Soybean oil emulsion phytosterols promote ROS accumulation and apoptosis-associated signaling in human liver cells

Total parenteral nutrition (TPN) is essential for patients who cannot tolerate enteral nutrition, but prolonged exposure to soybean oil-derived lipid emulsions (SOLEs) has been associated with parenteral nutrition-associated liver disease (PNALD). Sole contains phytosterols, including stigmasterol (ST), which may contribute to hepatocellular oxidative stress and apoptosis. This study investigated whether ST and SOLE promote oxidative injury, mitochondrial dysfunction, and apoptosis-related signaling in human L02 liver cells. Cells were exposed to ST, 0.5% SOLE, or their combination for 24 or 48 h. Cell viability, intracellular reactive oxygen species (ROS), mitochondrial membrane potential, apoptosis-related proteins, Nrf2/Keap1 signaling, and pathway-level molecular interactions were assessed using MTT and trypan blue assays, DCFH-DA fluorescence, JC-1 staining, Western blotting, immunofluorescence, and enrichment/network analyses. ST reduced L02 cell viability in a dose- and time-dependent manner and increased intracellular ROS accumulation. SOLE exposure further enhanced oxidative stress, whereas NAC partially attenuated ROS accumulation, supporting the contribution of oxidative stress to ST-induced cytotoxicity. ST also disrupted mitochondrial membrane potential and altered apoptosis-related markers, including caspase-3, PARP1, BAX/BCL2-associated signaling, and Nrf2/Keap1 stress-response regulation. Pathway enrichment and interaction network analyses further supported convergence of oxidative stress, mitochondrial dysfunction, DNA damage response, and apoptosis-related pathways. These findings provide mechanistic in vitro evidence that phytosterol-derived soybean oil emulsion can promote hepatocellular oxidative injury and mitochondrial apoptosis, supporting further investigation of phytosterol-reduced lipid formulations to improve hepatic safety during long-term PN.