Combined effects of vitamin D3 and dioxopiperidinamide derivative on lipid homeostasis, inflammatory pathways, and redox imbalance in non‐alcoholic fatty liver disease in vivo zebrafish model

Liver damage and metabolic dysfunctions, the defining features of non‐alcoholic fatty liver disease (NAFLD), are marked by inflammation, oxidative stress, and excessive hepatic fat accumulation. The current therapeutic approaches for NAFLD are limited, necessitating exploring novel treatment strategies. Dioxopiperidinamide derivatives, particularly DOPA‐33, have shown effective anti‐inflammatory and antioxidant properties, potentially offering therapeutic benefits against NAFLD. This study investigated the combined potential of vitamin D3 (Vit D3) and DOPA‐33 in treating NAFLD. The network pharmacology analysis identified key NAFLD targets modulated by Vit D3 and DOPA‐33, emphasizing their potential mechanisms of action. In NAFLD‐induced zebrafish models, Vit D3 and DOPA‐33 significantly reduced hepatic lipid accumulation, oxidative stress, and apoptosis, demonstrating superior efficacy over individual treatments. The treatment also lowered reactive oxygen species (ROS) levels, decreased liver damage, and enhanced antioxidant defense mechanisms. Moreover, behavioral analyses showed improved locomotion and reduced weight gain in treated zebrafish. Biochemical analyses revealed lower triglycerides (TG) and glucose levels with improved oxidative markers. Furthermore, histological analyses indicated reduced hepatic steatosis and inflammation, with decreased expression of lipogenesis‐related genes and inflammatory mediators. Finally, high‐performance liquid chromatography (HPLC) confirmed a significant reduction in hepatic cholesterol levels, indicating the effectiveness of the combination therapy in addressing key NAFLD‐related dyslipidemias. These findings suggest that Vit D3 + DOPA‐33 targets pathways involved in lipid metabolism, inflammation, and oxidative stress by offering a promising therapeutic approach for NAFLD.