Sclareol mitigates steatosis, inflammation, and fibrosis through the regulation of AMPK/SREBP1/NF-κB/TGF-β pathways in metabolic dysfunction-associated steatohepatitis

Metabolic dysfunction-associated steatohepatitis (MASH) is the progressive form of metabolic dysfunction-associated steatotic liver disease (MASLD). The increasing burden of MASH and its associated complications is challenging to cure. Our study aims to investigate the therapeutic potential of sclareol in MASH pathogenesis. Huh-7 cells were treated with various concentrations of free fatty acids (FFAs; 200-1200 µM), followed by treatment with sclareol (FFAs; 1200 µM + sclareol; 5 µM to 100 µM). An in vivo MASH model was developed using a high-fat diet and carbon tetrachloride (0.16 µg/kg every 15^th days) for 12 weeks, while control animals received a normal pellet diet. After model induction, animals were divided into four groups: Control, MASH, MASH + SLD (5 mg/kg/day, SLD: Sclareol low dose), and MASH + SHD (10 mg/kg/day, SHD: Sclareol high dose). Sclareol treatment was administered intraperitoneally for six weeks. In Huh-7 cells, co-treatment with sclareol, particularly at doses of 20, 40, and 80 µM, reduced reactive oxygen species and nitric oxide production while regulating lipogenic gene expression, thereby mitigating lipotoxicity. In vivo, sclareol alleviated dyslipidemia, hyperglycemia, liver injury, and oxidative stress in MASH animals. Histopathology confirmed that sclareol improved liver morphology by reducing nuclear infiltration, macro- and microsteatosis, hepatocyte ballooning, and fibrosis. GC-MS analysis revealed that sclareol intervention was associated with reduced fecal short-chain fatty acid (SCFA) levels. Interestingly, this reduction was accompanied by improvements in colon health. Furthermore, sclareol upregulated AMP-activated protein kinase and modulated the molecular expression of genes related to lipogenic, gluconeogenic, inflammation, fibrotic, and apoptotic pathways. This study is the first to report that sclareol prevents MASH progression by regulating oxidative stress, dyslipidemia, inflammation, fibrosis, and apoptosis.