Protective effects of Humanin on hypoxia-induced neuronal death

Yunqi Zhu; Yanli Li; Jingyi Liu; Xiaorong Yang; Ce Zhang

摘要：BACKGROUND: Humanin is a 24-amino acid peptide isolated from the brain of an Alzheimer's disease patient. Several studies have indicated that Humanin can protect cells against cytotoxicity induced by various insults.OBJECTIVE: To investigate the protective role of Humanin on hypoxia-induced neuronal death, and to determine the most appropriate therapeutic concentration of Humanin.DESIGN, TIME AND SETTING: Neuropathophysiological, randomized, controlled experiment, conducted at the Department of Physiology and Neurobiology, Shanxi Medical University, between March 2007 and October 2007.MATERIALS: Newborn Wistar rats, 5,5',6,6' tetrachloro-1,1',3,3'-tetraethyl- benzimidazolylcarbocyanine iodide (JC-1, USA), calcein-acetoxymethylester (calcein-AM, USA), and Humanin (Shanghai, China) were used in this study. METHODS: Primary cortical neurons were cultured with dulbecco's modified eagle's medium containing 15% fetal bovine serum. Cultures were divided into three groups: control, hypoxia, and hypoxia + Humanin. Various concentrations of Humanin (1, 10, and 20 μmol/L) were added to the cultures 16 hours prior to hypoxia induction. For hypoxic conditions, cells were maintained at 37 ℃ within an incubator chamber filled with 95% N2 and 5% CO2 for 24 hours. Cells in the control group were cultured in normal oxygen. MAIN OUTCOME MEASURES: Cell viability was determined through the use of the vital dye calcein-AM, and the number of live cells was determined. Mitochondrial membrane potential (ΔΨm) was assessed using the fluorescent probe JC-1. Mitochondrial permeability transition pore (mPTP) opening was determined with calcein-AM in the presence of cobalt chloride.RESULTS: (1) Cell viability: Hypoxia for 24 hours induced death in a large number of neurons. Pretreatment with 10 μmol/L and 20 μmol/L Humanin, 16 hours prior to hypoxia, protected cells against hypoxia. However, 1 μmol/L Humanin provided little protection. (2) ΔΨm: ΔΨm was reduced after 24-hour hypoxia, as assessed by JC-1 and a confocal microscope. Pretreatment with 20 μmol/L Humanin preserved the loss of ΔΨm. (3) mPTP: Hypoxia induced the opening of mPTP. Pretreatment with 20 μmol/L Humanin repressed the opening of mPTP, as most of the calcein fluorescence remained in the mitochondria.CONCLUSION: Humanin (20 μmol/L) protects neuronal cells from hypoxia-induced insults by inhibiting the opening of mPTP and preserving ΔΨm.