Activation of the Nrf2/GPX4 Signaling by Pratensein From Trifolium pretense Mitigates Ferroptosis in OGD/R-Insulted H9c2 Cardiomyocytes

Abstract

Background: Pratensein (PTS) is a type of flavonoid that has been identified in various plants, such as Trifolium pretense L., with a considerable cytoprotective effect against exogenous stimuli. However, the biological function of PTS in cardiomyocytes in response to ischemia-reperfusion (I/R) conditions is unclear. Purpose: In our study, we examined the function of PTS in the progression of myocardial infarction (MI). Methods: In this study, we established an oxygen-glucose deprivation/reoxygenation (OGD/R) model in H9c2 cells. The Cell Counting Kit-8 assay was used to assess the viability of H9c2 cells. The TdT-mediated dUTP-biotin nick end labeling and flow cytometry assays confirmed apoptosis of H9c2 cells. Reactive oxygen species (ROS), malondialdehyde (MDA), glutathione (GSH) content, and Fe2+ level were evaluated. Western blotting was used to detect relative protein expression. Results: We firstly found that PTS reduced apoptosis of H9c2 cells in response to OGD/R stimulation. PTS attenuates the increase in ROS and MDA production and the decrease in GSH content caused by OGD/R. The increased Fe2+ level in OGD/R-treated H9c2 cells was also restrained by PTS. For mechanism studies, we found that the decreased expression levels of Nrf2 and GPX4 in OGD/R-treated H9c2 cells were significantly elevated after PTS treatment. Knockdown of Nrf2 in H9c2 cells reversed the protective effect of PTS on ferroptosis in H9c2 cells induced by OGD/R, indicated by reduced cell viability, increased apoptotic cells and oxidation markers, and increased Fe2+ level. Conclusion: Based on these findings, we speculated that PTS may protect H9c2 cells from OGD/R-caused ferroptosis by modulating the Nrf2/GPX4 signaling.