Theaflavins counteract free fatty acid-driven oxidative-inflammatory injury in endothelial cells through Nrf2-NF-κB...

Chronically elevated circulating free fatty acids (FFAs) are key drivers of lipotoxic injury by triggering a self-amplifying oxidative stress-inflammation cascade, thereby contributing to metabolic and cardiovascular diseases. Vascular endothelial dysfunction represents an early and causal event in this process. Although black tea-derived theaflavins (TFs) possess antioxidant and anti-inflammatory properties, the subtype-specific and tissue-parallel effects of TFs against FFA-induced oxidative-inflammatory injury remain incompletely understood.

FFA-induced lipotoxicity models were established in vascular endothelial cells and hepatocytes to systematically compare the protective effects of four major TF subtypes: theaflavin (TF1), theaflavin-3-gallate (TF2A), theaflavin-3'-gallate (TF2B), and theaflavin-3, 3'-digallate (TFDG). Antioxidant capacity was assessed using both cell-free systems and oxidant-stimulated cellular models. Mechanistic studies were conducted to determine the involvement of the Nrf2-NF-κB axis.

All TF subtypes significantly attenuated FFA-induced ROS overproduction, lipid peroxidation, and inflammatory mediator release in both endothelial cells and hepatocytes, whereas no major changes in intracellular lipid accumulation were observed. Among the four subtypes, TF1 consistently exhibited the strongest and most reproducible cytoprotective effects. Further analyses demonstrated that TFs, particularly TF1, restored cellular redox homeostasis not only through direct free radical scavenging but, more importantly, through activation of endogenous antioxidant defenses. Transcriptomic and bioinformatics analyses indicated that TF1 enhanced antioxidant-related gene expression while suppressing inflammatory genes, with enrichment of oxidative stress-, atherosclerosis-, and NF-κB-related pathways and Nrf2 appearing as a key node. Molecular docking predicted a favorable TF1-Keap1 interaction. Consistently, TF1 promoted Nrf2 nuclear translocation and HO-1 expression, restored SOD2 and GPX1 expression, reduced mitochondrial ROS, and attenuated IκBα phosphorylation and NF-κB p65 nuclear accumulation. Silencing Nrf2 markedly weakened these protective effects, supporting an important role of Nrf2 in TF1-mediated endothelial protection.

TFs act as natural modulators of FFA-driven oxidative-inflammatory injury, with TF1 emerging as the most potent subtype. TF1 protects against FFA-induced endothelial dysfunction partly through an Nrf2-associated mechanism involving enhanced antioxidant responses and attenuation of NF-κB-related inflammatory signaling.