iProXProteomicsJinfu ZhouRattus Rattushttp://www.iprox.org/page/project.html?id=IPX0006285000zhou811203@fjmu.edu.cnMedical Genetic Diagnosis and Therapy Center, Fujian Maternity and Child Health Hospital College of Clinical Medicine for ObstetricsiProXFerroptosis is driven by iron‑dependent accumulation of lipid hydroperoxides, and hemolytic hyperbilirubinemia causes accumulation of unconjugated bilirubin and iron. The present study aimed to assess the role of ferroptosis in hemolytic hyperbilirubinemia‑induced brain damage (HHIBD). Rats were randomly divided into the control, phenylhydrazine (PHZ) and deferoxamine (DFO) + PHZ groups, with 12 rats in each group. Ferroptosis‑associated biochemical and protein indicators were measured in the brain tissue of rats. We also performed tandem mass tag‑labeled proteomic analysis. The levels of iron and malondialdehyde were significantly higher and levels of glutathione (GSH) and superoxide dismutase activity significantly lower in the brain tissues of the PHZ group compared with those in the control group. HHIBD also resulted in significant increases in the expression of the ferroptosis‑related proteins acyl‑CoA synthetase long‑chain family member 4, ferritin heavy chain 1 and transferrin receptor and divalent metal transporter 1, as well as a significant reduction in the expression of ferroptosis suppressor protein 1. Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis demonstrated that the differentially expressed proteins of rat brain tissues between the control and PHZ groups were significantly involved in ferroptosis, GSH metabolism and fatty acid biosynthesis pathways. Pretreatment with DFO induced antioxidant activity and alleviated lipid peroxidation‑mediated HHIBD. In addition, PC12 cells treated with ferric ammonium citrate showed shrinking mitochondria, high mitochondrial membrane density, and increased lipid reactive oxygen species and intracellular ferrous iron, which were antagonized by pretreatment with ferrostatin‑1 or DFO, which was reversed by pretreatment with ferrostatin‑1 or DFO. The present study demonstrated that ferroptosis is involved in HHIBD and provided novel insights into candidate proteins that are potentially involved in ferroptosis in the brain during hemolytic hyperbilirubinemia.Ferroptosis contributes to hemolytic hyperbilirubinemia‑induced brain damage in vivo and in vitro.Zhou Jinfu J, Lin Xinpei X, Liao Sining S, Li Guilin G, Tang Jianping J, Luo Jinying J, Zhang Chenran C, Wu Siying S, Xu Liangpu L, Li Huangyuan HfalseTandem mass tag-based quantitative proteomic analysis reveals proteins involved in hemolytic hyperbilirubinemia-induced brain injury in ratsHemolytic hyperbilirubinemia-induced brain injury (HHIBI) can cause neonatal death, and survivors often have neurological sequelae, including irreversible motor sensitive and cognitive abnormalities. However, the underlying mechanisms remain elusive. By identifying differentially abundant proteins (DAPs), using proteomics, in the brain tissue of HHIBI rats, we aimed to identify the underlying molecular mechanisms of HHIBI. Tandem mass tag (TMT) labelling and LC-MS/MS were performed, and thereafter bioinformatics methods were used to characterize the molecular and functional signatures of HHIBI. In total, 7104 proteins were identified and 6848 were quantified. We detected 138 upregulated and 242 downregulated DAPs. Further, we found that the upregulated DAPs are involved in complement and coagulation cascade pathways. In addition, downregulated DAPs are mainly involved in DNA replication, mismatch repair, base excision repair, and cell cycle pathways. The Protein-Protein Interactions (PPI) network revealed thaTue Apr 18 00:00:00 BST 2023PXD0416041011737937619