Role Of Nrf2/HO-1 And Bax/Bcl-2 Pathways In The Dexamethasone-induced Oxidative Stress Of HiPSC

Objective:Exploring the feasibility of SEF as hi PSC feeder layer,and laying a foundation for the further research on the effects of different feeder layers on the characteristics and stress capacity of stem cells.Investigating the relationship between dexamethasone(DEX)-induced hi PSC oxidative stress and Nrf2/HO-1 and Bax/Bcl-2 pathways,and providing a basis for exploring the antioxidative stress measures of pluripotent stem cells in vivo or in vitro.Method:SEF were isolated from 1-month-old sheep embryo,treated with mitomycin C and used as feeder cells for hi PSC culture in vitro.Then the effects of SEF and SNL feeder layer cells on hi PSC morphology and pluripotency indicators were compared.hi PSCs were treated with different concentrations of dexamethasone(12.5,25,37.5,and 50 ?mol/L)for 48 h and 72 h respectively.The oxidative stress status of hi PSC was analyzed by morphological observation,AP staining and ROS detection,and the apoptosis status of hi PSC was analyzed by TUNEL,mitochondrial membrane potential,and ATP detection.Furthermore,the expression levels of(p-)Nrf2,HO-1,Bax and Bcl-2 were detected by q RT-PCR,immunocytochemistry and Western blotting.Results:During the experimental period,being similar to hi PSCs cultured on the SNL feeder layer,hi PSCs cultured on the SEF feeder layer were able to rapidly proliferate and grow as colonies,appeared blue-purple after AP(a pluripotency indicator)staining,and could normally express c-Myc,Klf4,OCT4,SOX2,TRA-1-60 and SSEA-4 pluripotency factors.The treatment with different concentrations of dexamethasone(12.5,25,37.5,and 50 ?mol/L)for 48 h and 72 h resulted in the different degrees of damage and ROS accumulation in hi PSCs.The ROS level was the highest in hi PSCs at 50 ?mol/L DEX group.The treatment with dexamethasone for 48 h resulted in the significant decrease of mitochondrial membrane potential and ATP level in hi PSC,but no significant change for TUNEL staining.At 48 h after dexamethasone treatment for hi PSC,the expression of HO-1,Nrf2,Bax and Bcl-2 m RNA in most of treatment groups showed the up-regulation trend in different degrees,and there were significant difference at 50 ?mol/L DEX group.The protein expression levels of HO-1,Nrf2,p-Nrf2,Bax,and Bcl-2 in each treatment group showed increase trend in different degrees,and the differences were significant in 50?mol/L DEX group.Conclusion:The SEF feeder layer cells were successfully isolated and prepared,and were preliminarily confirmed as being used to culture hi PSC.The oxidative stress of hi PSCs was induced by dexamethasone treatment,and the involved mechanisms were related to Nrf2/HO-1 and Bax/Bcl-2pathways.