CAJ | 학술논문

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；Hoechst 33342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；Western blot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ICI 118,551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 ICI 118,551,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。
목적：연구β2-신상선소수체격동제clenbuterol대원대배양적심기세포결양/복양손상적작용급기시부여격활재관주손상만구격매( reperfusion injury salvage kinase, RISK)신호통로유관。방법장원대배양적신생Wistar대서유서심기세포분위8조,①정상배양조；②결양/복양( A/R)조；③clenbuterol(1μmol·L-1)+A/R；④ICI118,551(10μmol ·L-1)+clenbuterol(1μmol·L-1)+A/R조；⑤미탁락이metoprolol(10μmol·L-1)+ clenbuterol (1μmol·L-1)+A/R조；⑥ metoprolol(10μmol·L-1)+A/R조；⑦ PD98059(20μmol·L-1)+clenbuterol(1μmol·L-1)+A/R 조；⑧LY294002(10μmol·L-1)+clenbuterol(1μmol·L-1)+A/R조。채용MTT법측정각조세포존활솔；비색법검측심기세포배양액적유산탈경매( LDH)함량；Hoechst 33342형광염색법검측세포조망솔；분자탐침DCFH-DA검측세포내활성양적수평；Western blot검측심기세포결양/복양후ERK급p-ERK1/2단백적표체수평。결과여 A/R 조비교, clen-buterol+ A/R조명현증고세포존활솔,강저LDH 함량,강저세포조망솔, ROS산생감소, p-ERK1/2단백표체수평증고,이선택성β2수체조단제ICI 118,551가취소clenbuterol적상술작용,β1수체조단제Metoprolol대clenbuterol적작용무영향,PI3K억제제LY294002화ERK1/2억제제PD98059가조단clenbuterol대심기세포결양/복양손상적보호작용。결론 clenbuterol능구감경심기세포결양/복양손상,가입선택성β2수체조단제 ICI 118,551,PI3K 억제제 LY294002화ERK억제제PD98059균사clenbuterol적보호작용취소,표명clenbuterol가통과격동β2신상선소수체,격활RISK신호통로발휘항심기세포결양/복양손상적작용。
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.