Activation of extracellular signal-regulated kinase during silibinin-protected, isoproterenol-induced apoptosis in rat cardiac myocytes is tyrosine kinase pathway-mediated and protein kinase C-dependent.
OBJECTIVE
To investigate the mechanism of silibinin-protected isoproterenol-induced apoptosis in rat cardiac myocytes.


METHODS
The viability of rat cardiac myocytes was measured by MTT method. The apoptotic ratio was measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling. Protein kinase C (PKC) activity assay was carried out according to the instructions of the PepTag non-radioactive protein kinase C assay kit. Western blot analysis was used to evaluate the level of Ras, Raf-1 and mitogen-activated protein kinase (MAPK) expression.


RESULTS
The protective effects of silibinin were significantly suppressed by inhibitors, including genistein, manumycin A and GW5074 [inhibitors for protein tyrosine kinases (PTK), Ras and Raf-1, respectively]. The exposure of rat cardiac myocytes to isoproterenol alone caused decreased PKC activity, which was prevented by pretreatment with silibinin dose-dependently. Simultaneously, the increased expression of Ras and Raf-1 activated by silibinin were blocked by the PKC inhibitor, stauroporine. In addition, the extracellularly responsive kinase (ERK) inhibitor, PD98059, suppressed silibinin-protected apoptosis, whereas the p38 MAPK inhibitor, SB203580, protected cardiac myocytes from isoproterenol-induced injury, and the c-Jun N-terminal kinase (JNK) inhibitor, SP600125 had no protective effects. Furthermore, Western blot analysis showed that the expression of phosphorylated ERK was increased by silibinin, the expression of phosphorylated p38 MAPK was decreased and total ERK, p38, JNK and phosphorylated JNK MAPK did not change after treatment with both isoproterenol and silibinin. Furthermore, pretreatment of cardiac myocyte with PKC, Ras and Raf inhibitors significantly blocked ERK phosphorylation.


CONCLUSIONS
Silibinin is suggested to protect isoproterenol-induced rat cardiac myocyte apoptosis by activating the tyrosine kinase pathway, PKC and MAPK pathways.
Zhou
Bei
B
China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China. ikejimat@vip.sina.com
Wu
Li-Jun
LJ
Tashiro
Shin-Ichi
S
Onodera
Satoshi
S
Uchiumi
Fumiaki
F
Ikejima
Takashi
T
D000318:Adrenergic beta-Agonists / Q000494:pharmacology*; D000818:Animals; D000975:Antioxidants / Q000737:chemistry / Q000378:metabolism; D017209:Apoptosis / Q000187:drug effects*; D002478:Cells, Cultured; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D006801:Humans; D007545:Isoproterenol / Q000494:pharmacology*; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism; D015394:Molecular Structure; D032383:Myocytes, Cardiac / Q000166:cytology / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000502:physiology; D000077385:Silybin; D012838:Silymarin / Q000737:chemistry / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
D000318:Adrenergic beta-Agonists; D000975:Antioxidants; D004791:Enzyme Inhibitors; D012838:Silymarin; D000077385:Silybin; D011505:Protein-Tyrosine Kinases; D011493:Protein Kinase C; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D007545:Isoproterenol