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

Abstract

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.

CLC Number:

S859.796

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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Preliminary Research of Toxic Effects and Mechanisms of Salinomycin on Chicken Primary Cardiomyocytes

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