The Effect Of Selenium Against Oxidative Damage And Apoptosisof Primary Bovine Mammary Epithelial Cells Induced By Staphylococcus Aureus

Bovine mastitis is one of the important diseases after delivery,its main pathological feature is that pathogenic bacterium invade and survive by escaping from the immune system ofhost in the mammary gland,thus further accumulating immune cells and active oxygen free radicals(ROS)and releasing inflammatory factors.Each year this disease causeshuge economic losses for the development of the dairy industry.The essential trace element selenium(Se),in the form of selenoproteins,plays an important physiological role in anti-oxidation,anti-inflammation,immune regulation and metabolism.Previous studies in our laboratory found that selenium can increase the immunity of rats during breast feeding,improve the activity of antioxidant enzymes,inhibit inflammation and reduce inflammation injury.Therefore,in the current experiment,the anti-oxidation effect of se and the relationship between se and apoptosis signaling pathway was studied to provide the experimental date in the prevention and treatment of bovine mastitis.According to MOI=1:1,bovine mammary epithelial cells(BMECs)were infected with S.aureus for 0,1,2,4,6,8,10,12 and 24h.The cell vitality was measured by CCK8 assay.The degree of cell injury was measured by LDH test kit.The cell ROS level was measured by flow cytometry.The result showed that as time goes by,the cell vitality was significantly declined after S.aureus infection of BMECs.After infected 6,8 and I Oh,the cell vitality was only 80%?78.3%? 77.3%respectively;but the activity of LDH increased gradually,and after 6h,the activity increased significantly(p<0.01 or p<0.001);Flow cytometry results showed,since 4h,the ROS level of BMECs significantly increased(p<0.05),at 8h,ROS level peaked,after 8h,ROS level significantly decreased(p<0.05).Therefore,the oxidative stress of cells was induced by S.aureus invading at 6,8 and 10h at MOI = 1:1,and the processing time of oxidative stress model was determined.To explore the effect of se on oxidant stress factors,the cells were treated with se(2,4 and 8?M)for different time periods.The effects of se on SOD,CAT,GSH-PX,COX-2 and iNOS mRNA expression,the activity of T-AOC,SOD,CAT and GSH-PX and MDA level were tested.After S.aureus infection,the mRNA expression of SOD,CAT and GSH-Pxhad no significantly changes.After 6h infection,4 and 8?M of se could significantly increase the mRNA expression of SOD(p<0.05 or p<0.001),2 and 4?M of se could significantly increase the mRNA expression of CAT and GSH-Px(p<0.05 or p<0.001);After 8h infection,8?M of se could significantly increase the mRNA expression of SOD and CAT,2 and 8?M of se could significantly increase the mRNA expression of GSH-PX(p<0.01 or p<0.001).After S.aureus infection,the mRNA expression of COX-2 and iNOS was significantly increased(p<0.001).Se could significantly inhibit the mRNA expression of COX-2 and iNOS 6h after infection(p<0.001);After 8h infection,4?M of se could inhibit the mRNA expression of COX-2(p<0.001),se also could significantly inhibit the mRNA expression of iNOS(p<0.001);After 10h infection,2 and 8?M of se could significantly inhibit the mRNA expression of COX-2(p<0.001),4 and 8?M of se could significantly inhibit the expression of iNOS mRNA(p<0.001).After S.aureus infection,the activity of T-AOC,T-SOD,CAT was inhibited or significantly inhibited(p<0.05 or p<0.01),the MDA level was significantly increased(p<0.001).Se could significantly increase the activity of T-AOC,T-SOD,CAT and GSH-Px(p<0.01 or p<0.001)and significantly reduce MDA level(p<0.001).In addition to could causing oxidative stress,resulting in inflammatory response,S.aureus could also lead to apoptosis-related factors induced cell death,resulting in the incomplete tissue structure leading to as well as injury body.After S.aureus infection,TLR2 receptor adjusts the inflammatory signaling pathways,but also the apoptosis-related protein.To explore the regulatory effect of se on apoptosis signaling pathway,the key protein of PI3K/AKT/Bcl-2 was detected by Western Blot.The cells were randomly divided into 5 groups,the experimental groups were treated with three varying concentrations(2,4 and 8?M)of se for 12h before infection with S.aureus for 0.5h.While the positive control group was infected with S.aureus,the control group was left untreated.Then,the activation of TLR2,and AKT,and P-AKT,and Bcl-2,and Bax and cleaved Caspase-3 were measured.The result showed that,after stimulation with S.aureus for 0.5h,compared with the control group the expression of TLR2 and the phosphorylation of AKT were significantly increased.The ratio of Bax/Bcl-2 was significantlyincreased(p<0.001),the cleavage of Caspase-3 was significantly increased(p<0.001)to initiate the programmed cell death.Compared with positive control group,8?M of se could inhibit the expression of TLR2(p<0.05),and also could significantly increase the phosphorylation of AKT(p<0.01).2,4 and 8?M of se could significantly inhibite the ratio of Bax/Bcl-2(p<0.001)and finally inhibiting the cleavage of Caspase-3 to initiate programmed cell death,which protecte cells.In conclusion,se could play an role in antioxidation,cell protection and antibiosis by improving the activity of antioxidant enzyme and inhibiting apoptosis program.Our tests provided basic data and new direction for prevention of bovine mastitis.We wished that our results would provide theoretical guidance andhelp for the application of se in veterinary clinical and the development of protective drugs for S.aureus-induced mastitis.