[Suppressive effects of par-4 antisense oligodeoxynucleotide on up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects].
OBJECTIVE
To investigate the uppressive effects of par-4 antisense oligodeoxynucleotide on the up-regulation of intracellular calcium concentration in PC12 cell induced by glutamate and its anti-apoptosis effects.


METHODS
Cationic lipid-mediated par-4 antisense oligodeoxynucleotide (par-4-AS-ODN) was transfected into PC12 cells and followed by glutamate for treatment. Mismatch oligodeoxy-nucleotide (MS-ODN) was used as the control. Morphological assessment and evaluation of the anti-apoptosis effects of par-4-AS-ODN on PC12 cells were performed by laser scanning confocal microscopy by double staining of the cells with Hoechest 33258/propidium iodide (Hoe/PI) and flow cytometry respectively. The mRNA and protein levels of calpain 10 and par-4 were measured by RT-PCR and Western blot. Intracellular calcium concentration was determined by using laser scanning confocal microscope with Fura-2/AM as the fluorescent dye.


RESULTS
Par-4-AS-ODN repress the increase of par-4 protein in PC12 cell (52.3 +/- 5.0 vs 90.0 +/- 3.2, < 0.01). Par-4-AS-ODN significantly inhibited the apoptosis of PC12 cells induced by glutamate (53% vs 31%, < 0.01). Par-4-AS-ODN significantly suppress the up-regulation of intracellular calcium concentration in PC12 cells induced by glutamate (Rate of fluorescent density: 167.9 +/- 32.4 vs 228.8 +/- 36.8, < 0.01). Par-4-AS-ODN inhibited the increase of calpain 10 mRNA in PC12 cells induced by glutamate (46.3 +/- 3.7 vs 34.8 +/- 2.1, < 0.01).


CONCLUSIONS
par-4-AS-ODN enables to inhibit apoptosis of PC12 cells induced by glutamate. The mechanism of the inhibition may be closely related to suppression of the up-regulation of intracellular calcium concentration and calpain transcription expression.
Lu
Chao
C
Department of Pediatrics, First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, China. luchao8009@sohu.com
Chen
Ji-qing
JQ
Wu
Sheng-hua
SH
Zhao
Fei
F
Chi
Xia
X
Pan
Xiao-qin
XQ
Fei
Li
L
Guo
Mei
M
Huang
Song-ming
SM
Guo
Xi-rong
XR
Chen
Rong-hua
RH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins / Q000096:biosynthesis* / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D002154:Calpain / Q000096:biosynthesis / Q000235:genetics; D018698:Glutamic Acid / Q000494:pharmacology; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D016716:PC12 Cells; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D014162:Transfection; D015854:Up-Regulation
D051017:Apoptosis Regulatory Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D012333:RNA, Messenger; C101296:prostate apoptosis response-4 protein; D018698:Glutamic Acid; D002154:Calpain; C415197:calpain 10; D002118:Calcium