P43 | NEW INSIGHTS IN THE ACTIVATION OF FERROPTOSIS PROCESS DURING LIVER FIBROSIS

R. Mancinelli¹, S. Leone¹, A. Casini¹, R. Vaccaro¹, M. Tagliafierro¹, F.M. Bassi¹, E. Bocci¹, S. Vitale¹, A. Franchitto², L. Pannarale¹, P. Onori¹, E. Gaudio¹ | ¹Department of Anatomical, Histological, Forensic Medicine and Orthopaedics Sciences, Sapienza University of Rome, Italy; ²Division of Health Sciences, Department of Movement, Human and Health Sciences, University of Rome Foro Italico, Italy

Ferroptosis is an iron-dependent regulated cell death characterized by iron accumulation, lipid peroxidation, and production of ROS¹. The liver represents the primary site of iron-overload injury for its critical role in iron metabolism². Patients with chronic liver disease may exhibit hepatic and splenic iron overloading³. The involvement of ferroptosis in liver fibrosis was recently investigated at the level of Kupffer cells⁴, hepatocytes and hepatic stellate cells (HSCs)⁵, however its role in biliary epithelium is yet unknown. We investigated the possible role of ferroptosis in the bile duct ligated (BDL) rat and we highlighted the effects of ferroptosis inducers and inhibitors in primary murine cholangiocytes. In vivo, we evaluated the iron deposits through Perls Prussian blue and the presence of iron regulator proteins, such as Ferritin, glutathione peroxidase-4 (GPX4) and acyl-CoA synthetase long-chain family member 4 (ACLS4) through immunohistochemstry. In vitro, for each inducer and inhibitor we evaluated the toxicity, the effects in cellular morphology and the cell cycle perturbation. We found a decrease in the expression of both Ferritin forms (heavy and light chain). Moreover, ACLS4, a protein regulator of lipid peroxidation, increased in BDL model, whereas GPX4, that plays a crucial role against membrane lipid peroxidation, was reduced in the cholestatic model compared the control. We confirmed that erastin is a potent and specific inducer of ferroptosis also in our primary cholangiocytes. Whereas, we provided evidence that sorafenib and ellagic acid fails to trigger ferroptosis. In the other side, we found that ferrostatin-1 (Fer-1) is unable to inhibit ferroptosis, while β-Mercaptoethanol (βME) could protect cholangiocytes in ferroptosis-induced cell death. All these findings could be important to understand the role of ferroptotic modulators in treating liver fibrosis.