Research Of Toxic Effects And Machanisms Of Salinomycin On Chicken Primary Cardiomyocytes

Salinomycin is a monovalent carboxylic ionophore that has been used as an agricultural antibiotic to prevent coccidiosis in poultry.Overdosage or improper use in livestock can result in toxicosis.Target organs damaged by salinomycinwere identified to include the heart in poultry.The aim of the present study was to investigate the toxic effects and mechanism of salinomycin on chicken cardiomyocytes.We choosed the chicken primary cardiomyocytes as a model to study the toxic effect of salinomycin,detect the effect of salinomycin on cell viability and apoptosis,to explore the toxic machanisms of salinomycin on cardiomyocytes.The details aredivided into three parts as follows:1.Toxic effect of salinomycin on chicken primary cardiomyocytesThe cardiomyocytes isolated from 13d age specific pathogen free chick embryo were purified and cultured through differential adhesion with chemical purification method.The purity of cardiomyocytes was identified by α-actinin immunefluorescence staining.The chicken cardiomyocytes were exposed to salinomycin(1,5,10,20 and 50 μg·mL-1)for 24-72 h.Along with cell vialibity and cell morphological changes were detected by MTT,trypan blue assay and microscopy.After the treatment of salinomycin for 24 h,the cell culture supernatant were collected and the activities of lactate dehydrogenase(LDH)were detected by colorimetry method.The creatine kinase(CK)content of cardiomyocytes was determined by enzyme linked immunosorbent assay(ELISA)after incubation with salinomycin for 6-24 h.The results showed that the cardiomyocytes were triangular and irregularin control group,whereas,after treatment of salinomycin for 24 h,we observed cell density decrease gradually,vacuolization,granular sensation and shrinkage compared with controlsby morphological analysis.Many cells gradually became round and detached from the culture dish and floated in the culture solution.Salinomycin significantly inhibited cell viability and induced cell death in a concentration-and time-dependent manner.The release of LDH and CK content in cardiomyocytes exposed to salinomycin were significantly increased,respectively.These suggest that salinomycin inhibited cell viabilityand induced cell death.Salinomycin is cytotoxic inchicken cardiomyocytes.2.Salinomycin induces apoptosis in chicken primary cardiomyocytesAfter the treatment of salinomycin for 24 h,the morphologic variations of cell nucleus were observed by DAPI staining and apoptosis were analyzed by flow cytometryusing Annexin V-PI staining.The ultrastructural structures of cells were observed and imaged through transmission electron microscopy,mitochondrial membrane potential(MMP)were analyzed by JC-1 staining and flow cytometry,the levels of intracellular ROS were detected by DCFH-DA staining.The results showed an increase in the percentage of apoptotic cells compared with controlsin a concentration-dependent manner.The karyorrhexis and pyknosis of cardiomyocytes were observed in treatment group.When cardiomyocytes were treated with salinomycin for 24h,no significantly ultrastructural changes were observed in the cells of control group,the cells showed a normal ultrastructure with smooth rounded nucleus,intact nuclear membrane and integrated mitochondria with normal cristae,whereas,after incubation with salinomycin for 24 h,the mitochondrias were swollen and vacuolated,the cristae dissolved,blurred or even disappeared,organelles of cells were unclear,the cells were showing extensive vacuolation.The results showed that salinomycin induced the decline of MMP in a concentration-dependent manner,besides,the intracellular ROS levels were dramatically increased in cardiomyocytes treated with salinomycin for 24 h.These indicate salinomycin induced oxidative stress,mitochondrial damage and apoptosis in chicken cardiomyocytes.3.The mechanism of apoptosis in chicken primary cardiomyocytes induced by salinomycinAfter incubation with salinomycin for 24 h,We examined the activities of Caspase-3,Caspase-8 and Caspase-9 in chicken cardiomyocytes by chromatometry method.The transcription of apoptosis-related genes Caspase-3,Caspase-6,Caspase-9,Caspase-8,Bax,Bcl-2,Cyt-C,Apaf-1,Fas and Fas-L were determined by qRT-PCR.The results showed that salinomycin was able to significantly activate Caspase-3,Caspase-8 and Caspase-9 as a concentration-dependent manner.The transcription level of Caspase-3,Caspase-6,Caspase-9,Bax,Cyt-C and Apaf-1 genes were upregulated,Bcl-2 gene was downregulated.Morever,the transcription level of Caspase-8,Fas and Fas-L were also upregulated significantly.These indicate that salinomycin induced apoptosis in chicken cardiomyocytes via the mitochondrial pathway and the death receptor pathway.