The main active components of Prunella vulgaris L. alleviate myocardial ischemia-reperfusion injury by inhibiting oxidative stress and ferroptosis via the NRF2/GPX4 pathway

Abstract

Ethnopharmacological relevance: Prunella vulgaris L. (PV) is a widely distributed medicinal and edible plant used in traditional Chinese medicine for its anti-tumor, anti-inflammatory, anti-oxidant, hypoglycemic, and anti-hypertensive effects. Despite the numerous studies reporting on its cardiovascular protective effects, it is still unknown whether PV could relieve myocardial ischemia-reperfusion (MI/R) injury.

Aim of the study: To investigate the effects of PV on MI/R injury and explore the underlying mechanism of action.

Materials and methods: Sprague-Dawley rats were orally administrated with the aqueous extract of P. vulgaris for 7 days before MI/R injury was induced. Echocardiography, infarct staining, and TUNEL assay were used to evaluate the protective effect of P. vulgaris. H₂O₂- and RSL3-stimulated H9C2 rat myocardial cells were used to explore the underlying mechanism. Ultra-high-performance liquid chromatography/mass spectrometer analysis was used to identify the chemical constituents of P. vulgaris. AutoDock software was used to predict the binding affinity and the interactions between the main active compounds and Keap1. Nuclear factor erythroid 2-related factor 2 (Nrf2) knock-out mice were used to confirm whether the protective effect of P. vulgaris was mediated by Nrf2.

Results: P. vulgaris improved left ventricular systolic function and decreased the myocardial infarct area, which alleviated the MI/R injury. PV also increased the level of Nrf2 proteins and promoted the expression of HO-1, SOD, and GSH, thus upregulating the activity of the antioxidant system. The molecular docking simulations indicated that rosmarinic acid, salviaflaside, ursolic acid, and protocatechuic acid from P. vulgaris could strongly bind to Keap1 protein with good binding affinities. Additionally, ursolic acid was found to elevate NRF2 protein levels and promote NRF2 nuclear translocation. Moreover, the cardiac protective effect of PV or ursolic acid disappeared in NRF2-/- mice, indicating that this protective effect was mediated by NRF2. Also, PV increased the protein levels of GPX4 in MI/R rat or mice models, and this upregulation disappeared in NRF2-/- mice. Results from the RSL-3-induced ferroptosis H9C2 cell model showed that ursolic acid was the main active component of PV that protects cardiomyocytes against ferroptosis.

Conclusions: Collectively, the findings indicate that PV could alleviate MI/R injury by inhibiting oxidative stress and ferroptosis via the NRF2/GPX4 pathway, and ursolic acid is the main active component responsible for mediating both antioxidative and anti-ferroptosis effects, suggesting its potential use as a therapeutic agent against MI/R injury.

Keywords: Ferroptosis; GPX4; Myocardial ischemia-reperfusion injury; NRF2; Prunella vulgaris.