艾塞那肽减轻阿霉素引起的心肌细胞凋亡
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摘要
目的:探讨艾塞那肽是否对阿霉素引起的心肌细胞凋亡具有保护作用。方法:将体外培养的H9c2细胞分为空白组(单纯DMEM培养基)、阿霉素组(加入10μM阿霉素)、艾塞那肽组(阿霉素刺激前0.5h给予30nM艾塞那肽预处理)后继续培养24h。TUNEL法和流式细胞仪检测细胞凋亡情况,采用MTT法检测各组细胞的活性、酶标仪检测活性氧(ROS)水平、Caspase3、Caspase 9活性及线粒体膜电位(△Ψm),实时荧光定量PCR检测Bcl-2/Bax mRNA水平比值。结果:与空白组相比,阿霉素组细胞凋亡程度显著增加(流式细胞检测法:P<0.01;TUNEL法:P<0.01),H9c2心肌细胞活性(P<0.01),△Ψm(P<0.01)及Bcl-2/Bax比值(P<0.01)均显著降低,而细胞内ROS水平(P<0.01)及Caspase3、Caspase9活性(均为P<0.01)均显著增加。给予艾塞那肽预处理后能逆转上述变化,细胞凋亡程度显著下降(流式细胞检测法:P<0.01;TUNEL法:P<0.05),H9c2心肌细胞活性(P<0.05),△Ψm(P<0.01)及Bcl-2/Bax比值(P<0.01)均显著增加,细胞内ROS水平(P<0.05)及Caspase3、Caspase9(均为P<0.05)活性均显著降低。结论:艾塞那肽可能通过降低细胞内ROS水平,增加△Ψm,增加Bcl-2/Bax比值来改善阿霉素诱导的心肌细胞凋亡,发挥抗凋亡效应。
Objective:To investigate whether Exenatide could attenuate the cardiomyocyte apoptosis induced by Adriamycin.Methods:H9 c2 cells were cultured in vitro and divided into three groups:blank group(DMEM medium),adriamycin group(10μM adriamycin alone)and exenatide group(30 nM exenatide-pretreatment for 0.5 h before 10 nM adriamycin stimulation).Cells in each group were cultured for 24 hours.Cells apoptosis was detected by flow cytometry and TUNEL assay.MTT assay was used to detect the activity of cells in each group.The levels of reactive oxygen species(ROS),Caspase 3/9 and mitochondrial membrane potential(△Ψm)were detected by enzyme labeling instrument.The ratio of Bcl-2/Bax gene mRNA expression was detected by PCR.Results:The extent of apoptotic cells increased significantly(P<0.01 for TUNEL assay and P<0.01 for flow cytometry)in the adriamycin group compared with the blank group,and the activity of H9 c2 cells(P <0.01),△Ψm(P<0.01)and Bcl-2/Bax ratio(P<0.01)were significantly decreased,while the ROS level(P<0.01)and Caspase 3/9 activity(P <0.01)were significantly increased.After the administration of exenatide,all these changes were markedly reversed by exenatide.Conclusion:Exenatide may attenuate Adriamycin-induced cardiomyocyte apoptosis by decreasing ROS level and increasing △Ψm and Bcl-2/Bax ratio.
Objective:To investigate whether Exenatide could attenuate the cardiomyocyte apoptosis induced by Adriamycin.Methods:H9 c2 cells were cultured in vitro and divided into three groups:blank group(DMEM medium),adriamycin group(10μM adriamycin alone)and exenatide group(30 nM exenatide-pretreatment for 0.5 h before 10 nM adriamycin stimulation).Cells in each group were cultured for 24 hours.Cells apoptosis was detected by flow cytometry and TUNEL assay.MTT assay was used to detect the activity of cells in each group.The levels of reactive oxygen species(ROS),Caspase 3/9 and mitochondrial membrane potential(△Ψm)were detected by enzyme labeling instrument.The ratio of Bcl-2/Bax gene mRNA expression was detected by PCR.Results:The extent of apoptotic cells increased significantly(P<0.01 for TUNEL assay and P<0.01 for flow cytometry)in the adriamycin group compared with the blank group,and the activity of H9 c2 cells(P <0.01),△Ψm(P<0.01)and Bcl-2/Bax ratio(P<0.01)were significantly decreased,while the ROS level(P<0.01)and Caspase 3/9 activity(P <0.01)were significantly increased.After the administration of exenatide,all these changes were markedly reversed by exenatide.Conclusion:Exenatide may attenuate Adriamycin-induced cardiomyocyte apoptosis by decreasing ROS level and increasing △Ψm and Bcl-2/Bax ratio.
引文
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[2]Zhang YW,Shi J,Li YJ,et al.Cardiomyocyte death in doxorubicin-induced cardiotoxicity[J].Arch Immunol Ther Exp(Warsz),2009,57(6):435-445.
[3]Machida K,Cheng KT,Lai CK,et al.Hepatitis C virus triggers mitochondrial permeability transition with production of reactive oxygen species,leading to DNA damage and STAT3activation[J].J Virol,2006,80(14):7199-207.
[4]Gallego-Colon E,Klych-Ratuszny A,Kosowska,A,et al.Exenatide modulates metalloproteinase expression in human cardiac smooth muscle cells via the inhibition of Akt signaling pathway[J].Pharmacol Rep,2018,70(1):178-183.
[5]Chang G,Zhang D,Liu J,et al.Exenatide protects against hypoxia/reoxygenation-induced apoptosis by improving mitochondrial function in H9c2 cells[J].Exp Biol Med(Maywood),2014,239(4):414-422.
[6]Wang H,Bei Y,Lu Y,et al.Exercise Prevents Cardiac Injury and Improves Mitochondrial Biogenesis in Advanced Diabetic Cardiomyopathy with PGC-1alpha and Akt Activation[J].Cell Physiol Biochem,2015,35(6):2159-2168.
[7]Johnstone RW,AA Ruefli and SW Lowe.Apoptosis:a link between cancer genetics and chemotherapy[J].Cell,2002,108(2):153-164.
[8]Oliveira PJ,MS Santos,and KB Wallace.Doxorubicin-induced thiol-dependent alteration of cardiac mitochondrial permeability transition and respiration[J].Biochemistry(Mosc),2006,71(2):194-199.
[9]Green DR,G Kroemer.The pathophysiology of mitochondrial cell death[J].Science,2004,305(5684):626-629.
[10]Huang DC,A Strasser.BH3-Only proteins-essential initiators of apoptotic cell death[J].Cell,2000,103(6):839-842.
[11]Jean SR,Tulumello DV,Riganti C,et al.Mitochondrial targeting of doxorubicin eliminates nuclear effects associated with cardiotoxicity[J].ACS Chem Biol,2015,10(9):2007-2015.
[12]Granados-Principal S,El-Azem N,Pamplona R,et al.Hydroxytyrosol ameliorates oxidative stress and mitochondrial dysfunction in doxorubicin-induced cardiotoxicity in rats with breast cancer[J].Biochem Pharmacol,2014,90(1):25-33.
[13]Jia Y,Zuo D,Li Z,et al.Astragaloside IV inhibits doxorubicin-induced cardiomyocyte apoptosis mediated by mitochondrial apoptotic pathway via activating the PI3K/Akt pathway[J].Chem Pharm Bull(Tokyo),2014,62(1):45-53.