with S.aureus group.The gene expressions of NRF2,HO-1,GPX1,GPX4,SELENOP,and TRXR1 were significantly increased(p<0.05 or p<0.01),and KEAP1 gene expression was significantly decreased(p<0.01)in SeMet intervention group,compared with S.aureus group.Compared with the control group,the fluorescence intensity was declined in S.aureus group.And the fluorescence intensity was enhanced in SeMet intervention group,compared with S.aureus group.Finally,the gene expressions of TNF-α,IL-1β,IL-8,and the phosphorylation level of p65 and IκBαwere significantly decreased(p<0.01)in SeMet intervention group.In summary,SeMet enhanced the activities of antioxidant enzymes,and improved the antioxidant capacity of BMECs through regulation of gene expressions of selenoproteins and Nrf2 signaling pathway.When BMECs were invased by S.aureus,the NF-κB signaling pathway was activated.while the Nrf2 signaling pathway was activated and then inhibited.Meanwhile,the intervention of SeMet relieved the inflammation through the NF-κB signaling pathway.SeMet upregulated the gene expressions of selenoproteins and enhanced the activities of antioxidant enzymes in BMECs.Simultaneously,SeMet alleviated the oxidative stress caused by S.aureus in BMECs through Nrf2 signaling pathway.This study provided new ideas for the prevention and treatment of mastitis in dairy cows caused by S.aureus.