β_2-肾上腺素受体激动剂对大鼠心肌缺血/再灌注损伤的保护作用及机制的研究
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摘要
第一部分选择性β2受体激动剂对麻醉大鼠心肌缺血再灌注损伤的保护作用及其机制的研究
目的：许多研究表明,β2-受体激动剂Clenbuterol在缺血性心肌病模型中可降低心肌细胞凋亡,并在临床上已应用于心衰的治疗。本实验通过在体大鼠的心肌缺血／再灌损伤模型,观察Clenbuterol对心肌缺血再灌注损伤的保护作用,并探讨其机制。
方法：大鼠随机分6组：①假手术组；②缺血／再灌组(A/R)；③Clenbuterol+A/;④CI118,551+Clenbuterol+A/; (5)Metoprolol+ Clenbuterol+A/R;⑥Metoprolo+A/R.麻醉大鼠,采用结扎左冠状动脉前降支30min后再灌2h,建立急性心肌缺血再灌注损伤模型。
结果：与A/R比较,Clenbuterol组显著减少梗死面积,提高心肌舒张功能,降低血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)和脂质过氧化物丙二醛(MDA)的含量,并增加超氧化物酶(SOD)和Ca2+-ATPase的活性,减轻心肌细胞水肿、充血和损伤程度,抑制心肌细胞凋亡率。Clenbuterol还可促进缺血／再灌心肌组织ERK1／2磷酸化,提高缺血／再灌后心肌组织Bcl-2/Bax mRNA水平,降低caspase-3蛋白的表达,增强机体抗凋亡能力。以上作用均可被选择性β2受体阻断剂ICI118,551所取消。而Gi蛋白抑制剂百日咳毒素(PTX)可阻断clenbuterol咸少心肌梗死面积和改善心功能的作用。除了不能降低MDA含量和提高Ca2+-ATPase活性的作用外,β1受体阻断剂]metoprolol与clenbuterol对缺血再灌后心肌作用相似,但两者合用没有协同作用。
结论：Clenbuterol对缺血再灌损伤的心肌组织有显著的保护作用,其保护作用是通过激活β2受体-Gi-ERK1/2信号途径。β2-受体激动剂clenbuterol-与B1受体阻断剂1netoprololl司无协同作用。
第二部分β2-AR激动剂对心肌细胞缺氧/复氧损伤的影响
目的：探讨Clenbuterol对原代培养的心肌细胞缺氧／复氧后损伤的影响以及是否通过细胞外信号调节激酶(ERK)、磷酸肌醇3激酶(P13K)-蛋白激酶Akt途径。
方法：建立原代新生大鼠心肌细胞的缺氧／复氧模型,采用MTT比色法测细胞生存率；Hoechst33342荧光染色法和Annexin-PI双染法检测细胞凋亡率；以及检测细胞培养液中的LDH活性评价缺氧／复氧及相应处理后的细胞损伤程度；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Bcl-2、Bax、Caspase-3以及ERK及p-ERK1/2蛋白的表达。
结果：
1.缺氧／复氧组心肌细胞存活率较正常培养组显著下降,LDH活性,凋亡率增加,ROS含量增加；而Clenbuterol组、Metoprolol组及Clenbuterol+Metoprolol组的细胞存活率显著增高,ROS含量减少,但三者间无显著性差异；选择性β2受体阻断剂ICI118,551S可取消Clenbuterol的作用。
2.与缺氧／复氧组比较,Clenbuterol处理的缺氧／复氧后心肌细胞Bcl-2,p-ERK蛋白增加,而Bax,Caspase-3表达下降；而PI3K抑制剂LY294002和ERK抑制剂PD98059可使Clenbuterol+缺氧／复氧组的细胞存活率下降,凋亡细胞增多,Bax,Caspase-3表达增加,而Bcl-2表达降低；PD98059可使Clenbuterol+缺氧／复氧组p-ERK表达下降,但LY294002无此作用。
结论：Clenbuterol对缺氧／复氧后的心肌有保护作用,但与Metoprolol间无协同作用；并且ERK和PI3K/Akt途径也参与其中。
Objective:Considerable evidence indicates that theβ2-adrenoceptor agonist clenbuterol could decrease apoptosis in a rodent model of ischemic cardiomyopathy. In this study, we investigated the effects of clenbuterol on infarct size caused by myocardial ischemia/reperfusion (I/R) in anesthetized rats.
Methods:Rats were randomized to:1) sham group,2) I/R group,3) clenbuterol +I/R group,4) ICI 118551+clenbuterol+I/R group,5) metoprolol+clenbuterol+I/R group,6) metoprolol+I/R group,7) Pertussis toxin+clenbuterol+I/R group. Under anesthesia, left anterior descending (LAD) coronary artery was occluded for 30 min followed by reperfusion for 2 h.
Results:Compared with the control I/R group, clenbuterol (0.5mg-kg-1, i.p) had reduced infarct size, improved diastolic function and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activity, increased SOD activity, and decreased MDA level and LDH, CK release. Clenbuterol increased the phosphorylation of ERK1/2, which resulted in inhibition of myocardial apoptosis as evidenced by the reduction of TUNEL-positive staining, Bax/Bcl-2 mRNA and Caspase-3 protein expression. The G,-protein inhibitor pertussis toxin blocked the clenbuterol-induced improvement in cardiac function and infarct size. Pretreatment with ICI 118551 (a selectiveβ2-AR antagonist) inhibited the effects of clenbuterol mentioned above (P<0.05). Theβ1-AR agonist metoprolol had the similar effects compared with the clenbuterol-treated group but failed to reduce MDA and improve SERCA activity. As a combination therapy, there were no synergistic effects of metoprolol+clenbuterol treatment.
Conclusions:Clenbuterol pretreatment provides a significant cardioprotection mediated by theβ2-AR-Gi-protein signaling against ischemia/reperfusion injury. There are no synergistic effects in the combined use of clenbuterol and metoprolol.
Objective:To investigation the effect of clenbuterol on anoxia/reoxygenation (A/R) injury in neonatal rat cardiomyocytes. In addition, we have explored possible mechanisms for the effects.
Methods:To establish the model of anoxia/reoxygenation injury in primary neonatal cardiomyocytes. Cell viability was determined by the conventional MTT reduction assay; cell injury by lactate dehydrogenase(LDH) leakage; and cardiomyocyte apoptosis by Hoechst33342 and Annexin-PI staining; Intracellular reactive species(ROS) was monitored by the fluorescent DCFH-DA; Expression of apoptosis related factor Bcl-2 and Bax, phosphorylated ERK,caspase-3 were determined by western blot.
Results:Clenbuterol and metoprolol protected the cardiomyocytes from anoxia/reoxygenation injury, as evidenced by decreased LDH release and increased cell viability. This protective effect concomitantly decreased ROS production and apoptosis in cardiomyocytes. But selectiveβ2 receptor antagonist ICI 118,551 could abolish the effects of clenbuterol. There are no synergistic effects in the combined use of clenbuterol and metoprolol.
In clenbuterol-pretreated cardiomyocytes, we observed a decrease in the expression of pro-apoptotic protein Bax and caspase-3.We also found that clenbuterol pretreatment enhanced expression of anti-apoptotic protein Bcl-2 and activation of ERK concerning survival. The effects of clenbuterol were reversed by PI3K inhibitor LY294002 and ERK inhibitor PD98059. Only in in phosphorylated ERK1/2, there was no obvious difference between LY294002+clen+A/R group and clen+A/R group.
Conclusion:Clenbuterol exerts cardioprotective effect against Anoxia/reoxygenation injury by inhibiting oxidative stress and apoptosis. Clenbuterol pretreatment increase the Bcl-2/Bax ratio and phosphorylation of ERK, decrease caspase-3 express. The respective inhibitor of ERK and PI3K, PD98059 and LY294002, prevent the anti-apoptotic effect. But only PD98059 attenuated the increasing phosphorylation of ERK of clenbuterol. So the ERK and PI3K/Akt pathway may be involved in the cardioprotective effect of clenbuterol
目的：许多研究表明,β2-受体激动剂Clenbuterol在缺血性心肌病模型中可降低心肌细胞凋亡,并在临床上已应用于心衰的治疗。本实验通过在体大鼠的心肌缺血／再灌损伤模型,观察Clenbuterol对心肌缺血再灌注损伤的保护作用,并探讨其机制。
方法：大鼠随机分6组：①假手术组；②缺血／再灌组(A/R)；③Clenbuterol+A/;④CI118,551+Clenbuterol+A/; (5)Metoprolol+ Clenbuterol+A/R;⑥Metoprolo+A/R.麻醉大鼠,采用结扎左冠状动脉前降支30min后再灌2h,建立急性心肌缺血再灌注损伤模型。
结果：与A/R比较,Clenbuterol组显著减少梗死面积,提高心肌舒张功能,降低血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)和脂质过氧化物丙二醛(MDA)的含量,并增加超氧化物酶(SOD)和Ca2+-ATPase的活性,减轻心肌细胞水肿、充血和损伤程度,抑制心肌细胞凋亡率。Clenbuterol还可促进缺血／再灌心肌组织ERK1／2磷酸化,提高缺血／再灌后心肌组织Bcl-2/Bax mRNA水平,降低caspase-3蛋白的表达,增强机体抗凋亡能力。以上作用均可被选择性β2受体阻断剂ICI118,551所取消。而Gi蛋白抑制剂百日咳毒素(PTX)可阻断clenbuterol咸少心肌梗死面积和改善心功能的作用。除了不能降低MDA含量和提高Ca2+-ATPase活性的作用外,β1受体阻断剂]metoprolol与clenbuterol对缺血再灌后心肌作用相似,但两者合用没有协同作用。
结论：Clenbuterol对缺血再灌损伤的心肌组织有显著的保护作用,其保护作用是通过激活β2受体-Gi-ERK1/2信号途径。β2-受体激动剂clenbuterol-与B1受体阻断剂1netoprololl司无协同作用。
第二部分β2-AR激动剂对心肌细胞缺氧/复氧损伤的影响
目的：探讨Clenbuterol对原代培养的心肌细胞缺氧／复氧后损伤的影响以及是否通过细胞外信号调节激酶(ERK)、磷酸肌醇3激酶(P13K)-蛋白激酶Akt途径。
方法：建立原代新生大鼠心肌细胞的缺氧／复氧模型,采用MTT比色法测细胞生存率；Hoechst33342荧光染色法和Annexin-PI双染法检测细胞凋亡率；以及检测细胞培养液中的LDH活性评价缺氧／复氧及相应处理后的细胞损伤程度；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Bcl-2、Bax、Caspase-3以及ERK及p-ERK1/2蛋白的表达。
结果：
1.缺氧／复氧组心肌细胞存活率较正常培养组显著下降,LDH活性,凋亡率增加,ROS含量增加；而Clenbuterol组、Metoprolol组及Clenbuterol+Metoprolol组的细胞存活率显著增高,ROS含量减少,但三者间无显著性差异；选择性β2受体阻断剂ICI118,551S可取消Clenbuterol的作用。
2.与缺氧／复氧组比较,Clenbuterol处理的缺氧／复氧后心肌细胞Bcl-2,p-ERK蛋白增加,而Bax,Caspase-3表达下降；而PI3K抑制剂LY294002和ERK抑制剂PD98059可使Clenbuterol+缺氧／复氧组的细胞存活率下降,凋亡细胞增多,Bax,Caspase-3表达增加,而Bcl-2表达降低；PD98059可使Clenbuterol+缺氧／复氧组p-ERK表达下降,但LY294002无此作用。
结论：Clenbuterol对缺氧／复氧后的心肌有保护作用,但与Metoprolol间无协同作用；并且ERK和PI3K/Akt途径也参与其中。
Objective:Considerable evidence indicates that theβ2-adrenoceptor agonist clenbuterol could decrease apoptosis in a rodent model of ischemic cardiomyopathy. In this study, we investigated the effects of clenbuterol on infarct size caused by myocardial ischemia/reperfusion (I/R) in anesthetized rats.
Methods:Rats were randomized to:1) sham group,2) I/R group,3) clenbuterol +I/R group,4) ICI 118551+clenbuterol+I/R group,5) metoprolol+clenbuterol+I/R group,6) metoprolol+I/R group,7) Pertussis toxin+clenbuterol+I/R group. Under anesthesia, left anterior descending (LAD) coronary artery was occluded for 30 min followed by reperfusion for 2 h.
Results:Compared with the control I/R group, clenbuterol (0.5mg-kg-1, i.p) had reduced infarct size, improved diastolic function and sarcoplasmic/endoplasmic reticulum Ca2+-ATPase (SERCA) activity, increased SOD activity, and decreased MDA level and LDH, CK release. Clenbuterol increased the phosphorylation of ERK1/2, which resulted in inhibition of myocardial apoptosis as evidenced by the reduction of TUNEL-positive staining, Bax/Bcl-2 mRNA and Caspase-3 protein expression. The G,-protein inhibitor pertussis toxin blocked the clenbuterol-induced improvement in cardiac function and infarct size. Pretreatment with ICI 118551 (a selectiveβ2-AR antagonist) inhibited the effects of clenbuterol mentioned above (P<0.05). Theβ1-AR agonist metoprolol had the similar effects compared with the clenbuterol-treated group but failed to reduce MDA and improve SERCA activity. As a combination therapy, there were no synergistic effects of metoprolol+clenbuterol treatment.
Conclusions:Clenbuterol pretreatment provides a significant cardioprotection mediated by theβ2-AR-Gi-protein signaling against ischemia/reperfusion injury. There are no synergistic effects in the combined use of clenbuterol and metoprolol.
Objective:To investigation the effect of clenbuterol on anoxia/reoxygenation (A/R) injury in neonatal rat cardiomyocytes. In addition, we have explored possible mechanisms for the effects.
Methods:To establish the model of anoxia/reoxygenation injury in primary neonatal cardiomyocytes. Cell viability was determined by the conventional MTT reduction assay; cell injury by lactate dehydrogenase(LDH) leakage; and cardiomyocyte apoptosis by Hoechst33342 and Annexin-PI staining; Intracellular reactive species(ROS) was monitored by the fluorescent DCFH-DA; Expression of apoptosis related factor Bcl-2 and Bax, phosphorylated ERK,caspase-3 were determined by western blot.
Results:Clenbuterol and metoprolol protected the cardiomyocytes from anoxia/reoxygenation injury, as evidenced by decreased LDH release and increased cell viability. This protective effect concomitantly decreased ROS production and apoptosis in cardiomyocytes. But selectiveβ2 receptor antagonist ICI 118,551 could abolish the effects of clenbuterol. There are no synergistic effects in the combined use of clenbuterol and metoprolol.
In clenbuterol-pretreated cardiomyocytes, we observed a decrease in the expression of pro-apoptotic protein Bax and caspase-3.We also found that clenbuterol pretreatment enhanced expression of anti-apoptotic protein Bcl-2 and activation of ERK concerning survival. The effects of clenbuterol were reversed by PI3K inhibitor LY294002 and ERK inhibitor PD98059. Only in in phosphorylated ERK1/2, there was no obvious difference between LY294002+clen+A/R group and clen+A/R group.
Conclusion:Clenbuterol exerts cardioprotective effect against Anoxia/reoxygenation injury by inhibiting oxidative stress and apoptosis. Clenbuterol pretreatment increase the Bcl-2/Bax ratio and phosphorylation of ERK, decrease caspase-3 express. The respective inhibitor of ERK and PI3K, PD98059 and LY294002, prevent the anti-apoptotic effect. But only PD98059 attenuated the increasing phosphorylation of ERK of clenbuterol. So the ERK and PI3K/Akt pathway may be involved in the cardioprotective effect of clenbuterol
引文
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第一部分选择性β2受体激动剂对麻醉大鼠心肌缺血再灌注损伤的保护作用及其机制的研究
目的：许多研究表明,β2-受体激动剂Clenbuterol在缺血性心肌病模型中可降低心肌细胞凋亡,并在临床上已应用于心衰的治疗。本实验通过在体大鼠的心肌缺血／再灌损伤模型,观察Clenbuterol对心肌缺血再灌注损伤的保护作用,并探讨其机制。
方法：大鼠随机分6组：①假手术组；②缺血／再灌组(A/R)；③Clenbuterol+A/R; ④ICI118,551+Clenbuterol+A/R; (5)Metoprolol+ Clenbuterol+A/R; ⑥Metoprolo+A/R.麻醉大鼠,采用结扎左冠状动脉前降支30min后再灌2h,建立急性心肌缺血再灌注损伤模型。
结果：与A/R比较,Clenbuterol组显著减少梗死面积,提高心肌舒张功能,降低血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)和脂质过氧化物丙二醛(MDA)的含量,并增加超氧化物酶(SOD)和Ca2+-ATPase的活性,减轻心肌细胞水肿、充血和损伤程度,抑制心肌细胞凋亡率。Clenbuterol还可促进缺血／再灌心肌组织ERK1／2磷酸化,提高缺血／再灌后心肌组织Bcl-2/Bax mRNA水平,降低caspase-3
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3. Timmers, L. et al. Exenatide reduces infarct size and improves cardiac function in a porcine model of ischemia and reperfusion injury. J Am Coll Cardiol 53,501-10 (2009).
4. Salorinne, Y., Stenius, B., Tukiainen, P. & Poppius, H. Double-blind cross-over comparison of clenbuterol and salbutamol tablets in asthmatic out-patients. Eur J Clin Pharmacol 8,189-95 (1975).
5. Wong, K., Boheler, K. R., Bishop, J., Petrou, M. & Yacoub, M. H. Clenbuterol induces cardiac hypertrophy with normal functional, morphological and molecular features. Cardiovasc Res 37,115-22(1998).
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第一部分选择性β2受体激动剂对麻醉大鼠心肌缺血再灌注损伤的保护作用及其机制的研究
目的：许多研究表明,β2-受体激动剂Clenbuterol在缺血性心肌病模型中可降低心肌细胞凋亡,并在临床上已应用于心衰的治疗。本实验通过在体大鼠的心肌缺血／再灌损伤模型,观察Clenbuterol对心肌缺血再灌注损伤的保护作用,并探讨其机制。
方法：大鼠随机分6组：①假手术组；②缺血／再灌组(A/R)；③Clenbuterol+A/R; ④ICI118,551+Clenbuterol+A/R; (5)Metoprolol+ Clenbuterol+A/R; ⑥Metoprolo+A/R.麻醉大鼠,采用结扎左冠状动脉前降支30min后再灌2h,建立急性心肌缺血再灌注损伤模型。
结果：与A/R比较,Clenbuterol组显著减少梗死面积,提高心肌舒张功能,降低血清中乳酸脱氢酶(LDH)、肌酸激酶(CK)和脂质过氧化物丙二醛(MDA)的含量,并增加超氧化物酶(SOD)和Ca2+-ATPase的活性,减轻心肌细胞水肿、充血和损伤程度,抑制心肌细胞凋亡率。Clenbuterol还可促进缺血／再灌心肌组织ERK1／2磷酸化,提高缺血／再灌后心肌组织Bcl-2/Bax mRNA水平,降低caspase-3
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