Rhamnetin-3-O-α-L-Rhamnoside Attenuates Alcoholic Liver Injury in Mice via Regulation of the Autophagy/Ferroptosis Pathway

Abstract

Background: Rhamnetin-3-α-L-rhamnoside (ARR) is the principal monomer isolated from Loranthus tanakae Franch. and Sav, with the effect of prevention and treatment of inflammatory diseases. This study was to investigate the effects of ARR on acute alcoholic liver injury (ALI) in a mouse model and elucidate the mechanisms.

Methods: Balb/c mice were divided into six groups: naive control (NC), model control (MC), positive control (PC), ARR low-dose group (ARR-L), medium-dose group (ARR-M), and high-dose group (ARR-H). The treatment lasted for 11 days, with alcohol administration on the 9th and 11th day. Blood samples were taken 24 h postadministration for glutamic pyruvic transaminase (ALT) and glutamic oxaloacetic transaminase (AST) detection. Liver tissue samples underwent hematoxylin–eosin (H&E) staining, immunohistochemical analysis, and biochemical assays for glutathione (GSH), glutathione (MDA), superoxide dismutase (SOD), and tissue iron. Western blotting analysis was conducted for ferroptosis-related proteins including glutathione peroxidase 4 (GPX4), solute carrier family 7 member 11 (SLC7A11), and ferritin light chain (FTL) as well as autophagy-related proteins comprising benzyl chloride 1 (Beclin-1), autophagy-associated Gene 5 (ATG5), sequestosome 1 (P62), and microtubule associated protein 1 light chain 3 (LC3).

Results: H&E staining and analysis showed that alcohol caused liver cell damage, which was improved in the positive and ARR groups. The immunohistochemical analysis indicated decreased levels of heme oxygenase 1 (HO-1) and nuclear factor erythroid 2–related factor 2 (Nrf2) in the MC mice, whereas enhanced in the administration groups. Liver index, serum ALT, and AST significantly elevated in the MC group, which confirm successful modeling. Both positive drug and ARR ameliorated liver index, aminotransferase levels, oxidative stress, and tissue iron. Furthermore, western blotting revealed abnormal expression of ferroptosis- and autophagy-related proteins due to alcohol. In comparison with MC mice, administration groups showed significantly increased expressions of GPX4, SLC7A11, Beclin-1, ATG5, and LC3II/I and decreased expressions of FTL and P62.

Conclusion: ARR prevents acute ALI by inhibiting ferroptosis and inducing autophagy. These findings suggested that ARR may represent a natural therapeutic approach for this disease.