Acacetin alleviates sepsis-induced diaphragmatic dysfunction by inhibiting ferroptosis via the PI3K/AKT/NRF2 pathway

Abstract The mechanism underlying the protective effects of Acacetin (AC) against sepsis-induced diaphragmatic dysfunction (SIDD) remains to be elucidated. AC is present in several medicinal plants and has many pharmacological effects and has been reported to inhibit ferroptosis. This study aimed to investigate the role of AC in SIDD and clarify related mechanisms. In this study, SIDD models were established in vivo via cecal ligation and puncture (CLP) and in vitro using LPS-stimulated C2C12 cells. AC treatment significantly ameliorated diaphragmatic atrophy and ferroptosis in SIDD in vitro and in vivo. The protein expression levels of PI3K/AKT/NRF2 pathway components and atrophy-related markers were assessed by Western blotting, while the mRNA expression of atrophy-related genes was determined by RT-qPCR. Oxidative stress markers, JC-1 staining, C11-BODIPY staining, transmission electron microscopy, immunohistochemistry, immunofluorescence staining, and inflammatory cytokine assays were performed to assess oxidative stress and ferroptosis-related indicators, thereby verifying the protective role of AC in SIDD. Network pharmacology analysis predicted that AC may protect against SIDD by regulating the PI3K/AKT/NRF2 signaling pathway. Mechanistically, AC treatment restored the reduction of NRF2 expression and nuclear translocation in SIDD, as well as, the levels of downstream SLC7A11, HO-1 and GPX4. Notably, knockdown of NRF2 or administration of LY294002 in vitro, as well as ML385 treatment in vivo, abolished the protective effects of AC against SIDD. Collectively, these findings indicate that ferroptosis and oxidative stress are pivotal in SIDD, and AC was demonstrated to potentially alleviate SIDD by regulating the PI3K/AKT/NRF2 pathway.