Hepatology Basic Science

Introduction: The scarcity of donor livers has triggered efforts to expand the existing donor pool by using marginal donor livers, such as steatotic livers. However, steatotic donor livers are at a high risk of primary graft non-function, early allograft dysfunction, and graft loss due to their enhanced susceptibility to ischemia–reperfusion (I/R) injury. Necroptosis is a novel form of cell death and has been implicated in I/R injury. Receptor-interacting protein kinase 3 (RIPK3) is thought to be instrumental in the execution of necroptosis. However, necroptosis and RIPK3 have not been fully examined in steatotic liver undergoing I/R injury. In this study, we developed an in vitro hepatic steatosis model undergoing I/R injury to further study mechanisms of cell death. Methods: AML-12 cells were cultured in media containing increasing concentrations (0.25, 0.5, 1.0, 2.0) mM of free fatty acid (FFA) for 24 hours to induce hepatic steatosis. Further, FFA-treated cells were subjected to oxygen-glucose deprivation (OGD) conditions by culturing in glucose-free media under hypoxic conditions (1% O2, 5% CO2, and 94% N2) for 12 hours to mimic ischemia. Oil Red O staining was performed to assess the hepatocellular steatosis. Protein expression was detected by western blot analysis, and quantitative polymerase chain reaction was used for mRNA expression quantification. Cell viability was determined by CellTiter-Blue Cell Viability Assay (Promega). Results: A dose-dependent increase in fat accumulation was observed after 24 hours of FFA treatment. There was no significant decrease in cell viability after FFA exposure (P = 0.17). A concentration of 2 mM FFA was considered to be optimal as the cells maintained viability and FFA deposition even after 48 hours of FFA exposure. The hypoxia-sensitive genes, solute carrier family 2, facilitated glucose transporter member 1 (Slca1), and vascular endothelial growth factor (Vegf) were increased (2.6and 2.7-fold, respectively) after OGD. Treatment with FFA + OGD reduced cell viability after 12 hours of OGD (P < 0.01). RIPK3 protein was significantly upregulated in OGD-treated cells compared with control FFA-treated cells (2.4-fold; P = 0.01). Lack of cleaved-CASPASE3 expression indicated apoptosis was not an active pathway in our model. Nuclear factor NF-κB, which plays a role in regulating the DNA damage-repairing system, decreased in OGD-treated cells (4.6-fold). Similarly, phosphoglycerate mutase family 5 (Pgam5), a gene that protects the cells from necroptosis, was downregulated in OGD-treated cells (1.5-fold). Conclusion: Our findings suggest that necroptosis may contribute to I/R injury in our in vitro model. Future studies will investigate the use of RIPK3 inhibition during the re-oxygenation stage as a therapeutic agent to reduce the consequences of I/R injury. In conclusion, these findings suggest our model may be used to study the cell death pathways active during steatosis and hepatic I/R injury. H-Ferritin and iron activate inflammatory pathways in adipocytes LA JASKOWSKI,* KR BRIDLE,* LJ BRITTON,* A JAYACHANDRAN,* GA RAMM, DHG CRAWFORD* *Gallipoli Medical Research Institute, School of Clinical Medicine, University of Queensland, and QIMR Berghofer Medical Research Institute, Brisbane, Queensland, Australia