Mechanistic Framework for Nrf2-Mediated Cytoprotection by Nigerian Medicinal Plant Polyphenols: In Vitro and In Vivo Contributions

The Nrf2-Keap1-ARE signalling pathway is the principal cellular defence mechanism against oxidative stress and electrophilic toxicity, and its activation by dietary polyphenols is an area of intense pharmacological investigation globally. This dissertation develops a mechanistic framework for Nrf2-mediated cytoprotection by polyphenol-rich extracts from three Nigerian medicinal plants, Moringa oleifera, Garcinia kola, and Vernonia amygdalina, combining in vitro molecular mechanistic characterization with in vivo validation studies. The in vitro programme employs human hepatocellular carcinoma (HepG2) and human intestinal epithelial (Caco-2) cell models treated with standardized polyphenol fractions prepared by preparative HPLC. Nrf2 nuclear translocation was quantified by immunofluorescence confocal microscopy and Western blotting. ARE-driven reporter gene activation was measured in ARE-luciferase stably transfected HepG2 cells. Downstream Nrf2 target gene induction including HO-1, NQO1, GCLM, and GCLC was quantified by quantitative RT-PCR and Western blot. Keap1-Nrf2 protein-protein interaction disruption was assessed by co-immunoprecipitation. Molecular docking computations modelled binding interactions of the major identified polyphenols with Keap1 Kelch domain critical cysteines. In vivo validation in an acetaminophen hepatotoxicity model confirmed that Nrf2 pathway activation by the extracts correlated with hepatoprotective outcomes quantified biochemically and histopathologically. The dissertation establishes that these Nigerian plant polyphenols act as indirect Nrf2 activators through Keap1 cysteine modification, and constructs an original mechanistic framework distinguishing Keap1-dependent from transcriptional-level Nrf2 activation. Original theoretical contributions address the polyphenol structural determinants of differential Keap1 cysteine selectivity. Keywords: Nrf2 pathway, polyphenols, cytoprotection, medicinal plants, oxidative stress.