Integration of network pharmacology and proteomics analysis to identify key target pathways of Ginsenoside Re for myocardial ischemia.

• Re protects cardiomyocytes from oxidative stress with low toxicity. • Re regulates ROS level and reduces OGD- induced cardiomyocyte apoptosis. • Re reduces ischemia cardiac fibrosis, and improves myocardial status in mice. • MAPK, AKT and ferroptosis signaling are verified as key targets for Re against MI. Clinically, various diseases cause myocardial ischemia (MI), which further induces severe cardiac injury and leads to high mortality in patients. Ginsenoside Re , one of the major ginsenosides in ginseng, can regulate the level of oxidative stress in the injured myocardium. Thus, it may attenuate MI injury, but the related mechanism has not been comprehensively studied. This study aimed to investigate the anti-MI effect and comprehensively mechanisms of Ginsenoside Re. Oxygen–glucose deprivation (OGD), oxidative-induced cardiomyocyte injury, and isoproterenol-induced MI mice were used to explore their protective effect of Ginsenoside Re. An integrated approach of network pharmacology, molecular docking, and tandem mass tag proteomics was applied to determine the corresponding common potential targets of Ginsenoside Re against MI, such as target proteins and related pathways. The major anti-MI target proteins and related pathways were validated by immunofluorescence (IF) assay and Western blotting (WB). Ginsenoside Re (1.32–168.93 µM) had low toxicity to normal cardiomyocytes, and increased the survival of oxidative stress-injured (OGD-induced injury or H 2 O 2 -induced injury) cardiomyocytes in this concentration range. It regulated the reactive oxygen species (ROS) level in OGD-injured cardiomyocytes; stabilized the nuclear morphology, mitochondrial membrane potential (MMP), and mitochondrial function; and reduced apoptosis. Meanwhile, Ginsenoside Re (5–20 mg/kg) alleviated cardiac injury in MI mice and maintained cardiac function. Through network pharmacology and proteomics, the relevant mechanisms revealed several key pathways of Ginsenoside Re anti-MI, including inhibition of MAPK pathway protein phosphorylation, downregulation of phosphorylated PDPK1, AKT, and STAT3, and upregulation of TGF-β3, ferroptosis pathway (upregulation of GPX4 and downregulation of phosphorylation level of MDM2) and AMPK pathway (regulating the synthesis of cholesterol in the myocardium by downregulation of HMGCR). The key proteins of these target pathways were validated by IF and/or WB. Ginsenoside Re may target MAPK, AKT, ferroptosis pathways and AMPK pathway to prevent and/or treat MI injury and protect cardiomyocytes from oxidative damage. [Display omitted] [ABSTRACT FROM AUTHOR]