Gardenin A alleviates alcohol-induced oxidative stress and inflammation in HepG2 and Caco2 cells via AMPK/Nrf2 pathway

Chronic alcohol consumption triggers immune responses that lead to cell damage, contributing to alcohol-associated liver disease (ALD). Despite its prevalence, no FDA-approved treatment for ALD currently exists. This study explores the cytoprotective effects of Gardenin A (GarA), a polymethoxylated flavone, for protection against alcohol-induced oxidative stress and inflammation in HepG2 and Caco2 cell lines. GarA was isolated, characterized and, tested in-vitro, showing maximum cell viability at 10 μg/ml using MTT assays. Further, lipid accumulation assay, reactive oxygen species (ROS) estimation and nuclear morphology visualization was carried out using different staining techniques. RT-qPCR was employed to examine the expression of various pro- and anti-inflammatory cytokines, along with Cytochrome P4502E1 (CYP2E1) and Sterol regulatory element binding protein-2 (SREBP2) and tight junction genes crucial for gut barrier integrity. Moreover, ELISA was carried out for key protein targets such as AMPK (phosphorylated and total), TNFα, C5aR1, HO-1 and Nrf2. GarA caused a marked decrease in lipid droplets, ROS levels, and expression of pro-inflammatory cytokines. It showed anti-inflammatory and anti-oxidant activity and helped maintain the gut barrier and nuclear integrity. In-silico studies showed the conserved amino acid interaction and affinity of GarA with C5aR1, and TNFα, compared to the interactions with known inhibitors/activators, further corroborating the results. This study is the first to explore the effects of GarA on ALD, underscoring its potential as an anti-inflammatory and anti-oxidant agent targeting the AMPK/Nrf2 signaling pathway, suggesting its future as a promising therapeutic candidate for mitigating alcohol-induced liver and gut damage.