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Acta Pharmacologica Sinica 2007 June; 28 (6): 803-810; doi 10.1111/j.1745-7254.2007.00472.x |
| Original Article | [ Full text ] |
| 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 |
Bei ZHOU1, Li-jun WU2, Shin-ichi TASHIRO3, Satoshi ONODERA3, Fumiaki UCHIUMI4, Takashi IKEJIMA1,5 1China-Japan Research Institute of Medical and Pharmaceutical Sciences, Shenyang Pharmaceutical University, Shenyang 110016, China; 2Department of Phytochemistry, Shenyang Pharmaceutical University, Shenyang 110016, China; 3Department of Clinical and Biomedical Sciences, Showa Pharmaceutical University, Tokyo 194-8543, Japan; 4Department of Gene Regulation, Faculty of Pharmaceutical Sciences, Tokyo University of Science, Chiba 278-8510, Japan |
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.
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Keywords: silibinin; rat cardiac myocytes; tyrosine kinase; protein kinase C; mitogen-activated protein kinases |
| 1 This study was supported by the Korea Research Foundation Grant funded by Korean Government (MOEHRD; No KRF-2005-206-E00007). |
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