Enhanced hepatoprotective effects of fisetin-loaded liposomal nanoparticles against DEHP-induced liver injury in rats: modulation of oxidative stress, inflammation, apoptosis, and ferroptosis pathways.

Di-(2-ethylhexyl) phthalate (DEHP) is a widely used plasticizer with well-documented hepatotoxic effects mediated through oxidative stress, inflammation, apoptosis, and ferroptosis-related molecular alterations. This study investigated the hepatoprotective potential of free fisetin (FSN) and fisetin-loaded liposomal nanoparticles (FSN-LNPs) against DEHP-induced liver injury in rats, with special emphasis on the comparative efficacy of the nanoformulation. Sixty adult male Sprague Dawley rats were randomly allocated into six groups: control, FSN, FSN-LNPs, DEHP, DEHP + FSN, and DEHP + FSN-LNPs. Following 28 days of oral administration, liver function indices, lipid profile, oxidative stress markers, inflammatory mediators, apoptotic and ferroptosis-related biomarkers, along with histopathological and ultrastructural alterations, were evaluated. DEHP exposure significantly impaired liver function, as evidenced by elevated serum ALT, AST, and ALP levels, along with disrupted protein synthesis and dyslipidemia. These biochemical changes were accompanied by increased oxidative stress, NF-κB activation, elevated pro-inflammatory cytokines (TNF-α and IL-6), and activation of apoptotic (caspase-3, Bax) signaling, together with ferroptosis-associated molecular alterations characterized by increased ACSL4 expression, iron accumulation, and lipid ROS levels, accompanied by reduced GPX4 expression, alongside suppression of antioxidant defenses (SOD, CAT, GSH, Nrf2/HO-1). Histopathological and ultrastructural analyses further highlighted severe hepatic injury in DEHP-treated rats. Co-treatment with FSN alleviated these alterations, while FSN-LNPs demonstrated significantly superior protective effects across most evaluated parameters. Notably, FSN-LNPs restored several biomarkers to near-normal levels and markedly improved hepatic architecture and ultrastructure. In conclusion, FSN-LNPs provided enhanced hepatoprotection against DEHP-induced toxicity compared with free FSN, which was associated with attenuation of oxidative stress and inflammation and modulation of apoptosis- and ferroptosis-related pathways, likely due to improved bioavailability and cellular delivery. To the best of our knowledge, this is the first study to evaluate fisetin in a liposomal nanoparticle formulation for protection against DEHP-induced liver injury, highlighting its promising therapeutic potential while providing a foundation for future mechanistic and translational investigations.