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BACKGROUND: Aggregation of β-amyloid peptide (Aβ), excitatory intoxication, oxidation injury and inflammation reaction are generally regarded as the main pathogenesis for Alzheimer disease (AD). (–) clausenamide is characterized by promoting intelligent development, resisting oxidation, cleaning free radicals, resisting Aβ neurotoxicity and nerve cell apoptosis, inhibiting over phosphorylation of tau protein, and improving central cholinergic system. However, whether (– ) clausenamide has an effect on hippocampal neuron apoptosis or not need further study. OBJECTIVE: To observe the effect of (–) clausenamide on survival rate of hippocampal neurons due to sodium nitroprusside (SNP) and analyze the possible pathways. DESIGN: Contrast observation. SETTING: Institute of Biochemistry and Molecular Biology, Guangdong Medical College. MATERIALS: A total of 12 male SD rats of 24 hours old were provided by the Experimental Animal Center of Guangdong Medical College. The primer was synthesized by Beijing Huada Genetic Engineering Company and (–) clausenamide (99.6%) was provided by Pharmacological Department of Chinese Academy of Medical Sciences. SNP was provided by Sigma Company. METHODS: Bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 12th day, hippocampal neurons were pretreated with 0.2, 0.4, 0.8 and 1.6 μmol/L (–) clausenamide for 6 hours; the culture medium was gotten rid of and neurons were washed with non-serum DMEM solution for three times. In addition, non-serum DMEM solution was added with the above mentioned volume of (–) clausenamide and 50 μmol/L SNP to culture neurons for 24 hours and the collected cells were prepared for the experiment. Neurons were equally divided into control group (culture medium control), model group (SNP treatment) and experimental group [(–) clausenamide + SNP]. Experiment of each group was done for three times at least. Survival rate of cells was measured with MTT chromatometry; levels of mRNA of hippocampal neuron bcl-2 and bax gene were detected with reverse transcription polymerase chain reaction (RT-PCR); expression of hippocampal neuron Bcl-2 and Bax protein was measured with Western blot technique. MAIN OUTCOME MEASURES: ① Effect of (–) clausenamide on survival rate of SNP-induced hippocampal neuron apoptosis; ② bcl-2 and bax mRNA and protein expression of hippocampal neurons. RESULTS: ① Survival rate of hippocampal neurons: Survival rate of hippocampal neurons affected by 0.4–1.6 μmol/L (–) clausenamide was higher in the experimental group than the model group (P < 0.01), and the survival rate was increased with the larger volume of (–) clausenamide. Survival rate was the highest when hippocampal neurons were induced by 1.6 μmol/L, and it had obvious dosage dependence (P < 0.01). ② Expression of bcl-2 and bax mRNA: Hippocampal neurons were pretreated with 0.2–1.6 μmol/L (–) clausenamide for 6 hours in the experimental group and strap of PCR product of bcl-2 gene was brightened gradually. This suggested that, with the increase of concentration, expression of bcl-2 mRNA was increased simultaneously. However, when strap of PCR product of bax gene was darkened, expression of bax was decreased with the increase of concentration. ③ Expression of Bcl-2 and Bax protein: Hippocampal neurons were pretreated with 0.2–1.6 μmol/L (–) clausenamide for 6 hours in the experimental group and strap of PCR product of Bcl-2 protein was thickened gradually. This suggested that, with the increase of concentration, expression of Bcl-2 protein was increased simultaneously. However, when strap of PCR product of Bax protein was thinned, expression of Bax protein was decreased with the increase of concentration. CONCLUSION: (–) clausenamide can resist neurotoxic effect of SNP through dosage dependence, and the mechanism may be related to promoting expression of anti-apoptotic bcl-2 gene and inhibiting expression ofpro-apoptotic bax gene.
BACKGROUND: Aggregation of β-amyloid peptide (Aβ), excitatory intoxication, oxidation injury and inflammation reaction are generally regarded as the main pathogenesis for Alzheimer disease (AD). (-) clausenamide is characterized by promoting intelligent development, resisting oxidation, cleaning free Inhibits the phosphorylation of tau protein, and improving central cholinergic system. However, whether (-) clausenamide has an effect on hippocampal neuron apoptosis or not need further study. OBJECTIVE: To observe the effect of (-) clausenamide on survival rate of hippocampal neurons due to sodium nitroprusside (SNP) and analyze the possible pathways. DESIGN: Contrast observation. SETTING: Institute of Biochemistry and Molecular Biology, Guangdong Medical College. MATERIALS: A total of 12 male SD rats of 24 hours old were provided by the Experimental Animal Center of Guangdong Medical College. The primer was synt METHODS: Bilateral hippocampus was collected from newborn rats to establish single cell suspension. On the 12th day, hippocampal neurons were pretreated with 0.2, 0.4, 0.8 and 1.6 μmol / L (-) clausenamide for 6 hours; the culture medium was washed with and the neurons were washed with non-serum DMEM solution for three times. In addition, non-serum DMEM solution was added with the above volume of (-) clausenamide and 50 μmol / L SNP to culture neurons for 24 hours and the collected cells were prepared for the experiment. Neurons were equally divided into control group (culture medium control), model group (SNP treatment) and experimental group [(-) clausenamide + SNP]. Experiment of each group was done for three times at least. Survival rate of cells was measured with MTT chr omatometry; levelsof mRNA of hippocampal neuron bcl-2 and bax gene were detected with reverse transcription polymerase chain reaction (RT-PCR); expression of hippocampal neuron Bcl-2 and Bax protein was measured with Western blot technique. MAIN OUTCOME MEASURES: ① Effect of ( -) clausenamide on survival rate of SNP-induced hippocampal neuron apoptosis; ② bcl-2 and bax mRNA and protein expression of hippocampal neurons. RESULTS: ① Survival rate of hippocampal neurons: Survival rate of hippocampal neurons affected by 0.4-1.6 μmol / L (-) clausenamide was higher in the experimental group than the model group (P <0.01), and the survival rate was increased with the larger volume of (-) clausenamide. Survival rate was the highest when hippocampal neurons were induced by 1.6 μmol / (P <0.01) .②Expression of bcl-2 and bax mRNA: Hippocampal neurons were pretreated with 0.2-1.6 μmol / L clausenamide for 6 hours in the experimental group and strap of PCR product of bcl-2 gene was brightened gradually. This suggested that, with the increase of concentration, expression of bcl-2 mRNA was increased simultaneously. However, when strap of PCR product of bax gene was darkened, expression of bax was decreased with the increase of concentration. ③ Expression of Bcl-2 and Bax protein: Hippocampal neurons were pretreated with 0.2-1.6 μmol / L (-) clausenamide for 6 hours in the experimental group and strap of PCR product of Bcl-2 protein was thickened gradually . This suggested that, with the increase of concentration, expression of Bcl-2 protein was thinned, expression of Bax protein was decreased with the increase of concentration. CONCLUSION: (- ) clausenamide can resist neurotoxic effect of SNP through dosage dependence, and the mechanism may be related to promoting expression of anti-apoptotic bcl-2 gene and inhibiting expression of pro-apoptotic bax gen e.