Doxorubicin (DOX), a highly active chemotherapeutic drug, faces limitations in 
clinical application due to severe cardiotoxic effects (mainly through increased 
oxidative stress). Therefore, its effect is exacerbated in subjects with 
ischemic heart disease. We have recently reported that saffron extract (SAF), a 
natural compound mainly consisting of safranal and corcins, exerts a protective 
effect against DOX oxidative cytotoxicity in isolated rabbit hearts. Here, we 
aimed to investigate whether SAF exerts cardioprotection against combined 
ischemia-reperfusion (I/R) and DOX toxicity in H9c2 cardiomyocytes. H9c2 were 
subjected to simulated I/R, with or without DOX treatment at reperfusion, in the 
presence or absence of SAF prior to ischemia or at reperfusion. We evaluated the 
effects of these treatments by MTT, LDH and western blot analysis. Apoptosis was 
assessed by Hoechst 33258 staining, tetramethyl rhodamine methyl ester 
fluorescence and caspase activity. The results showed that I/R and DOX 
significantly decreased cardiomyocytes viability, inhibited reperfusion injury 
salvage kinase cardioprotective pathway, reduced contractile proteins 
(α-Actinine, Troponine C and MLC), increased caspase-3 expression and induced 
loss of mitochondrial membrane potential. These effects were remarkably 
inhibited by treatment with SAF (10 μg/mL) at reperfusion. SAF activated 
AKT/P70S6K and ERK1/2, restored contractile proteins expression, inhibited 
mitochondrial permeability transition pore and decreased caspase-3 activity. In 
conclusion, our findings indicate that SAF treatment exerted cardioprotection 
against I/R and DOX toxicity by reducing oxidative stress (LDH assay). Thereby, 
SAF offers a potential novel antioxidant therapeutic strategy to counteract I/R 
and DOX cardiotoxicity, paving the way for future clinical trials.