Xingfen Su,1 Handong Wang,2 Yuanxiang Presses Lin,1 Fuxiang Chen1 1Department of Neurosurgery, The First Affiliated Hospital of Fujian Medical University, Fuzhou 350005, Fujian, People’s Republic of China; 2Department of Neurosurgery, Jinling Hospital, School of Medicine, Nanjing University, Nanjing 210002, Jiangsu, People’s Republic of China Background: Intracerebral hemorrhage (ICH) is a devastating neurological injury associated with significant mortality.Necroptosis is a newly identified type of programmed necrosis initiated by the activation of tumor necrosis factor alpha.Evidences had demonstrated the importance of necroptosis in neuronal cell death.Necrostatin-1 is a specific inhibitor of necroptosis.The present study was carried out to explore whether RIP1/RIP3 pathways participate in hemin induced cell death in HT-22 hippocampal neuronal cells and investigate the potential neuroprotection of necrostatin-1 in hemin induced cell death in HT-22.
Methods: First, different concentrations of hemin (0, 25, 50, 100 µmol/L) were added to HT-22 cells.Propidium iodide (PI) positive cells and cell viability were measured at 24 hours after hemin treatment.Then, necrostatin-1, pan-caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone (z-VAD-fmk) and reactive oxygen species (ROS) scavenger butylated hydroxyanisole (BHA) were applied to hemin-treated HT-22 cells.PI positive cells and cell viability were measured at 24 hours after hemin treatment.MitoSox Red was used to indicate ROS level.
Last, Animatronic the effect of RIP3 in hemin induced HT-22 cell death was explored through RIP3 knockdown using siRNA.PI positive cells, cell viability and ROS lever were measured at 24 h after hemin treatment.Results: Hemin could induce a dose dependent cell death in HT22 neural cells.RIP1 specific inhibitor necrostatin-1 significantly inhibited cell death induced by hemin in HT-22 cells, greatly reducing PI positive cells, dramatically improving cell viability and decreasing ROS accumulation.BHA could significantly inhibit PI positive cells induced by hemin in HT-22 cells.
Furthermore, silencing of RIP3 using siRNA attenuated hemin induced cell death in HT-22 cells, greatly reducing PI positive cells, dramatically improving cell viability and decreasing ROS accumulation.Conclusion: These data revealed that RIP1/RIP3 might mediate hemin induced cell death in HT-22 cells, and necrostatin-1 played a neuroprotection role in hemin induced cell death in HT-22.RIP1 and RIP3 might represent novel therapeutic targets for ICH.Keywords: intracerebral hemorrhage, necroptosis, HT22, RIP1, RIP3, necrostatin-1.