中文摘要：

目的研究β2-肾上腺素受体激动剂clenbuterol对原代培养的心肌细胞缺氧/复氧损伤的作用及其是否与激活再灌注损伤挽救激酶（reperfusion injury salvage kinase,RISK）信号通路有关.方法将原代培养的新生Wistar大鼠乳鼠心肌细胞分为8组,①正常培养组;②缺氧/复氧（A/R）组;③clenbuterol（1μmol·L^-1）＋A/R;④ICI118,551（10μmol·L^-1）＋clenbuterol（1μmol·L^-1）＋A/R组;⑤美托洛尔metoprolol（10μmol·L^-1）＋ clenbuterol（1μmol·L^-1）＋A/R组;⑥ metoprolol（10μmol·L^-1）＋A/R组;⑦ PD98059（20μmol·L^-1）＋clenbuterol（1μmol·L^-1）＋A/R组;⑧LY294002（10μmol·L^-1）＋clenbuterol（1μmol·L^-1）＋A/R组.采用MTT法测定各组细胞存活率;比色法检测心肌细胞培养液的乳酸脱氢酶（LDH）含量;Hoechst33342荧光染色法检测细胞凋亡率;分子探针DCFH-DA检测细胞内活性氧的水平;Westernblot检测心肌细胞缺氧/复氧后ERK及p-ERK1/2蛋白的表达水平.结果与A/R组比较,clen-buterol＋A/R组明显增高细胞存活率,降低LDH含量,降低细胞凋亡率,ROS产生减少,p-ERK1/2蛋白表达水平增高,而选择性β2受体阻断剂ICI118,551可取消clenbuterol的上述作用,β1受体阻断剂Metoprolol对clenbuterol的作用无影响,PI3K抑制剂LY294002和ERK1/2抑制剂PD98059可阻断clenbuterol对心肌细胞缺氧/复氧损伤的保护作用.结论clenbuterol能够减轻心肌细胞缺氧/复氧损伤,加入选择性β2受体阻断剂ICI118,551,PI3K抑制剂LY294002和ERK抑制剂PD98059均使clenbuterol的保护作用取消,表明clenbuterol可通过激动β2肾上腺素受体,激活RISK信号通路发挥抗心肌细胞缺氧/复氧损伤的作用.

英文摘要：

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.