Cardiotoxicity Of Bufalin On Cardiomyocyte Apoptosis Induced By Endoplasmic Reticulum Stress

Backgrounds:Bufalin is a compound extracted from Chinese toad,its molecular formula C34H34O4,the relative molecular mass of 386.5,the melting point of 238-244?,not soluble in water.Modern pharmacological studies have shown that bufalin has a variety of pharmacological effects,mainly in the cardiac effect,anti-tumor effect,analgesic effect and increase the immune and so on,and has achieved satisfactory clinical efficacy.However,there are often clinical reports of poisoning due to overdosage or improper taking,and toxicity studies are not yet complete.How to avoid the toxic reaction while giving full play to the effectiveness of bufalin is an urgent problem to be solved.Understand the pharmacology of bufalin,toxic mechanism of action,for the guidance of clinical medication is of great significance.Apoptosis is crucial during heart development and has long been associated with many cardiovascular diseases such as ischemic heart disease,reperfusion injury,chemotherapy-induced cardiomyopathy and heart failure.At the same time,cardiomyocyte apoptosis has important pathophysiological consequences leading to abnormal myocardial function.A number of cardiovascular diseases have been reported,including myocardial infarction,end-stage heart failure,arrhythmogenic right ventricular dysplasia and doxorubicin-induced cardiomyopathy.Various stresses and conditions that trigger apoptosis are achieved by activating one or more signal transduction pathways that converge to activate a family of conserved aspartate-specific cysteine proteases called half Caspase.Caspases are expressed as inactive zymogens and are activated by dimerization or specific proteolytic cleavage in response to apoptotic stimuli.Activated cysteine proteases cleave and activate other family members and several key proteins that disrupt cells.Activation of caspases is a central cell apoptosis that can be activated by three different mechanisms:death receptor pathway,mitochondria release of cytochrome c and endoplasmic reticulum stress.Therefore,drug-induced cardiotoxicity does not rule out the possibility of apoptosis.The endoplasmic reticulum is the organelle responsible for protein folding and assembly,lipid and sterol biosynthesis,and free calcium storage.However,under physiological or pathological conditions,such as the limited ability of ERs to process proteins,the accumulation of unfolded and misfolded proteins induced by Ca²⁺depletion can lead to endoplasmic reticulum stress.In endoplasmic reticulum stress response,a series of homoeostatic responses to cellular activity,collectively referred to as unfolded protein responses?UPRs?,are maintained to maintain ER homeostasis.A major pathway of UPR is the up-regulation of ER chaperones,such as the glucose-regulated protein 78kDa?GRP78?,which can help repair unfolded proteins.Moderate ERS can relieve cell dysfunction and increase the chance of survival,while excessive and long-lasting ERS leads to apoptosis.Endoplasmic reticulum stress-induced apoptosis is triggered by the transcriptional induction of the C/EBP homolog?CHOP/GADDDD153?,the caspase-12-dependent pathway and the c-Jun NH2-terminal kinase1?JNK1?-dependent pathway Mediated.Caspase-12 activation induced by endoplasmic reticulum stress further triggers caspase-12 activation during caspase-induced apoptosis in the endoplasmic reticulum,caspase-12 is cleaved and specifically activated during endoplasmic reticulum stress 3 activation,eventually triggering apoptosis.Objective:Improve the primary myocardial cell extraction technology,shorten the experimental time and improve the purity of cells;observed by different concentrations of bufalin SD rat primary cardiomyocyte morphology,beating,calcium levels,apoptosis and endoplasmic reticulum Shock related indicators of the impact of buprenorphine cardiomyocyte cytotoxicity mechanism.Methods:1.Take the newborn 2-3 days SD rat rat in a sterile environment to take its heart,digested by digestive juice and differential adherence after primary cardiomyocytes obtained by cell observation and identification of cells to determine the nature of the cells Counts determine the purity of the cells and the cells obtained are used as a later experiment.2.Obtained in the cells by adding different concentrations of bufalin drugs,with CCK8 Determination of different concentrations of bufalin on cardiomyocyte activity in order to determine the concentration of bufalin for the next experiment,the concentration is divided into low,Medium and high three levels.3.The low,medium and high concentrations of bufalin added to the myocardial cells observed before and after dosing changes in cell morphology and the number of beating changes.4.Combined with Hochest33258 staining observed changes in cells and Caspase3 kit assay to determine whether there is apoptosis in primary cardiomyocytes after dosing.Protein expression of CHOP,Caspase and GRP78,a marker of endoplasmic reticulum stress-induced apoptosis,was detected by Western-blot.Results:1.Successfully improved the technology of primary cardiomyocyte extraction,shorten the experimental time to two hours?the experiment abroad usually takes overnight?,the purity of cells can generally reach more than70%to meet the experimental needs.2.After the addition of bufalin,the cell volume was reduced and the cytoplasm was denser;in the microscopic field,the beating count of four locations was counted by beating.The number of beating of cell clusters in the same area after the addition of drug was obviously accelerated,but the law was not obvious.Bufalin can significantly improve the primary cardiomyocyte beating frequency.3.Hochest staining showed that the cells appeared dense chromatin,staining deepened,the cell volume is reduced,the cytoplasm is more dense,eosinophilic staining increased,apoptotic bodies can be observed,suggesting that primary cardiomyocytes may exist apoptosis;after Caspase3 kit detection and found that caspase3 expression,suggesting that primary cardiac myocytes may exist apoptosis.Combining the two,bufalin can induce primary cardiomyocyte apoptosis in a concentration-dependent manner.4.After Western-blot analysis,it was found that the expression of CHOP,Caspase and GRP78 induced by endoplasmic reticulum stress increased with the increase of bufalin concentration,suggesting that bufalin induced cardiomyocyte apoptosis may occur Endoplasmic Reticulum stress related.Conclusion:1.Primary cardiomyocyte extraction,the use of step-by-step digestion method is more convenient than overnight digestion,no significant difference in cell purity;differential adherent 20min to 30min to remove most of the fibroblasts,leaving more active than Strong myocardial cells retained,cell purity was better than the differential adherent more than 1 hour.2.Bufalin can significantly inhibit cardiomyocyte activity,that is,has cytotoxicity;Bufalin can induce cardiomyocyte apoptosis,its role in inducing apoptosis has a concentration-dependent;Bufalin induced apoptosis may be caused by calcium Overloading stimulates cells to react with unfolded proteins,inducing endoplasmic reticulum stress and causing apoptosis.