盐霉素是一种常用的防治畜禽球虫病的聚醚类离子载体型抗生素。该类抗生素毒性较大、安全范围窄，若使用不当常造成畜禽中毒。对于家禽而言，盐霉素中毒主要损伤心肌。为探讨盐霉素对心肌细胞的毒性作用及其机制，本研究采取鸡原代心肌细胞作为模型，考察盐霉素的细胞毒性作用，检测其对心肌细胞活性、细胞存活率和细胞凋亡的影响，探究盐霉素致心肌细胞的毒性作用及其机制。具体分为以下几个部分： 1 盐霉素致鸡原代心肌细胞毒性作用 采取差速贴壁法结合5-溴脱氧尿嘧啶核苷（BRDU）纯化法，取13日龄SPF鸡胚的心室肌进行心肌细胞分离培养，并采用α-actinin抗体进行心肌细胞的免疫荧光鉴定。培养24 h，待心肌细胞贴壁后，在贴壁细胞中分别加入0（对照组）、1、5、10、20、50 μg·mL-1<上标!>盐霉素，分别继续培养24、48、72 h。培养结束后，于倒置光学显微镜下观察心肌细胞的形态，并用MTT比色法和台盼蓝染色法测定细胞活性及存活率。盐霉素处理心肌细胞24 h后，取细胞培养上清液，检测其中乳酸脱氢酶（LDH）的活性。裂解细胞，提取细胞总蛋白，采用ELISA方法，测定盐霉素处理6~24 h后心肌细胞肌酸激酶（CK）的含量。结果显示：对照组细胞贴壁牢固，多呈梭形、三角形、星形等不规则的形状，而且胞体大，胞浆丰富致密，细胞排列紧密，而处理组心肌细胞，贴壁细胞数量减少，细胞密度逐渐下降，1 μg·mL-1<上标!>即可观察到心肌细胞内出现空泡，细胞颗粒感增强，随着盐霉素浓度增加，部分心肌细胞逐渐圆缩，从培养皿上脱落并悬浮于培养液中；盐霉素处理心肌细胞24~72 h，心肌细胞活性和存活率显著下降，呈药物浓度和时间依赖性；盐霉素处理24 h后，心肌细胞LDH释放量和细胞内的CK含量呈药物浓度依赖性显著上升。结果表明，盐霉素抑制心肌细胞生长，降低细胞活性和存活率，造成心肌细胞损伤，导致心肌细胞死亡。盐霉素对鸡心肌细胞具有明显的细胞毒性作用。 2 盐霉素致鸡原代心肌细胞凋亡 0~50 μg·mL-1<上标!>盐霉素处理鸡原代心肌细胞24 h后，DAPI染色观察细胞核形态的变化，AnnexinV-FITC/PI双染通过流式细胞仪检测盐霉素对心肌细胞凋亡率的影响，通过透射电子显微镜观察盐霉素处理后心肌细胞超微结构的变化，JC-1荧光染料染色后经流式细胞仪检测心肌细胞线粒体膜电位的改变，荧光探针DCFH-DA染色经荧光显微镜和流式细胞仪检测细胞内活性氧（ROS）水平的变化。结果显示：盐霉素处理24 h后，与对照组相比，处理组心肌细胞凋亡率显著升高，呈浓度依赖性，且出现核碎裂、核浓缩现象；对照组细胞超微结构正常，细胞膜清晰完整，胞质内染色体丰富均匀，线粒体形态完整，线粒体嵴清晰，核膜完整，而处理组细胞内线粒体明显肿胀，呈现空泡化，线粒体嵴模糊、溶解甚至消失，胞质空泡增多，细胞器模糊不清；与正常对照组细胞相比，盐霉素处理组细胞的线粒体膜电位明显降低，细胞内ROS水平显著升高，呈药物浓度依赖性。结果表明，盐霉素能导致鸡心肌细胞的氧化应激，损伤心肌细胞线粒体的结构和功能，诱导细胞凋亡，从而导致其对鸡心肌细胞的毒性作用。 3 盐霉素致鸡原代心肌细胞凋亡的机制 不同浓度盐霉素处理鸡原代心肌细胞24 h，比色法检测心肌细胞Caspase-3、Caspase-9和Caspase-8活性的变化，实时荧光定量PCR法检测凋亡相关的Caspase-3、Caspase-6、Caspase-8、Caspase-9、Cyt-C、Bax、Bcl-2、Apaf-1、Fas和Fas-L基因mRNA的表达水平。结果显示：盐霉素导致鸡心肌细胞内的Caspase-3、Caspase-9和Caspase-8被激活，3种酶活性基本呈药物浓度依赖性显著升高；qRT-PCR结果显示，Caspase-3、Caspase-6以及线粒体凋亡途径中的促凋亡基因Caspase-9、Cyt-C、Apaf-1和Bax的表达均显著上调，而抑凋亡基因Bcl-2的表达显著下调，此外，死亡受体凋亡途径中的Caspase-8、Fas和Fas-L基因表达也显著上调。结果表明，线粒体途径和死亡受体途径可能共同介导了盐霉素诱导的心肌细胞凋亡，但具体机制仍需进一步研究。

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 cardiomyocytes The 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<上标!>) 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 cardiomyocytes After 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 24 h, 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 salinomycin After 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-1genes were upregulated, Bcl-2 gene was down- regulated. 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.