Inhibition of neuron apoptosis by acidic peptide / 中国组织工程研究

ABSTRACT

BACKGROUND: Excessive nitric oxide (NO) release can cause the occurrence and development of brain injury and senile dementia due to the apoptosis induction role of NO at high concentration to nerve cells. Therefore one strategy to prevent and treat senile dementia is inhibiting the apoptosis induced by NO.OBJECTIVE: To observe whether acidic peptide will inhibit the neuron apoptosis caused by NO. DESIGN: An cell and molecule observation experiment by comparisons. SETTING: Department of Biochemistry and Molecular Biology of Basic Medical College in Zhengzhou University and the Second Laboratory of Biological Active Peptide Institute in Zhengzhou University. MATERTALS The experiment was performed between May 2003 and May 2005, in the Second Laboratory of Biological Active Peptide Institute in Zhengzhou University and the cell culture room of Department of Biochemistry and Molecular Biology of Basic Medical College in Zhengzhou University. The newborn SD male rats within 24 hours after birth were provided by the Animal Center of Henan Province (410117).METHODS: On day 11 of primary cultures, hippocampus neurons of the newborn SD rats were pretreated with different dosages of acidic peptide for six hours. Sodium nitroprusside (SNP) of 50 μmol/L final concentration was added to the cells which were incubated for another 24 hours. Cells were collected and adopted in this experiment of five different groups, namely normal control group, group treated with SNP, group of SNP plus 0.037 5 mg/mL acidic peptide, group of SNP plus 0.075 mg/mL acidic peptide, group of SNP plus 0.15 mg/mL acidic peptide. The cell's survival rate wasmeasure by methyl thiazolyl (MTT) method; The neurofilament protein was stained with the method of immunohisto chemistry. The shape of apoptosis was display with acridine orange fluorescent stain. Then DNA ladder zone of apoptosis cells was analyzed with the method of agarose gel electrophoresis. Western Blot and absorbance scan were used to determine the expression level of Bcl-2 protein and Bax protein.MAIN OUTCOME MEASURES: ①Experimental result of cell survival rate with MTT method;②Observation results of nuclear type of apoptosis; ③DNA electrophoresis analysis of apoptosis; ④Western Blot analysis results of Bcl-2 protein and Bax protein.RESULTS: ①Neuron survival rate was 58.9％ for group treated with SNP, 70.0％ for group of SNP plus 0.037 mg/mL acidic peptide, 72.8％ for group of SNP plus 0.075 mg/mL acidic peptide, and 75.3％ for group of SNP plus 0.15 mg/mL acidic peptide. ②Observation results of nuclear type of apoptosis Significant characteristics of apoptosis were seen in group treated with SNP. The nucleus of hippocampus neuron treated with different concentrations of acidic peptide plus SNP was similar to that of normal control group in morphology. ③The results of DNA electrophoresis analysis of apoptosis Only the neuron DNA of group treated with SNP showed clear characteristic DNA ladder zone of apoptosis on agarose gel electrophoresis. ④Analysis results of Bcl-2 protein and Bax protein with Western Blot and absorbance scan The expression level of Bcl-2 protein in SNP treated group was decreased while that of Bcl-2 protein was increased. Bcl2 protein levels in acidic peptide plus SNP group were increased and Bax protein levels were decreased gradually with the increasing concentrations of acidic peptide compared with SNP treated group. CONCLUSION: Acidic peptide can inhibit neuron apoptosis, increase expression level of neuron Bcl-2 protein and decrease expression level of neuron Bax protein.