RIP1 and RIP3 mediate hemin-induced cell death in HT22 hippocampal neuronal cells
Authors Su X, Wang H, Lin Y, Chen F
Received 22 July 2018
Accepted for publication 27 September 2018
Published 15 November 2018 Volume 2018:14 Pages 3111—3119
Checked for plagiarism Yes
Review by Single-blind
Peer reviewers approved by Dr Andrew Yee
Peer reviewer comments 2
Editor who approved publication: Dr Yu-Ping Ning
Xingfen Su,1 Handong Wang,2 Yuanxiang 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, 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
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