Study On The Apoptosis/Autophagy Of Cardiomyocytes Induced By Cinobufagin

BackgroundVenenum bufonis is a very promising Chinese herbal medicine,its pharmacological effects clear,but because of its complex composition,mechanism of pharmacologic is diverse,the main active ingredient of toad base with toxicity,so the clinical use of relatively safe range become smaller.Repeatedly reported in clinical application of poisoning,allergies and other reports,of which a higher incidence of adverse cardiovascular events,which greatly restricted the toad as a drug development and clinical use.Cardiotoxicity of toad may be closely related to its chemical composition of the pharmacologically active ingredient toad steroid compounds and digitalis similarities.Cinobufagin,as one of the main components of toad,its efficacy has been elucidated and its clinical efficacy is multifaceted as the value of new drug development.At present,its research mainly focuses on its cardiac effects,anti-tumor and narcotic Pain and other pharmacological effects,and its toxicological effects,toxicokinetics and distribution in the body,absorption,metabolism and other research is relatively scarce.How to reduce its toxic effect,to maximize the medicinal value deserves our in-depth study.ObjectiveCinobufagin and Venenum bufonis has similar pharmacological,thus great as the value of new drug development.In this study,the primary cultured cardiomyocytes were extracted to establish an in vitro experimental model to observe whether Cinobufagin is toxic to primary cardiomyocytes,and to investigate the toxic effects of Cinobufagin on the heart and its related mechanisms from the perspective of autophagy and apoptosis.Methods1.The heart of neonatal rat was obtained by decapitation method.Purified primary cardiomyocytes were obtained by double enzyme digestion and differential adherent method.Cell viability was detected by trypan blue staining.The growth of cells and the number of beats were observed and recorded under the microscope.The growth curve of cardiomyocytes was detected by the method of MTT and the purity of cardiomyocytes was identified by immunofluorescence.2.Cardiomyocytes were treated with Cinobufagin for a certain period of time,the survival rate of cardiomyocytes was measured by thiazolyl blue method;the morphological changes of myocardial cells were observed by hematoxylin-eosin staining;the apoptosis of cells was observed by DAPI fluorescent staining;Cell apoptosis and necrosis were identified by cytometry.Fluorescence probe JC-1 was used to detect the change of mitochondrial membrane potential.Western Blot was used to detect the expression of Bcl-2 protein.3.Dansyl cadaverine staining and AO staining were to detect the formation of acid autophagic vacuoles;Western Blot to detect the LC3 protein expression;MTT assay detecct the autophagy inhibitors on the activity of myocardial cells.Results1.Primary cardiomyocytes isolation and culture The survival rate of isolated primary cardiomyocytes was about 70%-85%,and the proportion of primary cardiomyocytes in the total cells was large and irregular.After 12 h culture,cell adherence was observed.After 36 h,the cells showed irregular pulsation.48 h cells were cross-linked and clustered,and the cell pulsation increased.After 72 h,the pulsatility of clustered cells was accordance,with 50-110 /min pulsation.2.Cinobufagin induced cardiomyocyte apoptosis With different concentrations of Cinobufagin in primary myocardial cells cultured 24 h,microscope can be observed with the increase number of cell death with increase concentration of Cinobufagin,cell vacuoles and deformation;HE staining can observed in nuclear fragmentation and collapse;the results of MTT showed that the Cinobufagin has inhibitory effect on the proliferation of primary cardiac muscle cells.DAPI staining could observe the nuclear fragmentation of the cells treated with toad toxin above the concentration of 15μM and the formation of apoptotic bodies.The detection of mitochondrial membrane potential showed that the red orange fluorescence of the cardiac myocytes,in the Cinobufagin treated group was significantly higher than that in the blank control group.The results of Western blot showed that the expression of anti apoptotic protein Bcl-2 was lower than that in the blank group(P<0.01).3.Cinobufagin induces cardiomyocyte autophagy The result of AO and MDC staining indicated that Cinobufagin could induce the increase of autophagic vacuoles and autolysosome production in primary cardiac myocytes.The result of western blot showed that the expression of LC3 II protein was up-regulated and in peak at 4 hours and down later(P<0.05).ConclusionTo sum up,the toxicity of Cinobufagin on primary myocardial cells is concentration dependent,and large concentration can reduce cell viability and inhibit cell proliferation.Cinobufagin could induce apoptosis of primary cardiomyocytes through mitochondrial pathway,and induce autophagy in cardiomyocytes,which is one of the causes of myocardial damage.The Cinobufagin in vitro shows potential cardiac toxicity.