纳米氧化锌暴露对斑马鱼胚胎心脏发育的毒性效应和机制研究

Effect and mechanism of zinc oxide nanoparticle on cardiac development of zebrafish embryos

目的观察纳米氧化锌暴露对斑马鱼胚胎心脏发育及大鼠心肌细胞(H9c2)的毒性作用及可能机制。方法对纳米氧化锌材料进行表征化处理。斑马鱼胚胎在受精后4 h(4 hpf)暴露纳米氧化锌颗粒0.5,2.5,5.0和10.0 mg·L^(-1)24~96 h,计算胚胎存活率,并于胚胎17 hpf时间点,收集并用实时定量PCR测定中胚层脊索同源(noto),T盒16(tbx16),过短同源a(ta)和tbx6 m RNA表达;观察斑马鱼72 hpf心率。将心肌细胞标记红色荧光蛋白Tg(cmlc2:nuc-ds Red)的斑马鱼交配取胚胎,染毒纳米氧化锌2.5和5.0 mg·L^(-1),计数72 hpf胚胎心肌细胞数目。大鼠心肌细胞(H9c2)与纳米氧化锌0,0.1,0.5,1,5.0和10.0 mg·L^(-1)共培养24 h,Alamar Blue方法检测细胞活力,透射电子显微镜观察线粒体超微结构,化学发光法和Seahorse仪分别检测细胞ATP和耗氧率水平。结果纳米氧化锌在双蒸水中的粒径分布为(331±3)nm。48 hpf的斑马鱼胚胎纳米氧化锌LC50为21.81 mg·L^(-1)。暴露纳米氧化锌2.5 mg·L^(-1)后,17 hpf斑马鱼胚胎的心脏发育关键基因noto,ta和tbx6表达降低(P<0.05)。72 hpf斑马鱼暴露纳米氧化锌5.0 mg·L^(-1),心率为153 min-1,与正常对照组相比降低12.6%(P<0.01),心肌细胞数目减少15.5%(P<0.01)。纳米氧化锌0.5 mg·L^(-1)可引起大鼠心肌细胞(H9c2)细胞活力降低(P<0.05)以及线粒体出现空泡化现象。与细胞对照组相比,细胞ATP减少25.7%(P<0.05),耗氧率减少27.2%(P<0.01)。结论低剂量纳米氧化锌暴露对斑马鱼心脏发育有一定影响,主要表现为心率降低和心肌细胞数目减少,这些变化可能与心脏发育相关基因表达减少以及细胞线粒体结构和功能损伤有关。

OBJECTIVE To explore the toxic effects of zinc oxide nanoparticls（Zn O-NPs） on cardiac development of zebrafish embryos and rat myocardial cell lines（H9c2）, as well as potential molecular mechanisms. METHODS Zn O-NPs were characterized. Zebrafish embryos were exposed to different doses of Zn O-NPs（0, 0.5, 2.5, 5.0 and 10.0 mg·L-1） for 24 to 96 h at 4 h post fertilization（4 hpf）. The embryo mortality was observed. The expressions of notochord homeobox（noto）, T-box 6（tbx16）, T, brachyury homolog a（ta）, and tbx6 which were related to cardiac mesoderm were investigated using real-time PCR at 17 hpf. The heart rate and number of cardiomyocytes of embryos[Tg（cmlc2：nucds Red）]exposed to 0, 2.5 and 5 mg·L-1Zn O-NPs were detected at 72 hpf. Rat myocardial cell lines（H9c2） were treated with Zn O-NPs（0.1, 0.5, 1.0, 5.0 and 10.0 mg · L-1） for 24 h. Cell viability was measured with Alamar Blue method. Mitochondrial ultrastructure was observed by transmission electron microscopy. Cellular ATP was detected using chemiluminescence, and oxygen consumption rate（OCR）was examined with Seahorse instrument. RESULTS The particle size of Zn O-NPs was（331±3）nm.The Zn O-NPs LC50 of zebrafish embryos at 48 hpf was 21.81 mg·L-1. The m RNA expressions of noto,ta and tbx6 were reduced after Zn O-NPs 2.5 mg·L-1treatment at 17 hpf. The heart rate of 72 hpf zebrafish was 153 min-1in the Zn O-NPs 5 mg·L-1group, 12.6% lower than that in the cell vehicle group（P〈0.01）, and the number of cardiomyocytes decreased by 15.5%（P〈0.01） compared with the cell vehicle group. Reduced cell viability and mitochondrial vacuolation were observed in H9c2 after Zn O-NPs0.5 mg ·L-1exposure. Compared with the cell vehicle group, the cell ATP decreased by 25.7%（P〈0.05）, and OCR decreased by 27.2%（P〈0.01）. CONCLUSION Low-dose Zn O-NPs exposure has effect on the cardiac development of zebrafish, mainly due to reduced heart rate and decreased number of cardiomyocytes. These changes may be related to the decreased expressions of cardiac development-related genes and the impairment of mitochondrial structure and function.