盐霉素对鸡原代心肌细胞的毒性作用及其机制的初步研究

doi:10.11843/j.issn.0366-6964.2017.04.018

摘要/Abstract

摘要：

初步探讨盐霉素对鸡原代心肌细胞的毒性作用及其毒性机制。采用差速贴壁法结合化学纯化法分离鸡原代心肌细胞，应用免疫荧光法鉴定心肌细胞纯度，给予1、5、10、20、50 μg·mL^-1盐霉素处理后，观察心肌细胞形态，采用MTT法测定盐霉素对心肌细胞活性的影响；比色法检测心肌细胞乳酸脱氢酶（LDH）释放、肌酸激酶（CK）活性；流式细胞术分析盐霉素对心肌细胞凋亡及线粒体膜电位的影响；电子显微镜观察盐霉素对心肌细胞超微结构的影响；荧光显微镜观察盐霉素对心肌细胞氧化应激的影响；比色法测定盐霉素对心肌细胞Caspase-3、Caspase-8和Caspase-9蛋白酶活性的影响；qRT-PCR检测Caspase-3、Caspase-8、Caspase-9、细胞色素C（Cyt-C）、Bax及Bcl-2 mRNA的转录水平。结果表明：分离所得鸡原代心肌细胞形态良好，纯度大于90%；盐霉素以显著的浓度-时间依赖性方式抑制心肌细胞生长并诱导细胞死亡；盐霉素导致心肌细胞LDH释放极显著增加（P<0.01），细胞内CK酶活性增强，呈浓度依赖性（P<0.01）；盐霉素导致心肌细胞凋亡率极显著升高（P<0.01），线粒体膜电位（ΔΨm）明显下降（P<0.01）；电子显微镜观察显示盐霉素导致心肌细胞线粒体严重肿胀、空泡化，嵴溶解甚至消失；盐霉素导致心肌细胞内活性氧（ROS）显著升高；细胞质Caspase-3、Caspase-8和Caspase-9蛋白酶活性均极显著升高（P<0.01）； qRT-PCR结果表明凋亡相关基因Caspase-3、Caspase-8、Caspase-9、Cyt-C及Bax mRNA转录上调，Bcl-2 mRNA转录下调（P<0.01）。盐霉素可以通过细胞凋亡引起鸡心肌细胞死亡，线粒体途径和死亡受体途径可能共同参与了盐霉素诱导的鸡心肌细胞凋亡。

Abstract:

The aim of the present study was to investigate the toxic effects and mechanism of salinomycin on chicken cardiomyocytes. The cardiomyocytes were purified and cultured through differential adhesion and inhibition of 5-bromodeoxyuridine (Brdu). The purity of cardiomyocytes was identified by α-actinin immunofluorescence staining. The chicken cardiomyocytes were exposed to 1, 5, 10, 20 and 50 μg·mL^-1 of salinomycin for 24-72 h. Cell vialibity and cell morphological changes were measured by MTT and microscopy. Membrane integrity (LDH assay) and creatine kinase (CK) activity were determined by colorimetric method. Apoptosis and mitochondrial membrane potential (MMP) were analyzed using flow cytometry. The ultrastructural structures of cells were observed and imaged through transmission electron microscopy and the levels of intracellular ROS were observed by fluorescence microscope. The activities of Caspase-3, Caspase-8 and Caspase-9 were determined by colorimetry. The transcription of several apoptosis-related genes, Bcl-2, Bax, Cytochrome-C (Cyt-C), Caspase-3, Caspase-8 and Caspase-9 mRNA were determined by qRT-PCR. Our result showed that cardiomyocytes purity was more than 90%. Compared with control group, salinomycin significantly inhibited cell growth in a concentration- and time-dependent manner (P<0.01). The release of LDH and CK activity in cardiomyocytes exposed to salinomycin were significantly increased, respectively (P<0.01). Salinomycin induced apoptosis and disrupted mitochondrial function by decreasing mitochondrial membrane potential (MMP) in a concentration-dependent manner. We also found that mitochondrias of cardiomyocytes were severely swollen, vacuoles and the cristae dissolved or even disappeared through transmission electron microscopy. Salinomycin induced the oxidative stress of cardiomyocytes, the levels of intracellular ROS were increased. Activities of Caspase-3, Caspase-8 and Caspase-9 were increased significantly (P<0.01). The relative transcription of Bax, Caspase-3, Caspase-8, Caspase-9 and Cyt-C mRNA were all upregulated (P<0.01), however, the transcription of Bcl-2 mRNA was downregulated (P<0.01). In summary, salinomycin caused chicken cardiomyocytes death through apoptosis, which might involve mitochondial pathway and death-receptor pathway.

中图分类号:

S859.796

郑亚妮, 高修歌, 刘晓晓, 滕佩, 季辉, 江善祥. 盐霉素对鸡原代心肌细胞的毒性作用及其机制的初步研究[J]. 畜牧兽医学报, 2017, 48(4): 740-751.

ZHENG Ya-ni, GAO Xiu-ge, LIU Xiao-xiao, TENG Pei, JI Hui, JIANG Shan-xiang. Preliminary Research of Toxic Effects and Mechanisms of Salinomycin on Chicken Primary Cardiomyocytes[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(4): 740-751.

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