Thymoquinone inhibits adipocyte development in 3T3-L1 cells system by modulating the AKT and AMPK signaling pathways

Obesity, a major global health concern, leads to various metabolic disorders. Adipogenesis, the process of preadipocytes differentiating into mature adipocytes, is key to excessive lipid accumulation. This study examined the anti-obesity effects of thymoquinone (TQ), a compound from Nigella sativa seeds, using 3T3-L1 pre-adipocytes in vitro. Cell viability was assessed with the neutral red assay. Lipid accumulation and GPDH activity were evaluated using oil red O staining and the Wise and Green method, respectively. Reactive oxygen species levels were quantified with the DCFH-DA method, while immunofluorescence staining was employed to examine the nuclear localization of C/EBPβ. The qRT-PCR and western blot assays were used to analyze the adipogenic and lipogenic markers. TQ inhibited lipid accumulation at concentrations of 6–30 µM without affecting cell viability. It also suppressed adipogenic transcription factors (PPARγ, C/EBPα, C/EBPβ, and SREBP-1c) and lipogenic genes ( AdipoQ, FABP4, FAS, and ACC1 ). Additionally, TQ inhibited the AKT pathway and enhanced AMPK phosphorylation. The results suggest that thymoquinone has anti-obesity properties by inhibiting adipogenic transcription factors and lipogenic genes through the regulation of AKT and AMPK pathways. Consequently, it could serve as a potential therapeutic agent for obesity. • Thymoquinone prevents lipid buildup in 3T3-L1 preadipocytes without cytotoxicity. • Thymoquinone inhibits key adipogenic transcription factors and lipogenic genes. • Thymoquinone affects the AKT and AMPK signaling pathways.