Protective effect of ultra low molecular weight heparin on glutamate-induced apoptosis in cortical cells.
OBJECTIVE
To investigate the effect of ultra low molecular weight heparin (ULMWH) on glutamate induced apoptosis in rat cortical cells and to explore the possible mechanisms.


METHODS
Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was first analyzed with Hoechst 33258 and then confirmed by DNA fragmentation. The concentration of free intracellular calcium ([Ca(2+)](i)) was determined with fura-2/AM fluorometry. The expression of Bcl-2 family protein and caspase-3 were evaluated with Western blot.


RESULTS
Typical apoptotic morphological change in rat cortical cells treated with 100 micromol/L glutamate for 24h was detected by Hoechst 33258 staining, which was then confirmed by the DNA ladder of agarose gel electrophoresis. The apoptotic rate of the glutamate treated cells was up to 33.21%, and 24 h of treatment with glutamate increased [Ca(2+)](i), down-regulated Bcl-2 expression, up-regulated Bax expression, and increased caspase-3 activation in rat cortical cells. Our research demonstrated that ULMWH pretreatment can prevent the glutamate-induced apoptosis, attenuate the increase of [Ca(2+)](i) not only in medium containing Ca(2+) but also in Ca(2+)-free medium, up-regulate the expression of Bcl-2, down-regulate the expression of Bax, and decrease caspase-3 activation.


CONCLUSIONS
ULMWH has neuroprotective capacity to antagonize glutamate-induced apoptosis in cortical cells, through decrease of Ca(2+) release and modulation of apoptotic processes.
Yu
Tian-Gui
TG
Pharmacological Institute of New Drugs, School of Pharmacy, Shandong University, 44 Wen Hua Xi Road, Jinan 250012, PR China.
Zhang
Qing-Zhu
QZ
Zhang
Zhi-Guo
ZG
Wang
Wei-Wei
WW
Ji
Sheng-Li
SL
Du
Guan-Hua
GH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism; D053148:Caspase 3 / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D053938:DNA Fragmentation / Q000187:drug effects; D018698:Glutamic Acid / Q000494:pharmacology*; D006495:Heparin, Low-Molecular-Weight / Q000494:pharmacology*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D017208:Rats, Wistar; D051028:bcl-2-Associated X Protein / Q000378:metabolism
D006495:Heparin, Low-Molecular-Weight; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein; D018698:Glutamic Acid; D053148:Caspase 3; D002118:Calcium