Feed

Diabetic liver injury (DLI), a common complication of diabetes mellitus, is characterized by metabolic disturbances that induce hepatic dysfunction and histopathological alterations such as steatosis, inflammation, and fibrosis. These alterations may progress to cirrhosis with limited effective therapies. Total flavones from Abelmoschus manihot (L.) Medik. [Malvaceae] extract (TFA), a traditional bioactive compound, has attracted attention due to its potent anti-inflammatory and metabolic regulatory properties. However, the capacity of TFA to ameliorate DLI and its underlying mechanism remain unknown.

This study aimed to investigate the hepatoprotective effects of TFA against DLI and to elucidate its underlying mechanism, with a particular focus on its association with the regulation of the PI3K/AKT/Nrf2 signaling pathway and ferroptosis.

Ultra-high-performance liquid chromatography coupled with quadrupole Orbitrap high-resolution mass spectrometry (UHPLC-Q-Orbitrap HRMS) was employed to identify TFA metabolites. DLI mouse model was established by high-fat diet (HFD) feeding and streptozotocin (STZ) injection. The mice were randomly allocated into four groups (n = 8): Control, Model, TFA-L, and TFA-H. TFA was administered daily by oral gavage for 12 consecutive weeks. In vitro hepatocyte model was induced with high glucose (HG) and oleic acid/palmitic acid (OA/PA). To systematically clarify the pharmacodynamic basis and molecular mechanisms underlying which TFA ameliorates DLI via inhibiting ferroptosis through the PI3K/AKT/Nrf2 pathway, integrated multi-omics strategies including network pharmacology, transcriptomics, and metabolomics were coupled with molecular docking, cellular thermal shift assay (CETSA), and experimental validations-including reactive oxygen species (ROS) detection, superoxide dismutase (SOD), glutathione (GSH), and oxidized glutathione (GSSG) determination, ferric/ferrous ion assay, and lipid peroxidation detection.

A total of 56 metabolites in TFA were identified using UHPLC-Q-Orbitrap HRMS. TFA significantly ameliorated metabolic disorders in HFD/STZ-induced DLI mice, as evidenced by reduced body weight, improved glycemic control, attenuated dyslipidemia, and decreased hepatic steatosis and injury markers (p< 0.05). Concomitantly, histopathological and immunohistochemical analyses revealed substantial alleviation of liver damage and fibrosis. Furthermore, integrated network pharmacology, transcriptomics, and metabolomics suggested that TFA exerts multi-target effects, potentially involving the PI3K/AKT/Nrf2 pathway-an axis that regulates amino acid and lipid metabolism, while also suppressing ferroptosis-related molecular programs to mitigate metabolic and hepatic disturbances. In line with this, molecular docking and CETSA suggested potential binding interactions between key TFA components and core targets of the PI3K/AKT/Nrf2 pathway as well as ferroptosis regulators. Notably, both in vivo and in vitro experiments consistently demonstrated that TFA alleviates oxidative stress, inflammation, and critically, ferroptosis in hepatic cells-manifested by reduced lipid peroxidation, restored GSH homeostasis, and balanced iron metabolism (p< 0.05). Importantly, these protective effects were abrogated by the PI3K inhibitor LY294002, ferroptosis inducer Erastin, and si-Nrf2, thereby confirming that TFA suppresses ferroptosis via a mechanism dependent on PI3K/AKT/Nrf2 pathway to exert its hepatoprotective effects against DLI (p< 0.05).

TFA exerts hepatoprotective effects against DLI through multi-target regulation, primarily involving the PI3K/AKT/Nrf2 pathway to suppress ferroptosis while concurrently ameliorating metabolic dysfunction, oxidative stress, and inflammation. These findings not only elucidate a novel mechanism of action but also position TFA as a promising therapeutic candidate worthy of further development for the clinical management of DLI.