Qishen granule alleviates doxorubicin-induced cardiotoxicity by suppressing ferroptosis via nuclear erythroid factor 2-related factor 2 (Nrf2) pathway

Ethnopharmacological relevance: The clinical usage of doxorubicin (DOX) is greatly constrained because of its side effects, especially cardiotoxicity. Studies have suggested that ferroptosis of cardiomyocytes is one of the important causes of doxorubicin-induced cardiotoxicity (DIC). Up-regulating Nuclear erythroid factor 2-related factor 2 (Nrf2) is a potential way to prevent ferroptosis associated with DIC. Qishen granules (QSG) has been shown cardioprotective effects on various cardiovascular diseases, including DIC. However, the mechanism of QSG to prevent and treat DIC are not fully understood. Aim of the study: The main purpose of this work is to probe whether QSG can mitigate DIC by inhibiting ferroptosis, and whether QSG suppresses ferroptosis via Nrf2 pathway. Materials and methods: The effects of QSG on mitigating DIC and the potential targets of QSG were investigated in a DIC mice model. The cardiac function of mice was monitored by echocardiography. Transmission electron microscopy was used to assess mitochondrial damage. ROS content was measured by dihydroethidium (DHE) staining. The glutathione (GSH) and malondialdehyde (MDA) levels in cardiac tissue were detected by kits to evaluate cellular oxidative stress. The accumulation and nuclear translocation of Nrf2 was detected by immunofluorescence. Ferroptosis analysis was determined by tissue iron content and key proteins in Nrf2 pathway were measured by western blotting. The anti-oxidant and anti-ferroptosis mechanisms of QSG were explored in vitro studies. Delivery of Nrf2 small interfering RNA (siRNA) to H9c2 cells aimed to investigate whether QSG could prevent DIC through Nrf2 signaling pathway. The protective effects of QSG on mito-chondrial function and free iron were measured by MitoSOXTM Red and FerroOrange staining assays, respectively. Results: In vivo, QSG could improve heart function and suppress ferroptosis in DIC mice. DOX-induced ROS production decreased after QSG treatment. The accumulation of free iron and MDA induced by DOX was suppressed with QSG treatment. The level of GSH increased after QSG intervention. QSG also protected against DOXinduced mitochondrial structural damage. Meanwhile, QSG promoted the expression of Nrf2 pathway-related proteins, thereby resisting ferroptosis. In vitro, , QSG exerted anti-oxidant and anti-ferroptosis effects. QSG promoted the nuclear-translocation of Nrf2. In addition, interference with Nrf2 attenuated the regulatory effect of QSG on free iron content and mitochondrial ROS production. Moreover, Nrf2 knockdown weakened the antiferroptosis effects of QSG and inhibited the expressions of key proteins in Nrf2 pathway. Conclusion: The results of this study first illustrated that QSG could alleviate DIC by suppressing ferroptosis via Nrf2 pathway. Nrf2 may be a potential key target for QSG to prevent and treat DIC.