Study On Maduramicin-induced Toxicity And Toxic Mechanism In Primary Chicken Myocardial Cells

Maduramicin,an excellent ionophore antibiotic,is extensively used to control coccidiosis in poultry for it has many advantages such as broad spectrum of anti-coccidiosis,high titer,low cost,low price,growth promotion and coccidiosis is not susceptible to resistance.Due to maduramicin has a narrow safety range and the recommended dose and the toxic dose of maduramicin is very near,toxicity incidents have been reported in target animal broiler chicken and non-target animals such as swine,cattle,sheep,rabbit as well as human being who consumed animal products or food-contaminated maduramicin.Target organs damaged by toxic dose of maduramicin are identified to contain the cardiac and skeletal muscles in most reported species.However,the most published literatures related to intoxication reports and the summary of the clinical and histological data.To date,the mechanisms of maduramicin induced cardiotoxicity and muscle toxicity remain unclear in chicken and other animals.To investigate the mechanism of maduramicin-induced myocardial damage in chicken will provide a theoretical support for the prevention and control of maduramicin intoxication.Consequently,this study will provide reference basis for rational use of maduramicin.In the present study,to reveal maduramicin-induced cardiotoxicity in chicken,primary chicken myocardial cells were used as an in vitro model together with transcriptomics and bioinformatics analysis.Our findings indicate that inflammatory reaction,apoptosis,cytoplasmic ions imbalance and cytoplasm vacuolization may be its main toxic mechanism.Further confirmatory experiments proved that maduramicin-induced severe myocardial damage are mediated by a novel cell death named methuosis and apoptosis.The details are as follows:1.Transcriptomics analysis maduramicin-induced cytotoxicity of primary chicken myocardial cellsTo understand the underlying mechanisms of maduramicin-induced toxicity in chicken myocardial cells,RNA-seq and bioinformatics analysis were performed by using primary chicken myocardial cells as an in vitro model in this chapter.A series of concentrations of maduramicin(0,0.05,0.1,0.5,1,5 ?g/mL)were exposed to chicken myocardial cells at 24 h,48 h and 72 h,respectively.After that,CCK-8 assay was used to evaluate cytotoxicity ratio for seeking the best exposure scheme of maduramicin.Chicken myocardial cells were treated with 5 ?g/mL of maduramicin and vehicle control for 24 h,and followed with isolation of total RNA for sequencing by using Illumina Hiseq 4000 platform.To obtain the clean reads and differential expressed genes,the raw reads were filtered and then mapped to the reference genome along with calculation of gene expression by using FPKM method.The verification,enrichment analysis and function annotation of maduramicin-induced differential expressed genes were carrying out by RT-qPCR,GO and KEGG database,respectively.The results showed that enough high quality reads were produced by RNA-seq analysis.Compared with the control group,a total of 1442 differential expressed genes were identified,among which 810 genes were up-regulated and the rest 632 genes were down-regulated by 5 ?g/mL of maduramicin treatment for 24 h.RT-qPCR results were significantly positive correlation with RNA-Seq data,indicating the high accuracy and quality of RNA-seq analysis.GO enrichment analysis indicated that most differential expressed genes were related to multiple biological functional items such as cytoplasm,energy metabolism and cytoplasmic transport.KEGG annotation suggested that cytokine-cytokine receptor interaction,apoptosis,calcium signal and cytoplasmic vacuolization were key metabolic pathways intensively associated with maduramicin-induced cytotoxicity.Taken together,the mechanisms of maduramicin-induced cardiotoxicity are related to multiple pathways in chicken.2.Verification of transcriptomics analysisTo verify the accuracy of RNA-seq and bioinformatics analysis results in Chapter 2 and reveal the mechanisms of maduramicin-induced cardiotoxicity in chicken,a series of experiments were carried out using primary chicken myocardial cells.Chicken myocardial cells were exposed to maduramicin(0-5 ?g/mL)for 12 h or 24 h and followed by ELISA,flow cytometry,western blotting,colorimetry and transmission electron microscope observation for analysing release of inflammation factors,apoptosis rate,apoptotic proteins,cytoplasmic calcium,Na+-K+/Ca2+-ATPase and cytoplasm vacuolization,respectively.The obtained results showed that pro-inflammatory cytokines including tumor necrosis factor alpha(TNF-?)and interleukin-8(IL-8)were elevated by maduramcin exposure.Intracellular calcium level and Ca2+-ATPase activity were increased significantly after maduramicin treatment.Maduramicin induced a significant increase of cell apoptosis in a concentration-dependent manner and activated caspase-3 activity significantly.Massive vacuoles characterized with a single membrane,various sizes,phase-lucent and non cytoplasmic contents were found in the cytoplasm by morphology and transmission electron microscopy observation.In summary,pro-inflammation,apoptosis,cytoplasmic ion imbalance and vacuolization contribute to maduramicin-induced cardiotoxicity in chicken.3.Apoptotic mechanism induced by maduramicin in chicken primary myocardial cellsTo investigate the role and the underlying mechanisms of apoptosis in maduramicin-induced cardiotoxicity,primary chicken myocardial cells were isolated to use as an in vitro model.Prior to or maduramicin(0?5 ?g/mL)treatment for 24 h-72h,N-benzyloxycarbonyl-Val-Ala-Asp(O-Me)fluoromethyl ketone(z-VAD-fink 20 ?M)and N-acetyl-cysteine(NAC 500 mM)were incubated with cells for 1h,followed by microscope observation,MTT assay,DAPI staining,flow cytometry,RT-qPCR,colorimetry,JC-1 staining and DCFA-DA staining.Our results showed that maduramicin caused morphological changes and a decrease in the viability of chicken myocardial cells.Annexin V-FITC/PI and 4',6-diamidino-2-phenylindole(DAPI)staining showed a significant increase in the number of apoptotic cells.Furthermore,caspases-3/8/9 were activated at the gene and protein levels and this was accompanied by the upregulation of apoptosis-related genes,including bcl-2,bax,and cytochrome C.Treatment with the pan-caspase inhibitor z-VAD-fink ameliorated the apoptosis rather than cytotoxicity.Furthermore,intracellular Ca2+ and reactive oxygen species(ROS)were elevated,whereas mitochondrial membrane potential(MMP)and intracellular glutathione(GSH)decreased with exposure to maduramicin.The antioxidant N-acetyl-cysteine(NAC)had no significant effect on maduramicin-induced cytotoxicity and apoptosis.Taken together,our findings demonstrate that maduramicin is toxic to primary chicken myocardial cells via caspase dependent and independent apoptotic pathways.4.Methuosis and its mechanism induced by maduramicin in chicken primary myocardial cellsTo understand the morphological characteristics and molecular mechanism of non-apoptotic cell death induced by maduramicin,primary chicken myocardial cells were chosen as in vitro model.Chicken myocardial cells were treated with various concentrations of maduramicin(0?5 ?g/mL)for 12 h?72 h and observed under microscope and transmission electron microscope.After 5 ?g/mL maduramicin exposed to cells for 12 h,laser confocal microscope was used for understand the dynamics of vacuole formation.Dextran 488-FITC,ER-Tracker,Mito-Tracker and Lyso Tracker staining were performed to investigate the relationship between maduramicin-induced vacuoles with organelles.Chicken myocardial cells,pretreated with or without z-VAD-fink,3-methyladenine(3-MA),chloroquine(CQ)or bafilomycin A1 for 1 h,were treated with a series of maduramicin(0?5 ?g/mL)for 12 h or 24 h,followed by microscope and transmission electron microscope observation and CCK-8 assay.The gene and protein expression of LC3,p62,H-Ras,K-Ras and Rac 1 were analysis by RT-qPCR and western blot after maduramicin treatment for 24 h.The results showed that maduramicin induced cytoplasmic vacuolization in a time and concentration-dependent manner.Maduramicin-induced vacuoles were differed with the characteristics of apoptosis or autophagy and were dependent with macropinosome rather than the swelling of organelles such as endoplasmic reticulum,mitochondria and lysosome.The cytoplasmic vacuolization and cytotoxicity induced by maduramicin were not affected by z-VAD-fmk,3-MA and CQ but bafilomycin Al.The expression of LC3-?/? and p62 were altered by maduramicin treatment,while maduramicin-induced cytoplasmic vacuolization was not amelirateed through down-regulated these proteins.Maduramicin significantly affected the expression of K-Ras,and Rac 1 protein.Collectively,these findings suggested that maduramicin-induced cell death is dependent on macropinosome mediated non-apoptotic cell death,which is relate to H+-ATPase and Ras-Racl pathway.Taken together,our findings indicated that maduramicin.induced severely damage and significantly altered the transcriptional profile of primary chicken myocardial cells.The differential expressed genes were mainly relate to inflammatory reaction,apoptosis,cytoplasm ion imbanlance and cytoplasmic vacuolization.Additionally,caspase-dependent and independent apoptosis pathway was involved in maduramicin-induced cardiotoxicity in chiken.Interestingly,a novel cell death type-methuosis has played a dominant role in maduramicin caused cardiotoxicity in primary chicken.Collectively,our findings provide a systematic explanation of maduramicin-induced cardiotoxicity in chicken and give a support for the prevention and control of maduramicin intoxication.