Enhanced hepatoprotective effects of empagliflozin and vitamin D dual therapy against metabolic dysfunction‐associated steatohepatitis in mice by boosted modulation of metabolic, oxidative stress, and inflammatory pathways

Although single treatment with sodium-glucose cotransporter-2 inhibitors (SGLT2i) or vitamin D₃ (VD₃) inhibited metabolic dysfunction-associated steatohepatitis (MASH) development in diabetic patients, their combination has not been explored previously. Hence, this study investigated the hepatoprotective effects of SGLT2i (empagliflozin) and/or VD₃ against MASH in type 2 diabetic mice. Forty Mice were assigned into negative (NC) and positive (PC) controls, SGLT2i, VD₃, and SGLT2i + VD₃ groups. All animals, except the NC group, received high-fructose/high-fat diet (8 weeks) followed by diabetes induction. Diabetic mice then received another cycle of high-fructose/high-fat diet (4 weeks) followed by 8 weeks of treatment (five times/week) with SGLT2i (5.1 mg/kg/day) and/or VD₃ (410 IU/Kg/day). The PC group demonstrated hyperglycaemia, dyslipidaemia, elevated liver enzymes, and increased non-alcoholic fatty liver disease activity score (NAS) with fibrosis. Hepatic glucose transporting molecule (SGLT2) with lipogenesis (SREBP-1/PPARγ), oxidative stress (MDA/H₂O₂), inflammation (IL1β/IL6/TNF-α), fibrosis (TGF-β1/α-SMA), and apoptosis (TUNEL/Caspase-3) markers alongside the PI3K/AKT/mTOR pathway increased in the PC group. Conversely, hepatic insulin-dependent glucose transporter (GLUT4), lipolytic (PPARα/INSIG1), antioxidant (GSH/GPx1/SOD1/CAT), and anti-inflammatory (IL-10) molecules with the inhibitor of PI3K/AKT/mTOR pathway (PTEN) decreased in the PC group. Whilst SGLT2i monotherapy outperformed VD₃, their combination showed the best attenuation of hyperglycaemia, dyslipidaemia, and fibrosis with the strongest modulation of hepatic glucose-transporting and lipid-regulatory molecules, PI3K/AKT/mTOR pathway, and markers of oxidative stress, inflammation, fibrosis, and apoptosis. This study is the first to reveal boosted hepatoprotection for SGLT2i and VD₃ co-therapy against diabetes-induced MASH, possibly via enhanced metabolic control and modulation of hepatic PI3K/AKT/mTOR, anti-inflammatory, anti-oxidative, and anti-fibrotic pathways.