[Effect of antisense oligonucleotide against Smac/DIABLO on inhibition of hydrogen peroxide induced myocardial apoptosis of neonatal rats].
OBJECTIVE
To investigate the effects of antisense phosphorothioate oligonucleotides against Smac/DIABLO (asODN) on hydrogen peroxide (H2O2) induced myocardial apoptosis in neonatal rats.


METHODS
Primary myocardial cells from neonatal rats were cultured in vitro, and randomly divided into A (normal control, without transfection), B (with treatment of single liposome), C (with transfection of scrODN), D (with transfection of asODN), E (with H2O2, stimulation), F (with H2O2 stimulation after scrODN transfection), and G (with H2O2 stimulation after asODN transfection) groups. The expression of asODN mRNA and protein were determined with RT-PCR and Western blotting, respectively. The changes in cellular nuclear morphology were observed with 33258 fluorescent staining, and the percentage of nuclear apoptosis was calculated. DNA fragmentation was observed by agarose gel electrophoresis. Activation of caspase-3 and caspase-9 were evaluated by caspase colorimetric analysis kit.


RESULTS
The expression of Smac/DIABLO mRNA and protein was obviously inhibited by asODN, which was about 80% percent lower than the protein level in A,B and C groups, but there was no difference noted among A,B and C groups( P > 0.05). Not only the nuclear apoptotic percentage, but also the activity of caspase-3 and caspase-9 in A, C and D groups were in very low levels. But these indices in G group 24 hours after H2O2 stimulation were obviously lower than that in E and F groups [the nuclear apoptotic percentage were (19 +/- 5) %, (52 +/- 3) %, (55 +/- 5) %, respectively, P < 0.01)]. The DNA ladders in G group were also decreased markedly.


CONCLUSIONS
Myocardial apoptosis induced by H2O2 can be inhibited by asODN in rat.
Liang
Peng-fei
PF
Departnent of Burns and Plastic Surgery, Xiangya Hospital, Central Southern University, Changsha 410008, PR China.
Huang
Xiao-yuan
XY
Long
Jian-hong
JH
Xiao
Mu-zhang
MZ
Yang
Xing-hua
XH
Zhang
Pi-hong
PH
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D053148:Caspase 3 / Q000378:metabolism; D053453:Caspase 9 / Q000378:metabolism; D002478:Cells, Cultured; D006861:Hydrogen Peroxide; D024101:Mitochondrial Proteins / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects* / Q000378:metabolism; D020319:Oligodeoxyribonucleotides, Antisense / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar
D051017:Apoptosis Regulatory Proteins; D002352:Carrier Proteins; C489013:DIABLO protein, rat; D024101:Mitochondrial Proteins; D020319:Oligodeoxyribonucleotides, Antisense; D006861:Hydrogen Peroxide; D053148:Caspase 3; D053453:Caspase 9