金属硫蛋白间接调控线粒体解偶联蛋白3抵抗阿霉素心肌毒性的作用机制研究  

Mechanism of metallothionein against doxorubicin-induced cardiac toxicity through regulation of uncoupling protein 3

作　　者：梁宝璐[1] 荆黎[1] 彭双清 LIANG Bao-lu;JING Li;PENG Shuang-qing(School of Public Health,Capital Medical University,Beijing 100069,China;Institute of Disease Control and Prevention,Academy of Military Medical Sciences,Beijing 100071,China)

机构地区：[1]首都医科大学公共卫生学院,北京100069 [2]解放军疾病预防控制中心,北京100071

出　　处：《毒理学杂志》2022年第3期218-223,230,共7页Journal of Toxicology

基　　金：国家自然科学基金(81202608)。

摘　　要：目的探讨金属硫蛋白(metallothionein,MT)和线粒体解偶联蛋白3(uncoupling protein 3,UCP3)抗阿霉素(doxorubicin,Dox)心脏毒性的作用及具体分子机制。方法体内实验以MT敲除型(MT^(-/-))小鼠和MT野生型(MT^(+/+))小鼠为动物模型并通过腹腔注射给予Dox(15 mg/kg·bw);体外实验采用出生1~3 d的两种小鼠的心脏构建心肌细胞体外原代培养模型,并给于Dox(1μmol/L)和NAC(5 mmol/L)处理。通过苏木素-伊红(HE)染色法检测Dox对小鼠心脏组织的损伤作用;免疫组织化学染色法检测MT的表达水平;细胞色素C还原测定法检测O_(2)^(-)的含量;TBARS法测定MDA的含量;RT-qPCR试验方法检测UCP3基因表达水平;Western blot方法检测UCP3蛋白表达水平;免疫共沉淀方法检测MT与UCP3的相互作用方式。结果Dox可导致心脏组织病理学改变,氧化损伤,超氧阴离子产生增多,抑制UCP3基因和蛋白表达(P<0.05,P<0.01),但是MT并不能通过与UCP3直接结合而发挥作用。NAC可抑制Dox导致的两种心肌细胞中UCP3基因表达水平的下降(P<0.05),但是对UCP3蛋白表达水平的下降没有抑制作用。结论MT除了可通过清除氧自由基抵抗Dox对UCP3基因表达的抑制作用外还可通过其他途径调控UCP3的蛋白表达水平。Objective To investigate the effect and molecular mechanism of metallothionein(MT)and uncoupling protein 3(UCP3)against doxorubicin(Dox)-induced cardiotoxicity.Methods In vivo,MT-I/II null(MT^(-/-))mice and wild-type(MT^(+/+))mice were given a single dose of Dox intraperitoneally(15 mg/kg·bw);In vitro,cardiomyocytes were prepared from MT^(-/-)and MT^(+/+) neonatal mice and were pretreated with NAC(5 mmol/L)followed by exposure to Dox(1μmol/L).The Dox-induced cardiac morphological damage was detected by HE staining;MT expression was detected by immunohistochemical staining;the level of O_(2)^(-)was detected by Cytochrome C reduction assay;the MDA content was detected by TBARS method;RT-qPCR assay was used to detect UCP3 gene expression;Western blot method was used to detect UCP3 protein expression;the interaction between MT and UCP3 was detected by co-immunoprecipitation method.Results Dox caused cardiac histopathological changes and oxidative damage,increased superoxide anion production and suppressed UCP3 mRNA and protein expression in the hearts of mice(P<0.05,P<0.01).Moreover,MT did not directly bind with UCP3.NAC could inhibit the decrease of UCP3 gene expression level in both cardiomyocytes caused by Dox(P<0.05).But there was no inhibition on reduction of UCP3 protein expression level in cardiomyocytes.Conclusion MT can inhibit the decrease of UCP3 mRNA expression induced by Dox through scavenging oxygen radicals and regulate the protein expression of UCPs by other regulatory mechanisms.

关 键 词：阿霉素 金属硫蛋白 线粒体解偶联蛋白3 氧化应激 

分 类 号：R114[医药卫生—卫生毒理学] R99[医药卫生—公共卫生与预防医学]