The Role Of Nrf2-ARE Pathways In Oxidative Damage Of Bovine Mammary Epithelial Cells

The present study was divided into three parts to examine the role of Nrf2-ARE pathways in oxidative damage of bovine mammary epithelial cells.The experiment 1 was conducted to established the oxidative stress injury model with the bovine mammary epithelial cell(BMEC)induced by hydrogen peroxide(H2O2),and screen the appropriate dose and time of H2O2.The experiment 2 was conducted to determine the function that Nrf2-ARE pathway protected BMEC against oxidative stress injury in dairy cows by transfection of Nrf2-siRNA interference fragment or over expression plasmid pCMV6-XL5-Nrf2 to downregulate or upregulate expression of Nrf2 in control model and oxidative stress injury model individually.The experiment 3 was established the molecular mechanism of activation obstacles of Nrf2-ARE pathway in the periparturient dairy cow mammary cells induced oxidative stress by the interference test in control model and oxidative stress injury model individually.The results of this study showed as follows:(1)BMEC was damaged by H2O2 under oxidative stress,and reduced the number and viability of BMEC.the reason is that H2O2 increase ROS,MDA,Isoprostane,PC,8-OHdG concent and decrease antioxidant activity in cells,the redundant ROS caused intracellular lipid peroxidation by attacking the breast cells,and protein and DNA of intracellular were also caused oxidative damage by H2O2.(2)the cell vitality damage induced by H2O2 was related with the time and dose of H2O2.The results of effect of H2O2 on BMEC proliferation rate and antioxidant index showed,BMEC was obvious damaged by oxidative stress When the concentration and time of H2O2 was respectively 600mol/L and 6h,which could be used as the optimal conditions to establish the oxidative stress injury model.(3)Nrf2-siRNA3 interference fragment can be used as the optimal transfection fragment because transfection effect of Nrf2-siRNA interference fragment was more than 50%.Nrf2 played an important role in the Nrf2-ARE pathways by upregulating and downregulating Nrf2;Nrf2-ARE antioxidant pathway was one of the major defense mechanism of BMEC against oxidative damage,and plays an important role in protecting BMEC from oxidative damage.HO-1 played an important role in protecting BMEC against oxidative damage by Nrf2-ARE pathways,which as a Nrf2-ARE pathways downstream gene was a main gene for BMEC playing the antioxidant function.(4)Oxidative stress played an important role in mastitis of perinatal dairy cows,the results showed that it could reduce the oxidative damage of BMEC by activating Nrf2 transcription or elevating HO-1 expression level.The Nrf2 transcription activity declined in the xidative stress condition,which showed that Nrf2 had not been activated in the oxidative stress condition.(5)The main reason of Nrf2 induced activation barrier in the oxidative stress condition was that ATM protein kinase expression was abnormal,which reduced the Nrf2 phosphorylation and nuclear,reduced the combination of Nrf2 and ARE,and further reduced the expression of antioxidant genes HO-1,NQO1,GCLC,GCLM of Nrf2-ARE pathway downstream,and eventually resulted the apoptosis or necrosis of BMEC.Taking all these results into consideration,Nrf2-ARE pathway is a major defense mechanism of BMEC against oxidative damage,which could protect the BMEC from oxidative damage.But Nrf2 transcription activity declined in the oxidative stress condition,which resulted in Nrf2 activation barrier.The main reason of Nrf2 induced activation barrier in the oxidative stress condition was that ATM protein kinase expression was abnormal,which reduced the Nrf2 phosphorylation and nuclear,reduced the combination of Nrf2 and ARE,and further reduced the expression of antioxidant genes HO-1,NQO1,GCLC,GCLM of Nrf2-ARE pathway downstream,and eventually resulted the apoptosis or necrosis of BMEC.