Role Of Nrf2/HO-1 Signal Pathway In The Oxidative Stress Of Human Induced Pluripotent Stem Cells

Objective:Oxidative stress is a key problem encountered in the cultivation and application of induced pluripotent stem cell(i PSC).Exploring its mechanism has great theoretical and practical meaning in i PSC.At present,there are relatively few studies on human i PSC(hi PSC)oxidative stress and the role of Nrf2/HO-1 signaling pathway wherein is still unclear.Therefore,this experiment aimed to establish the hi PSC oxidative stress model by different concentrations of H₂O₂,detect the antioxidant capacity of hi PSC and study the role of Nrf2/HO-1 signaling pathway in hi PSC oxidative stress,which will provide the basis for preventing and curing the oxidative stress damage.Method:In this experiment,hi PSCs were treated with different concentrations of H₂O₂(100,200,300,400 ?M),and then the degree of oxidative damage was observed and the pluripotency was tested by alkaline phosphatase kit(AP)after 4 h and 24 h respectively.The reactive oxygen species(ROS)level was detected using the superoxide anion fluorescent probe,and the expression levels of(p-)Nrf2 and HO-1proteins and m RNAs were detected by immunocytochemistry,western blotting and q RT-PCR,respectively.Results: The treating with different concentrations of H₂O₂(100,200,300,400 ?M)for 4 h/24 h resulted in different degrees of morphology changes in hi PSC.There are slight changes of morphology and pluripotency in hi PSC at 100 ?M H₂O₂ group,while significant apoptosis and damage in hi PSC at 300 ?M and 400 ?M H₂O₂ groups.H₂O₂ increased ROS and reduced AP in a concentration-dependent manner.The results of immunocytochemistry,western blotting and q RT-PCR showed that the expression levels of Nrf2,HO-1 and p-Nrf2 in H₂O₂ treatment groups increased to different degrees and most of which reached significant levels.The expression levels of Nrf2 m RNA and p-Nrf2 protein in 100 ?M H₂O₂ group were the highest,while the expression levels of HO-1 m RNA and HO-1 protein in 200 ?M H₂O₂ group were the highest.It indicated that lower levels of ROS could stimulate the expression of Nrf2 and further activated the expression of HO-1 to remove excess ROS and maintain the oxidation/antioxidant balance of the hi PSCs.However,under the condition of over high peroxide,the production of too much ROS lead to the damage of hi PSCs and affected the upregulation of Nrf2 expression,thus the higher level of HO-1 needed to be activated by other mechanisms,which was necessary for quickly removing ROS and resisting thedamage.The promoter prediction results showed that the transcription of HO-1 gene was regulated by many other transcriptional factors besides Nrf2.Conclusion: This experiment successfully constructed hi PSC oxidative stress model,which confirmed that different concentrations of H₂O₂ could induce the expression of Nrf2 and HO-1 in hi PSC to counteract the adverse effects of excessive ROS production.However,it was found that the expression of Nrf2 or HO-1 didn't increase in a H₂O₂ concentration-dependent manner and many potential motifs were predicted on the promoter of HO-1 gene,which indicated that the regulation mechanism of hi PSC oxidative stress also involved the interaction of other related pathways.The complicated regulatory network needs to be further explored.