Role of RISK signal pathway in reducing clenbuterol-induced cardiomycytes A/R injury of neonatal rat / 中国药理学通报

ABSTRACT

Aims To study the effects of clenbuterol on anoxia/reoxygenation( A/R) injury in neonatal Wistar rat cardiomyocytes and to explore whether its mecha-nism is related to reperfusion injury salvage kinase ( RISK) or not. Methods The cultured primary neo-natal cardiomyocytes were randomly divided into eight groups ①normal culture group; ②anoxia/reoxygen-ation( A/R) group;③ clenbuterol ( 1 μmol · L-1 ) +A/R;④ICI118,551(10 μmol·L-1) + clenbuterol ( 1 μmol · L-1 ) + A/R; ⑤Metoprolol ( 10μmol · L-1 ) + clenbuterol(1μmol·L-1 ) + A/R group;⑥Metoprolol ( 10 μmol · L-1 ) + A/R group; ⑦PD98059 ( 20 μmol · L-1 ) + clenbuterol ( 1 μmol · L-1 ) + A/R group;⑧ LY294002(10 μmol·L-1 ) +clenbuterol(1 μmol · L-1 ) + A/R group. Cell via-bility was determined by the conventional MTT reduc-tion assay. The content of LDH in cultured medium was measured with colorimetry. Cardiomyocyte apopto-sis was determined by Hoechst33342 . Intracellular re-active species( ROS) were monitored by the fluorescent DCFH-DA. Total ERK2 and phosphorylated ERK were detected by western blot. Results Compared with A/R group, clenbuterol significantly increased vaibility of cells, reduced LDH release, lowered the rate of apop-tosis and ROS production. When addedβ2 receptor an-tagonist ICI118 , 551 , PI3 K inhibitor LY294002 and ERK inhibitor PD98059 , the effects of clenbuterol a-bove were inhibited; but β1 receptor antagonist Meto-prolol protected the cardiomyocytes from A/R injury, as evidenced by decreased LDH release and increased cell viability. There were no synergistic effects in the combined use of clenbuterol and Metoprolol. Conclu-sion clenbuterol exerts cardioprotective effects against A/R injury by inhibiting oxidative stress and apopto-sis. The protection of clenbuterol is inhibited by ICI118 , 551 , LY294002 and PD98059 . clenbuterol protects cardiomyocytes against A/R injury via RISK pathway by activation of β2 receptor.