Biomarker-guided analysis of metabolic and inflammatory pathway modulation by Nigella sativa bioactive constituents

Abstract

Nigella sativa is a medicinal plant rich in bioactive constituents, particularly thymoquinone, with reported metabolic and anti-inflammatory activities. However, mechanistic evidence linking its phytochemical composition to defined molecular biomarkers remains limited. This study adopted a biomarker-centered approach to evaluate the effects of Nigella sativa ethanolic extract and thymoquinone on metabolic, inflammatory, and oxidative stress pathways using RAW 264.7 macrophages and HepG2 hepatocytes as complementary in vitro models of immunometabolic regulation. Cells were exposed to non-cytotoxic concentrations of the extract (25–100 μg/mL) and thymoquinone (10–25 μg/mL) following lipopolysaccharide stimulation in RAW 264.7 macrophages or high-glucose challenge in HepG2 hepatocytes. In RAW 264.7 cells, treatment significantly reduced the secretion of TNF-α, IL-6, and IL-1β and suppressed NF-κB protein expression. Oxidative stress was attenuated through a marked decrease in intracellular reactive oxygen species and upregulation of Nrf2 protein levels. In HepG2 cells, high-glucose–induced impairment of metabolic signaling was reversed by restoration of AMPK activation, GLUT4 expression, and PPARγ levels. Molecular docking analysis predicted stable interactions of thymoquinone with NF-κB, AMPK, Nrf2, and PPARγ, supporting its multi-target potential. The study provides mechanistic insight into immunometabolic pathway regulation and supports the use of biomarker-guided strategies for the translational evaluation of plant-derived pharmaceutical leads.