Esculetin, a natural dihydroxy coumarin derived from the Chinese herbal medicine Cortex Fraxini, has demonstrated significant pharmacological activities, including anticancer properties. Ferroptosis, an iron-dependent form of regulated cell death, has garnered considerable attention due to its lethal effect on tumor cells. However, the exact role of ferroptosis in esculetin-mediated anti-hepatocellular carcinoma (HCC) effects remains poorly understood. This study investigated the impact of esculetin on HCC cells both in vitro and in vivo. The findings indicate that esculetin effectively inhibited the growth of HCC cells. Importantly, esculetin promoted the accumulation of intracellular Fe²⁺, leading to an increase in ROS production through the Fenton reaction. This event subsequently induced lipid peroxidation (LPO) and triggered ferroptosis within the HCC cells. The occurrence of ferroptosis was confirmed by the elevation of malondialdehyde (MDA) levels, the depletion of glutathione peroxidase (GSH-Px) activity, and the disruption of mitochondrial morphology. Notably, the inhibitor of ferroptosis, ferrostatin-1 (Fer-1), attenuated the anti-tumor effect of esculetin in HCC cells. Furthermore, the findings revealed that esculetin inhibited the Nrf2-xCT/GPx4 axis signaling in HCC cells. Overexpression of Nrf2 upregulated the expression of downstream SLC7A11 and GPX4, consequently alleviating esculetin-induced ferroptosis. In conclusion, this study suggests that esculetin exerts an anti-HCC effect by inhibiting the activity of the Nrf2-xCT/GPx4 axis, thereby triggering ferroptosis in HCC cells. These findings may contribute to the potential clinical use of esculetin as a candidate for HCC treatment.
Umbelliferones/administration & dosage* ; Ferroptosis/drug effects* ; Carcinoma, Hepatocellular/physiopathology* ; NF-E2-Related Factor 2/genetics* ; Humans ; Liver Neoplasms/physiopathology* ; Phospholipid Hydroperoxide Glutathione Peroxidase/genetics* ; Animals ; Cell Line, Tumor ; Mice ; Amino Acid Transport System y+/genetics* ; Mice, Inbred BALB C ; Male ; Signal Transduction/drug effects* ; Lipid Peroxidation/drug effects* ; Reactive Oxygen Species/metabolism* ; Mice, Nude

Objective To investigate the effect of Ba Bao Dan(BBD)on cardiac injury induced by doxorubicin in zebrafish.Methods We induced a zebrafish myocardial injury model using the chemotherapeutic drug,doxorubicin.We then examined the effects of different concentrations of BBD on pericardial edema and heart rate under an in vivo microscope.We also examined the inhibitory effects of BBD on neutrophil infiltration in the heart in Tg(mpx:EGFP)transgenic zebrafish.The impacts of BBD on superoxide dismutase,catalase,and malondialdehyde were observed.mRNA expression levels of ferroptosis-related factors,including glutathione peroxidase 4a(gpx4a),prostaglandin-endoperoxide synthase 2(ptgs2),arachidonate 5-lipoxygenase(alox5a),and acyl-CoA synthetase long-chain family member 4(acsl4)were determined by Real-time quantitative polymerase chain reaction.The accumulation of ferrous ions in zebrafish heart was assessed using a fluorescent probe for ferrous ions.Results BBD alleviated doxorubicin-induced pericardial edema and bradycardia in zebrafish,reduced neutrophil infiltration in the heart(P＜0.05),decreased malondialdehyde concentration(P＜0.05),and enhanced the activities of superoxide dismutase and catalase(P＜0.05).BBD also significantly inhibited ferroptosis,reduced the accumulation of ferrous ions in the zebrafish heart,suppressed the expression of ptgs2,alox5a,and acsl4(P＜0.05),and promoted the expression of gpx4a(P＜0.05).Conclusions BBD can attenuate doxorubicin-induced zebrafish myocardial injury and improve cardiac function by inhibiting lipid peroxidation and regulating ferroptosis.