Exploration Of The Anti-Oxidant Properties Of Human Embryonic Stem Cells

Purpose:It is not fully elucidated about the cellular anti-oxidant properties of human embryonic stem cells?hESCs?.Here we investigated and compared the different responses against oxidative stress and the underlying mechanisms between human embryonic stem cells?hESCs?and human corneal epithelial cells?HCE?using two different oxidative stress models.Methods:We applied two oxidative stress models:H₂O₂-induced and 4-Hydroxynone al?HNE?-induced to cultured human embryonic stem cells?hESCs?and human corneal epithelial?HCE?cells.We measured the cell viability by cell counting kit-8 assays of ESCs,and human gastric epithelial?GES-1?cells.The production of endogenous ROS in the cells were detected by flow cytometry;the level of—a ROS marker:3-nitrotyrosine?3-NT?was examined by Western Blot.Wethen detected the expression of major oxidase and antioxidant enzymes such as NOX4,SOD,catalase, GSTP,and PRDX1,and the levels of major factors of autophagy,such as Beclinl,Atg5-12,LC3-II/LC3-Iand P62,by real-time quantitative PCR?qRT-PCR?enzyme activity kit and Western Blot were detected by real-time quantitative PCR?qRT-PCR?,enzyme activity kit and Western Blot.We also verified the NOX4 changes induced by H₂O₂ in hESCs using NOX4 agonist—phorbol-12-myristate-13-acetate?TPA?.Results:It was demonstrated that H1 had a strong cell viability under the oxidative stress induced by H₂O₂ and HNE,while the cell viabilities of HCE,HUVECs and GES-1 cells were significantly suppressed under the same stress condition.The production of ROS was not significantly changed in H1 after treated with H₂O₂,while the production of ROS was increased in HCE under the same stress condition.It was shown that the level of 3-NT,an oxidative stress markers in H1 cells was decreased after exposed to H₂O₂,but increased in HCE cells.It was also shown that the gene expression and protein levels of antioxidants:SOD and Catalase kits were up-regulated in H1 and down-regulated in HCE after treatment with H₂O₂.Meanwhile the gene expression and protein levels of antioxidants:GSTP and PRDX1 were up-regulated in H1 but decreased in HCE after treatment of both H₂O₂ and HNE.On the other hand,the levels of autophagy markers,such as Beclinl,Atg5-12 and LC3-?/LC3-I were increased in the H1,and the expression of P62 protein was decreased after exposed to H₂O₂ and HNE for 4 hours,indicating that autophagy in H1 was activated at early stage of oxidative stress,whereas autophagy of HCE cells is not activated or inhibited.Conclusion:Human embryonic stem cells?ESCs?has significant antioxidant properties under oxidative stress induced by H₂O₂ and HNE comparing with human corneal epithelial cells?HCE?.The underlying mechanisms are through up-regulation of the related antioxidant enzymes and activation of autophagy at early stage.The novel evidence suggests that human embryonic stem cells have strong antioxidant capability,and helps to better understand the cellular characteristics,differentiation,and function of stem cells as well as potential clinical application of stem cells.