Verbenalin attenuates hepatic damage and mitochondrial dysfunction in alcohol-associated steatohepatitis by regulating MDMX/PPARα-mediated ferroptosis.

ETHNOPHARMACOLOGICAL RELEVANCE: Verbenalin is a major compound in Verbena officinalis L. Verbena officinalis L was first recorded in the 'Supplementary Records of Famous Physicians.' Verbenalin (VE) is its active constituent and has been found to have many biological effects, including anti-obesity, anti-inflammatory, and antioxidant activities, removing jaundice, and treating malaria. It could treat lump accumulation, dysmenorrhea, throat obstruction, edema, jaundice, and malaria. Palmitic acid (PA), oleic acid (OA), ethanol, and acetaminophen liver injuries have been proven to benefit from verbenalin. AIM OF THE STUDY: To study the effects of verbenalin on the prevention of alcoholic steatohepatitis (ASH) through the regulation of oxidative stress and mitochondrial dysfunction by regulating MDMX (Murine double minute X)/PPARα (Peroxisome proliferator-activated receptor alpha)-mediated ferroptosis. MATERIAL AND METHODS: C57BL/6 mice treated with alcohol followed by the Gao-Binge protocol were administered verbenalin by gavage simultaneously. The mitochondrial mass and morphology were visualized using TEM. AML-12 cells were stimulated with ethanol to mimic ASH in vitro. Western blotting, co-immunoprecipitation, and kit determination were simultaneously performed. The target protein of verbenalin was identified by molecular docking, and cellular thermal shift assay (CETSA) further confirmed its interactions. RESULTS: Verbenalin alleviates oxidative stress and ferroptosis in alcohol-associated steatohepatitis. To elucidate the molecular mechanism by which verbenalin inhibits abnormal mitochondrial dysfunction, molecular docking was performed, and MDMX was identified as the target protein of verbenalin. CETSA assays revealed a specific interaction between MDMX and verbenalin. Co-immunoprecipitation demonstrated that PPARα played a critical role in promoting the ability of MDMX to affect ferroptosis. Verbenalin regulates MDMX/PPARα-mediated ferroptosis in AML-12 cells. CONCLUSION: Verbenalin regulates ferroptosis and highlights the therapeutic potential of verbenalin and ferroptosis inhibition in reducing alcoholic steatohepatitis.

Hastatoside attenuatescarbon tetrachloride-induced liver fibrosis by targeting glycogen synthase kinase-3ß.

BACKGROUND: Hastatoside is an iridoid glycoside extracted from the herb, Verbena officinalis, that exerts various pharmacological effects, including anti-inflammatory, sleep-promoting, and analgesic effects. However, only a few studies have reported the efficacy of hastatoside in liver fibrosis. Liver fibrosis is a pathophysiological process, and its persistence can seriously affect the quality of life and well-being of the patients. HYPOTHESIS/PURPOSE: This study aimed to investigate the role of hastatoside on liver fibrosis and its possible underlying mechanisms. METHODS: C57BL/6 J mice with carbon tetrachloride (CCl4)-induced hepatic fibrosis were used as the in vivo models. Histological features of the liver were observed using Masson's trichrome and hematoxylin-eosin staining. Alanine aminotransferase and aspartate aminotransferase levels and the hepatic fibrosis indices (type 3 procollagen, laminin, and hyaluronic acid) were measured using corresponding assay kits. LX-2 human hepatic stellate cells (HSCs) stimulated with the transforming growth factor ß1 were used as the vitro models. Transfection of the glycogen synthase kinase (GSK)-3ß small interfering RNA (siRNA) and ß-catenin plasmids was also performed in vitro. Protein levels of GSK-3ß, phospho-GSK-3ß (Ser 9), α-smooth muscle actin, collagen type I alpha 1, c-Myc, cyclin D1, and ß-catenin were determined via western blotting. Moreover, the p-GSK-3ß:GSK-3ß ratio was calculated to determine the GSK-3ß activity. RESULTS: Hastatoside prevented CCl4-induced liver injury and histological damage. It inhibited the upregulation of α-SMA and Col1α1 levels in a CCl4-induced mouse hepatic fibrosis model. In vitro, hastatoside inhibited the proliferation and activation of HSCs by decreasing the expression levels of cyclin D1 and c-Myc and the proportion of LX-2 cells activated in the G0/G1 phase. Molecular docking results showed that hastatoside bound to GSK-3ß. Hastatoside significantly increased the GSK-3ß activity and inhibited the downstream effector expression of ß-catenin. CONCLUSION: These findings suggest that hastatoside can bind to GSK-3ß and promote its activity, while inhibiting the GSK-3ß downstream effector expression of ß-catenin, thereby inhibiting the activation and proliferation of HSCs, which further prevents the development of liver fibrosis. These results provide innovative insights into the underlying liver fibrosis. Moreover, hastatoside is a potential anti-fibrosis monomer that can potentially be used for the treatment of liver fibrosis.