ABSTRACT
BACKGROUND: β-amyloid has been proved to be capable of inducing cell apoptosis and play a vital role in Alzheimer disease (AD).OBJECTIVE: To probe into olanzapine's protective effect and mechanism of PC12 cell apoptosis induced by β-amyloid 25-35.SETTING: Department of Neurology, Southern Building of the General Hospital of Chinese PLA.DESIGN: Randomized design.MATERIALS: This experiment was carried out in the Neuropsychopathic Research Institute, Medical College of the University of Saskatchewan(Canada), between May 2002 and March 2003.METHODS: P12 cells were cultured with RPMI1640 culture medium.100 μL cell suspension was inoculated in each well of 96-well culture plate, and 5 mL suspension was inoculated in 25 cm2 culture bottle covered with collagen and cultured for 24 hours, then with additional 50 μmol/L and 100 μmol/L olanzapine, respectively, for 24 hours, and β-amyloid 25-35 of different concentrations (0.01 μmol/L, 2 μmol/L and 20 μmol/L) for 24 hours. PC12 cell apoptosis was induced by β-amyloid 25-35 in 96-well culture plate and cells were harvested to assay their survival rate with MTF colorimetric assay. PC12 cells in 25-cm2 culture bottles were also harvested to detect the effect of olanzapine on Bax and caspase-3 expression in PC12 cells using Western blot assay.MAIN OUTCOME MEASURES: ① Cell survival rate; ② the expression of Bax and caspase-3 in PC12 cells.RESULTS: ① Cell survival rate: cell activity was found declined from 75% to 35% in PC12 cells induced by β-amyloid 25-35, but obviously increased in PC12 cells due to pretreatment with olanzapine of 50 μmol/L and 100 μmol/L. ② Olanzapine's effects on Bax expression in PC12 cell apoptosis induced by β-amyloid 25-35: Bax expression increased in PC12cells due to exposure to β-amyloid 25-35 of 0.01 μmol/L, 2 μmol/L and 20 μmol/L, but it could be suppressed if pretreated with olanzapine of 50 μmol/L. ③ Effect of olanzapine on caspase-3 expression in PC12apoptotic cells induced by β-amyloid 25-35: There was no change in PC12cells induced by 0.001 μmol/L or 0.01 μmol/L of β-amyloid, as well as in PC12 cells pretreated with 50 μmol/L olanzapine. However, caspase-3 expression obviously increased in PC12 cells exposed to 2 μmol/L and 20 μmol/L of β-amyloid 25-35, and it could be suppressed by pretreatment with 50 μmol/L of olanzapine.CONCLUSION: ① β-amyloid 25-35 can induce the high expression of cell apoptosis related Bax and caspase-3 in vitro cultured PC12 cells. ②Olanzapine can reduce the expression, thus enhancing the survival rate of PC 12 cells.
ABSTRACT
The neurofilament proteins (NFPs) from different neuronal tissues including Alzheimer and Huntington disease gray matter, rat brain gray, white matter and spinal cord were separated biochemically into two major fractions. A systematic investigation on the distribution, expression and phosphorylation of NFPs in those fractions was undertaken in the present study. It was found that only non-phosphorylated NF-H and NF-M, but not NF-L subunit were detected in Alzheimer brain gray matter high speed supernatant, whereas all neurofilament subunits including non-phosphorylated and phosphorylated were measured in high speed pellet fraction of the same tissue. The hyperphosphorylation of NF-H and NF-M in Alzheimer brain was shown by phosphorylation dependent monoclonal antibodies SMI31 and SMI34. This hyperphosphorylation was confirmed by non-phosphorylation dependent antibody SMI32 with dephosphosphorylation of the samples. Furthermore, an increased amount of NF-H, NH-M and NF-L, detected by SMI33 and NR4 respectively, was also observed in Alzheimer samples, in which the elevation in NF-L was significant. A significantly different immunoblot patterns in distribution, expression and phosphorylation were determined in various position of the neural system and alternative fractions. To our best knowledge, this is the first data shown definite abnormality of NFPs in Alzheimer disease. The information obtained in the present study will be extremely valuable in further study of the proteins both in physiological and pathological conditions.
ABSTRACT
Objective To investigate the mechanism of the protective effects of olanzapine against apoptosis of PC12 cells induced by ?-amyloid peptide 25-35 (A?_ 25-35 ). Methods Based on the model of apoptosis of PC12 cells induced by A?_ 25-35 , cell viability was determined by MTT assay. The expressions of Bax, Caspase-3 of PC12 cells induced by A?_ 25-35 and olanzapine were assessed by Western blot. Results 10 -14 -10 -5 mol/L A?_ 25-35 lowered the cell viability of PC12 cells, 50?mol/L and 100?mol/L olanzapine pretreatment enhanced the cell viability of PC12 cells, and there was significant difference compared with olanzapine non-pretreated groups (P