Role of NF-κB signaling pathway in HO-induced oxidative stress of hiPSCs.

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 HO-induced developmental damage by network toxicology analysis. Then, the oxidative stress model was established with different concentrations of HO 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 HO 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 HO 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 HO concentration. The expression levels of P65, P50, IκB, SOD2, and FHC mRNA were upregulated in most HO-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 HO. The protein levels of P65, p-P65, P50, p-P50, IκB, p-IκB, SOD2, and FHC were upregulated in most HO-treated groups. However, the upregulation induced by 400 μM HO could be reversed by BAY 11-7082, except for IκB and SOD2. In conclusion, HO 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.