Dapagliflozin activates the RAP1B/NRF2/GPX4 signaling and promotes mitochondrial biogenesis to alleviate vascular endothelial ferroptosis

Abstract

Vascular endothelial ferroptosis is a key mechanism underlying endothelial injury and atherosclerotic plaque formation. Dapagliflozin, an essential medication in the management of heart failure, has been shown to delay atherosclerosis progression. However, the underlying mechanisms remain unclear. This study aimed to elucidate the molecular pathways whereby dapagliflozin inhibits vascular endothelial ferroptosis. We utilized human umbilical vein endothelial cells (HUVECs) to construct a cell model of atherosclerosis combined with ferroptosis. Dapagliflozin significantly decreased the iron and malondialdehyde levels and the release of inflammatory factors in HUVECs treated with oxidized low-density lipoprotein or Erastin but increased the superoxide dismutase activity and the reduced glutathione / oxidized glutathione ratio. Results from transmission electron microscopy indicated that dapagliflozin alleviated the mitochondrial shrinkage and the reduction in the number of cristae in these HUVECs. RNA sequencing revealed that dapagliflozin upregulates RAP1B. In vitro experiments showed that RAP1B upregulates NRF2 and promotes its nuclear translocation, activating the xCT/GPX4 signaling pathway and inhibiting lipid peroxidation. Additionally, dapagliflozin induces mitochondrial biogenesis and enhances oxidative phosphorylation through the RAP1B/NRF2 pathway, reducing iron overload and excessive production of mitochondrial reactive oxygen species, ultimately mitigating ferroptosis. At the animal level, we constructed an atherosclerosis model by using Apoe^−/−; Rap1b^−/− double-knockout mice. Rap1b knockout blocked the inhibitory effects of dapagliflozin on atherosclerotic plaque formation and ferroptosis activation. We confirmed in vivo that dapagliflozin upregulates GPX4 and key factors of mitochondrial biogenesis via RAP1B, promoting oxidative phosphorylation. When mitochondrial oxidative phosphorylation was pharmacologically inhibited, ferroptosis was reactivated, promoting atherosclerotic plaque formation. In conclusion, this study demonstrated that dapagliflozin activates the RAP1B/NRF2/GPX4 signaling pathway and promotes mitochondrial biogenesis, thereby alleviating vascular endothelial ferroptosis.