Targeting DDAH II with Puerarin ameliorates doxorubicin-induced endothelial damage and ferroptosis through ADMA/NOS dysregulation rescue.

INTRODUCTION: The ADMA/DDAHII/eNOS/NO pathway plays a pivotal role in maintaining the vascular endothelial structure and functional integrity, which might become a target attacked doxorubicin (Dox)-induced endotheliotoxicity (DIE). Puerarin (Pue) derived from an active monomer in traditional Chinese medicine has many bioactivities. However, whether it can protect the vascular endothelium against DIE injury and the underlying mechanism remain unclear. OBJECTIVES: We explored that the effects and mechanism of Pue protect the vascular endothelium against DIE injury. METHODS: First, conducting a bioinformatics analysis integrated with network pharmacology focused on Pue protection, potential genes, and related pathways involved in DIE damage. Then, using differential proteomics, molecular docking, molecular dynamics simulation, microscale thermophoresis, cellular thermal shift assay, and drug affinity responsive target stability analysis explored the interaction between Pue and DDAHII. Next, DIE and Pue treatment (Pue-pre) models are established using mice and human umbilical vein endothelial cells (HUVECs). Through a variety of cellular, molecular, enzymatic, functional and morphological indicators as well as related tool drugs, explored that Pue-Pre protects vascular endothelial cells and ameliorates ferroptosis induced by DIE damage and its mechanism. RESULTS: Pue could directly target binding to DDAH II and increase its expression and bioactivity, p-eNOS/eNOS and NO levels, decrease ADMA accumulation, activate adaptive autophagy, and inhibit ROS overgeneration, Fe2+ content, and abnormal lipid metabolism, which could improve mitochondrial function, and end the vicious cycle against DIE damage, whereas, pAD/DDAHⅡ-shRNA, erastin (a ferroptosis inducer), and 3-methyladenine (an autophagic inhibitor) could reverse the effects. CONCLUSIONS: Ferroptosis, apoptosis, and autophagy are involved in DIE damage. Pue could bind to DDAH II, regulate the ADMA/eNOS/NO pathway, activate adaptive autophagy, and protect the vascular endothelium against DIE-induced apoptosis and ferroptosis. Pue provides multiple protective effects against DIE damage.