氧化和抗氧化之间的平衡对胚胎的发育至关重要。胚胎干细胞(ESCs)在体内遇到严重的氧化应激,对早期胚胎发育是有害的。诱导多能干细胞(iPSCs)与ESCs非常相似,是替代ESCs进行研究和治疗的重要细胞来源。研究表明,iPSCs具有较好的抗氧化应激能力,但其机制尚不清楚.在这项研究中,通过网络毒理学分析,我们预测NF-κB通路可能参与H2O2诱导的发育损伤。然后,采用不同浓度的H2O2建立氧化应激模型,探讨NF-κB通路在人诱导多能干细胞(hiPSCs)氧化应激中的作用机制。结果表明:随着H2O2浓度的增加,ROS水平逐渐升高,导致hiPSC的损伤程度增加;然而,仅400μMH2O2组MDA含量明显较高;100μM组SOD2和T-AOC等抗氧化指标活性明显上调,随着H2O2浓度的增加,大多数抗氧化指标都有不同程度的下调趋势。P65、P50、IκB的表达水平,SOD2和FHCmRNA在大多数H2O2治疗组中上调,显示剂量依赖性关系。在随后的实验中,IκB-α磷酸化的抑制剂,Bay11-7082,逆转了P65,IκB的上调,和400μMH2O2诱导的FHCmRNA表达。P65,p-P65,P50,p-P50,IκB,p-IκB,在大多数H2O2处理组中,SOD2和FHC上调。然而,除IκB和SOD2外,BAY11-7082可以逆转400μMH2O2诱导的上调。总之,H2O2可以促进NF-κB的表达和磷酸化,从而上调其下游抗氧化基因的表达,以最大程度地减少氧化应激对hiPSCs的损伤。这些结果有助于对iPSCs抗氧化机制的基本理解,并将进一步促进iPSCs的应用。为控制胚胎发育早期遇到的氧化应激提供参考。
The balance between oxidation and antioxidation is crucial for the development of embryo. It is harmful to the early embryonic development if embryonic stem cells (ESCs) encounter the serious oxidative stress in vivo. Induced pluripotent stem cells (iPSCs) are very similar to ESCs and are the important cell source to replace ESCs for research and therapy. Studies show that iPSCs have better resistant ability to oxidative stress, but the involved mechanism remains unclear. In this study, we predicted that the NF-κB pathway might be involved in H2O2-induced developmental damage by network toxicology analysis. Then, the oxidative stress model was established with different concentrations of H2O2 to investigate the mechanism of NF-κB pathway in oxidative stress of human induced pluripotent stem cells (hiPSCs). The results showed as follows: With the increase of H2O2 concentration, the ROS level gradually went up leading to an increasing damage degree of hiPSCs; however, the MDA content was obviously high only in the 400 μM H2O2 group; the activities of some antioxidant indexes such as SOD2 and T-AOC were significantly upregulated in the 100 μM group, while most of antioxidant indexes showed downregulated tendency to different degrees with the increase of H2O2 concentration. The expression levels of P65, P50, IκB, SOD2, and FHC mRNA were upregulated in most H2O2-treated groups, showing a dose-dependent relationship. In subsequent experiments, the inhibitor of IκB-α phosphorylation, Bay11-7082, reversed the upregulation of P65, IκB, and FHC mRNA expression induced by 400 μM H2O2. The protein levels of P65, p-P65, P50, p-P50, IκB, p-IκB, SOD2, and FHC were upregulated in most H2O2-treated groups. However, the upregulation induced by 400 μM H2O2 could be reversed by BAY 11-7082, except for IκB and SOD2. In conclusion, H2O2 could promote the expressions and phosphorylations of NF-κB that could upregulate the expressions of its downstream antioxidant genes to minimize the damage of hiPSCs caused by oxidative stress. These results contribute to a fundamental understanding of the antioxidant mechanism of iPSCs and will further facilitate the application of iPSCs, as well as provide a reference for controlling the oxidative stress encountered in the early development stage of embryo.