Dahuang Gancao Decoction Suppresses Ferroptosis via SIRT3-Mediated NRF2 Deacetylation in Acute Kidney Injury

Background: Acute kidney injury (AKI) is a leading complication of sepsis, necessitating the exploration of novel treatment modalities. The traditional Chinese medicine Dahuang Gancao Decoction (DHGC) has potential in the treatment of sepsis-associated acute kidney injury (SA-AKI). However, its specific mechanisms require further investigation. Purpose: This study aimed to validate the antiferroptotic effects of DHGC in the treatment of SA-AKI in vivo and in vitro . Methods: C57BL/6 mice and C57BL/6-background sirtuin-3 (SIRT3) -/- mice were induced with Reagents Lipopolysaccharide (LPS), while normal and si-SIRT3 human proximal tubular epithelial (HK-2) cells were induced with erastin to establish a ferroptosis phenotype, followed by DHGC treatment. First, the DHGC components were identified via liquid chromatography-tandem mass spectrometry (LC‒MS/MS), and the potential active components were further screened through comparison with the high-performance liquid chromatography (HPLC) reference standards. Ferroptosis levels and the protective mechanisms of DHGC in vivo and in vitro were evaluated using biochemical assays, transmission electron microscopy, immunohistochemistry, immunofluorescence, and Cell Counting Kit-8 (CCK-8) assays. Key pathways were identified by RNA-seq and validated via enzyme-linked immunosorbent assay (ELISA) and western blotting. Finally, the potential active components were subjected to molecular docking with SIRT3 to select candidate monomers for subsequent in vitro functional validation. Results: LPS was used to induce SA-AKI in mice, and erastin was used to induce ferroptosis in HK-2 cells. DHGC can improve renal function, increase cell viability, and reverse abnormal changes in key ferroptosis indicators. Mechanistically, the expression levels of SIRT3/nuclear factor erythroid-2 related factor 2 (NRF2) pathway-associated proteins were downregulated, and NRF2 nuclear translocation was restricted after modeling; in contrast, DHGC upregulated the expression of SIRT3/NRF2 pathway-related proteins, promoted NRF2 nuclear translocation, and activated downstream glutathione peroxidase 4 (GPX4) expression. Importantly, DHGC failed to improve the above indicators in SIRT3-deficient models. Potential active components of DHGC were screened via HPLC reference standard comparison, and subsequent molecular docking with SIRT3 identified candidate monomers. These monomers exerted ferroptosis-inhibiting effects consistent with those of the parent compound DHGC. Conclusions: The present study offers the first demonstration that DHGC retards SA-AKI progression through stimulating SIRT3-driven deacetylation of NRF2, promoting NRF2 nuclear translocation, and inhibiting ferroptosis. These findings suggest that DHGC is a potential therapeutic candidate for SA-AKI, warranting further clinical investigation.