氯氮平通过诱导斑马鱼胚胎心脏细胞凋亡引起心脏毒性
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摘要
目的观察氯氮平对斑马鱼胚胎心脏的毒性,研究其毒性作用的机制。方法以24 hpf(hours post fertilization)斑马鱼胚胎为研究模型,以不同浓度的氯氮平(12.5、25、37.5、50、62.5μmol·L~(-1))进行处理,于处理24、48、72 h后观察斑马鱼的死亡率、心率和心脏形态变化。采用吖啶橙染色初步考察氯氮平产生心脏毒性的作用机制;利用实时定量PCR检测与凋亡相关的基因Bcl-2、Bax、caspase-9、caspase-3的表达。结果氯氮平可引起斑马鱼出现体长明显缩短、心包水肿、心脏变小、心率下降等现象,这些具有剂量依赖性。吖啶橙染色结果显示,氯氮平可剂量依赖性地诱导斑马鱼心脏细胞发生凋亡。给药处理72 h后,斑马鱼胚胎细胞抗凋亡基因Bcl-2的表达水平降低,促凋亡基因Bax的表达明显升高,Bcl-2/Bax表达比值明显下降;caspase-9和caspase-3表达均明显增加。结论氯氮平可导致斑马鱼胚胎产生心脏毒性,其毒性作用可能与其诱导胚胎细胞发生凋亡有关。
Aim To observe the toxic effects of clozapine on zebrafish embryos and investigate the possible mechanisms underlying its cardiac toxicity. Methods Zebrafish of 24 hpf(hours post fertilization) were exposed to different concentrations of clozapine. After treatment for 24,48 and 72 h,the heart rates of the zebrafish embryos were examined and morphological changes of the heart were determined. The mechanism of clozapine induced cardiac toxicity was preliminarily explored by means of acridine orange(AO) staining.The mRNA levels of Bcl-2,Bax,caspase-9,caspase-3 were detected by real-time PCR analysis. Results Clozapine induced the decrease of body length,formation of pericardial edema,and decrease of heart rates of the embryos in a time and dose-dependent manner.AO staining showed that clozapine induced cell apoptosis of the cardiomyocytes. Real-time PCR analysis showed that the anti-apoptotic gene Bcl-2 was downregulated and pro-apoptosis gene Bax was up-regulated at 72 h post treatment,resulting in the decreased expression ratio of Bcl-2/Bax. In addition, caspase-9 and caspase-3 were induced significantly by clozapine.Conclusion Clozapine caused cardiac toxicity in zebrafish embryos by inducing cell apoptosis.
Aim To observe the toxic effects of clozapine on zebrafish embryos and investigate the possible mechanisms underlying its cardiac toxicity. Methods Zebrafish of 24 hpf(hours post fertilization) were exposed to different concentrations of clozapine. After treatment for 24,48 and 72 h,the heart rates of the zebrafish embryos were examined and morphological changes of the heart were determined. The mechanism of clozapine induced cardiac toxicity was preliminarily explored by means of acridine orange(AO) staining.The mRNA levels of Bcl-2,Bax,caspase-9,caspase-3 were detected by real-time PCR analysis. Results Clozapine induced the decrease of body length,formation of pericardial edema,and decrease of heart rates of the embryos in a time and dose-dependent manner.AO staining showed that clozapine induced cell apoptosis of the cardiomyocytes. Real-time PCR analysis showed that the anti-apoptotic gene Bcl-2 was downregulated and pro-apoptosis gene Bax was up-regulated at 72 h post treatment,resulting in the decreased expression ratio of Bcl-2/Bax. In addition, caspase-9 and caspase-3 were induced significantly by clozapine.Conclusion Clozapine caused cardiac toxicity in zebrafish embryos by inducing cell apoptosis.
引文
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[2]Elman I,Goldstein D S,Eisenhofer G,et al.Mechanism of peripheral noradrenergic stimulation by clozapine[J].Neuropsychopharmacology,1999,20:29-34.
[3]Layland J J,Liew D,Prior D L.Clozapine-induced cardiotoxicity:a clinical update[J].Med J Aust,2009,190(4):190-2.
[4]王雪,刘可春,王希敏,等.大黄酸对马兜铃酸A引起的斑马鱼肾脏损伤的保护作用[J].中国药理学通报,2016,32(3):361-5.[4]Wang X,Liu K C,Wang X M,et al.Protective effect of rhein on aristolochic acid-induced renal injury in zebrafish[J].Chin Pharmacol Bull,2016,32(3):361-5.
[5]许冰洁,张立将,李春启,等.斑马鱼胚胎评价5种药物的发育毒性与模型验证[J].中国药理学通报,2016,32(1):74-9.[5]Xu B J,Zhang L J,Li C Q,et al.Model validation and evaluation of developmental toxicity of five drugs using zebrafish embryos[J].Chin Pharmacol Bull,2016,32(1):74-9.
[6]Mc Grath P,Li C Q.Zebrafish:a predictive model for assessing drug-induced toxicity[J].Drug Discov Today,2008,13(9):394-401.
[7]Westerfield M,Wegner J,Jegalian B G.Specific activation of mammalian Hox promoters in mosaic transgenic zebrafish[J].Genes Dev,1992,6(4):591-8.
[8]罗涛,万普蓉,张咏梅,等.氯氮平急性中毒的临床表现和处理[J].临床精神医学杂志,2017,27(1):68-9.[8]Luo T,Wan P R,Zhang Y M,et al.Clinical manifestation and management of acute poisoning by clozapine[J].J Clin Psychiatry,2017,27(1):68-9.
[9]张洁,张宝生,田韶辉,等.长期服用氯氮平对患者心肌酶的影响[J].中国民族民间医药,2014,23(7):88-9.[9]Zhang J,Zhang B S,Tian S H,et al.Long-term use of clozapine myocardial enzymes in patients with the result analysis[J].Chin J Ethnomed Ethnopharm,2014,23(7):88-9.
[10]赵芳霞,王怀海,何宏.氯氮平致心包与胸腔积液1例[J].临床精神医学杂志,2010,20(6):382.[10]Zhao F X,Wang H H,He H.1 case of pericardial effusion and pleural effusion caused by clozapine[J].J Clin Psychiatry,2010,20(6):382.
[11]韩利文,赵亮,楚杰,等.采用GC-MS代谢组学技术表征阿司咪唑诱导斑马鱼心脏毒性的内源性代谢物[J].中国药学杂志,2015,50(1):45-50.[11]Han L W,Zhao L,Chu J,et al.Endogenous metabolites investigation of cadiotoxicity induced by astemizole on zebrafish using GC-MS metabonomics[J].Chin Pharm J,2015,50(1):45-50.
[12]吴晓敏,何秋霞,韩利文,等.阿司咪唑对斑马鱼心脏毒性的初步研究[J].中国药理学通报,2013,29(9):1251-4.[12]Wu X M,He Q X,Han L W,et al.Preliminary study of astemizole’s cardiotoxicity to zebrafish[J].Chin Pharmacol Bull,2013,29(9):1251-4.
[13]Lee S H,Kim H R,Han R X,et al.Cardiovascular risk assessment of atypical antipsychotic drugs in a zebrafish model[J].J Appl Toxicol,2013,33(6):466-70.
[14]Abdel-Wahab B A,Metwally M E.Clozapine-induced cardiotoxicity:role of oxidative stress,tumour necrosis factor alpha and NF-κB[J].Cardiovasc Toxicol,2015,15(4):355-65.
[15]郑锡基,马继红,赵雪庆,等.氯氮平致严重心脏毒性反应2例[J].临床精神医学杂志,2002,12(1):62.[15]Zheng X J,Ma J H,Zhao X Q,et al.2 cases of severe cardiac toxicity induced by clozapine[J].J Clin Psychiatry,2002,12(1):62.