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- benzimidazolylcarbo- cyanine 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 （△Ψm） 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. Pre- treatment 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） △Ψm： △Ψm was re-duced after 24-hour hypoxia, as assessed by JC-1 and a confocal microscope. Pretreatment with 20 μmol/L Humanin preserved the loss of △Ψm. （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 in- hibiting the opening of mPTP and preserving △Ψm.
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- benzimidazolylcarbo- cyanine 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 （△Ψm） 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. Pre- treatment 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） △Ψm： △Ψm was re-duced after 24-hour hypoxia, as assessed by JC-1 and a confocal microscope. Pretreatment with 20 μmol/L Humanin preserved the loss of △Ψm. （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 in- hibiting the opening of mPTP and preserving △Ψm.
基金
the National Natural Science Foundation of China, No. 30572085
