Title : Investigation of bergapten’s pharmacological activity against doxorubicin induced cardiotoxicity: supporting evidence from cellular and animal models
Abstract:
Introduction: Doxorubicin (DOX) is a broadly used anthracycline anticancer drug, but its therapeutic application is limited by cumulative, dose?dependent cardiotoxicity that may culminate in heart failure. Oxidative stress, mitochondrial impairment and activation of apoptotic pathways are well?characterised contributors to DOX?mediated cardiac damage. Bergapten (BeG), a citrus?derived furanocoumarin, exhibits antioxidant and cytoprotective activities in several non?cardiac experimental systems; however, its capacity to counteract DOX?induced cardiotoxicity and the molecular mechanisms involved remain insufficiently defined. We therefore examined whether BeG attenuates DOX?associated cardiac injury by modulating oxidative stress and apoptosis through the NOX4/Nrf2/HO?1 signalling axis.
Methods: We used a combination of in vitro and in vivo approaches to determine the preventive action of BeG against DOX induced cardiotoxicity without compromising the anti-cancer activity of DOX. H9c2 cardiomyoblasts were exposed to DOX in the presence or absence of BeG, and Nrf2 dependence was probed using siRNA?mediated Nrf2 knockdown to determine whether BeG?mediated protection persisted. Cellular endpoints included quantification of reactive oxygen species (ROS), assessment of mitochondrial membrane potential, and evaluation of apoptotic indices. We used multiple cancer cell line to examine the anti-cancer potential of DOX alone and with combination of BeG. In vivo, a rodent model of DOX?induced cardiotoxicity was treated with BeG, and outcomes encompassed antioxidant enzyme activities, expression of NOX4, Nrf2 and HO?1, lipid peroxidation, caspase?3 protein levels, histopathological changes, TUNEL staining, and echocardiographic parameters. Statistical analyses were conducted using suitable parametric or non?parametric tests, with significance defined as p<0.05.
Results: BeG co-treatment markedly attenuated DOX-induced ROS generation, preserved mitochondrial membrane potential, and reduced cardiomyocyte apoptosis in H9c2 cells. Mechanistically, BeG downregulated NOX4 while concurrently upregulating Nrf2 and its downstream target HO-1. siRNA-mediated silencing of Nrf2 abrogated BeG-induced HO-1 upregulation and ROS suppression in DOX-treated H9c2 cells, confirming that Nrf2 is indispensable for BeG-mediated cytoprotection. Notably, in cancer cell models, BeG did not diminish the cytotoxic efficacy of DOX, suggesting that BeG may mitigate DOX-induced cardiotoxicity without compromising its antitumour activity. In vivo, BeG treatment restored cardiac antioxidant enzyme activities, reduced lipid peroxidation, suppressed pro-apoptotic caspase-3 activation, and elevated anti-apoptotic Bcl-2 levels. Histopathological analysis demonstrated attenuation of myocardial structural damage and cardiomyocyte degeneration, accompanied by a significant reduction in TUNEL-positive apoptotic cells. Echocardiographic assessment further revealed improvement in cardiac functional parameters. Cardiac tissue from BeG-treated animals exhibited reduced NOX4 expression, enhanced Nrf2 and HO-1 protein levels, and decreased caspase-3 activation relative to DOX-only controls.
Conclusions: These combined in vitro and in vivo data reveal BeG as a promising cardioprotective agent capable of mitigating DOX-induced cardiac injury. BeG exerts its protective effects by concurrently suppressing oxidative stress and inhibiting apoptotic signalling through precise modulation of the NOX4/Nrf2/HO-1 axis. Importantly, BeG intervention preserved the cytotoxic efficacy of DOX in cancer cell models, indicating that BeG may attenuate DOX-associated systemic toxicity without compromising its antitumour activity. These data support further preclinical and translational evaluation of BeG as a potential adjunctive therapeutic strategy to preserve cardiac function during DOX-based chemotherapy regimens.
