Neohesperidin Alleviates Ferroptosis in Doxorubicin-Induced Cardiotoxicity via Mettl3-m⁶A-Nrf2 Signaling

Doxorubicin (DOX) has antitumor efficacy, but its clinical application is restricted by multiple side effects, especially cardiotoxicity. Neohesperidin, a flavonoid in citrus fruits, has various health benefits. The aim of this study was to explore the cardioprotective effects and mechanisms of neohesperidin against DOX-induced cardiotoxicity (DIC). The study used DOX-treated mice to evaluate cardiac function and cardiomyocyte injury. In addition, primary cardiomyocytes treated with DOX were used to measure cell viability and death. FIN56, a ferroptosis inhibitor, was used to test the relationship between neohesperidin's beneficial effect and ferroptosis. Mechanistic investigations were carried out to explore the role of N6-methyladenosine (m⁶A) levels, nuclear factor erythroid-2-related factor 2 (Nrf2) expression, and Methyltransferase-like 3 (Mettl3) in the cardioprotective effect of neohesperidin. Overexpression and knockdown of Mettl3 were also performed to confirm its role in the cardioprotective effect. Neohesperidin attenuated cardiac function and cardiomyocyte injury in DOX-treated mice. It alleviated DOX-induced oxidative stress by reducing reactive oxygen species and malondialdehyde levels and increasing superoxide dismutase and glutathione levels, restoring ATP production. In DOX-treated primary cardiomyocytes, neohesperidin increased cell viability and decreased cell death. FIN56 abolished the beneficial effect of neohesperidin. Mechanistically, neohesperidin decreased m⁶A levels, leading to increased Nrf2 expression. Nrf2 activation was related to the inhibition of Mettl3, an m⁶A "writer," which protected cardiomyocytes against DOX-induced ferroptosis. Overexpressing Mettl3 reversed the cardioprotective effects of neohesperidin and diminished its benefits on ferroptosis. These findings suggest that neohesperidin exerts a cardioprotective effect against DIC by inhibiting ferroptosis. This effect is achieved by regulating the Mettl3-m⁶A-Nrf2 signaling pathway, indicating the therapeutic potential of neohesperidin.