Study On Preventive Of Buyanghuanwu Decoction Medicated Serum On Doxorubicin Induced Cardiomyocyte Apoptosis

BackgroundApoptosis is programmed cell death, it is a prestored death procedure in cell triggered by factors of vitra-in vivo. Research proves that cardiomyocyte apoptosis plays an important role in cardiovascular diseases, such as myocardial ischemia/ reperfusion injury, heart failure, myocardial infarction, ventricular remodeling after myocardial infarction, etc. Therefore, inhibition of cardiomyocyte apoptosis can improve prognosis of cardiovascular disease effectively.Doxorubicin(DOX) is one kind of anthracyline antibiotics, it is one kind of antitumor drug with high performance and broad-spectrum. All anthracyline antibiotics have cardio toxicity, and DOX have serious cardio toxicity, cardiomyocyte apoptosis plays an important role in cardio toxicity caused by DOX. In recent years, model of cardiomyocyte apoptosis induced by DOX has been widespreadly used in research of cardiomyocyte apoptosis.Modern empirical study of Buyanghuanwu Decoction Medicated Serum (BDMS) proves that BDMS has therapeutical effect on many cerebrovascular disease and cardiovascular diseases. BDMS can inhibit cerebral ischemical reperfusion injury, protect endothelium function of mouse suffering form atherosclerosis, inhibit the development of atherosclerosis, improve patients, left ventricular systolic and diastolic function who suffers from chronic heart failure, inhibit cardiomyocyte apoptosis induced by hypoxia-reoxygenation.Medicated serum refers to giving animals a certain amount of medicine, after a period of time, the blood-collecting was made through aorta abdominalis, then centrifuged blood at a revolution, then the serum was collected.The effect of chinese materia medica can be directly observed through culture cells by using the medicated serum, and pharmacological mechanism can be explaind through genes, gene product, drug receptors, enzyme activity et al. by using the instrument of cytology and molecular biology. Secondly, culturing cells by the medicated serum can make the modern science and technology and traditional chinese medicine have a workable combining site, promote the technological progress of TCM. At present, chinese medicine serum pharmacology has already been a general method in vitro research of pharmacological experiments of traditional chinese medicines, and widely used in Chinese traditional medicine and its preparations.There is no report of the research of the preventive effect of BDMS on Doxorubicin induced cardiomyocyte apoptosis in home and abroad. In this research, we will investigate the effect of BDMS on cardiomyocyte apoptosis induced by Doxorubicin.ObjectiveTo investigate the preventive effect of BDMS on Doxorubicin induced neonatal rat cardiomyocyte apoptosis. Methods1. The Preparation of BDMSConcentrate BDMS at a concentration of containing dried medicinal herb 1g/ml, 13.6g/kg-d herb medicine decoction administered orally, continuous administration for 7 days. When the rats were administered for the last time, then after 1h, the blood-collecting was made through aorta abdominalis under the condition of anesthesia. Laid the blood at room temperature for 3～4h, then centrifuged at a revolution of 3000rpm/min, then the serum was collected.2. Neonatal cardiomyocyte culturesPrimary cultures of 1-3 day old neonatal Wistar rat myocytes were prepared and cut into 1mm×1mm×1mm size. Minced ventricular myocardium was placed into D-Hanks’ salt solution. The cells were dissociated by a trypsin (0.01%) digestion in D-Hanks’ salt solution. After each of five to six successive 6 min incubations, the dissociated cells were mixed with DMEM containing 10% FBS and were centrifuged and pooled. The dissociated cells were enriched for cardiomyocytes by the technique of differential adhesion for 90 min and plated at a concentration of 2×105 cells or 2×10 cells in 6-well plates or 96-well plates. The cardiomyocytes were re-cultured for 48h～72h in a humidified 95% air and 5% CO2 atmosphere at 37℃. After 48h-72h incubation in DMEM containing 10% FBS, the attached cells were rinsed and maintained in DMEM containing2% FBS for 24h. After 24h of serum starvation, cardiomyocytes weretreated with various agents.3. Observation of different concentrations of Doxorubicin on cardiomyocytes activity by CCK-8 colorimetricAdjusted the density of cardiomyocytes to 2×104/ml with 10% FBS containing DMEM medium, inoculated with the 96-well plates, each plant hole 200μl.After incubate 48h～72h. After 24h of serum starvatiocells with DMEM containing 2% FBS, the cardiomyocytes were randomly divided into 5 groups:control group (control), DOX 0.5uM Group, DOX luM Group, DOX 2uM Group, DOX 2uM Group, each group 6 holes. CCK-8 cell viability assay was measured after incubate 24 hours.4. Observation of different times of Doxorubicin on cardiomyocytes injury by CCK-8Based on the results of aforementioned experiment, we choose 2uM as the final concentration, observation of different times of Doxorubicin on cardiomyocytes activity. Adjusted the density of cardiomyocytes to 2×104/ml with 10% FBS containing DMEM medium, inoculated with the 96-well plates, each plant hole 200μl.After incubate 48h～72h. After 24h of serum starvatiocells with DMEM containing 2% FBS, the cardiomyocytes were randomly divided into 5 groups: control group (control), Dox 6h Group, Dox 12h Group, Dox 24h Group, Dox 48h Group, each group 6 holes. The cell viability assay was measured by CCK-8.5. Use Hoechst 33258 Staining to assay cardiomyocytes apoptosis induced by DOXAdjusted the density of cardiomyocytes to 2×105/ml with 10% FBS containing DMEM medium, inoculated with the 6-well plates, each plant hole 2ml.After incubate 48h～72h. After 24h of serum starvatiocells with DMEM containing 2% FBS, the cardiomyocytes were randomly divided into 2 groups:control group (control), DOX Group. Hoechst 33258 Staining was used to assay cardiomyocytes apoptosis after incubate 24 hours.6. Observation of BDMS on cardiomyocytes activity by CCK-8.Adjusted the density of cardiomyocytes to 2×104/ml with 10% FBS containing DMEM medium, inoculated with the 96-well plates, each plant hole 200μl.After incubate 48h-72h. After 24h of serum starvatiocells with DMEM containing 2% FBS, the cardiomyocytes were randomly divided into 2 groups:(1)control group: 10% normal serum; (2) 10% BDMS Group, each group 6 holes. CCK-8 cell viability assay was measured after incubate 24 hours.7. Observation of BDMS on DOX induced cardiomyocytes injury intervention role by CCK-8Adjusted the density of cardiomyocytes to 2×104/ml with 10% FBS containing DMEM medium, inoculated with the 96-well plates, each plant hole 200μl.After incubate 48h～72h. After 24h of serum starvatiocells with DMEM containing 2% FBS, the cardiomyocytes were randomly divided into 3 groups:(1) control group: 10% normal serum; (2) 10% BDMS+2uM DOX; (3) 10% normal serum+2uM DOX, each group 6 holes. CCK-8 cell viability assay was measured after incubate 24 hours.8. Use Hoechst 33258 Staining to assay the preventive effect of BDMS on cardiomyocytes apoptosis induced by DOXAdjusted the density of cardiomyocytes to 2×105/ml with 10% FBS containing DMEM medium, inoculated with the 6-well plates, each plant hole 2ml.After incubate 48h-72h. After 24h of serum starvatiocells with DMEM containing 2% FBS, the cardiomyocytes were randomly divided into 3 groups:(1) control group 10% normal serum; (2) 10% BDMS+2uM DOX; (3) 10% normal serum+2uM DOX. Hoechst 33258 Staining was used to assay cardiomyocytes apoptosis after incubate 24 hours.9. Western-Blot analysisTo explore the molecular mechanisms of the antihypertrophic effect of total flavones from Psidium guajava leaves, we determined whether total flavones from Psidium guajava leaves inhibits signaling through AT1-PKC pathway. After various treatments, myocytes were harvested and lysed in 150μl NP-40 lysis buffer. The protein concentration was determined by the BCA method. Equal protein was loaded in each lane, resolved by SDS-PAGE, blotted on nitrocellulose membrane. Membranes were blocked in 5% nonfat milk powder in Tris-buffered saline (TBS)/0.1% Tween-20 for 90min at room temperature, and then incubated with specific antibodies in 5% BSA in TBS for overnight. Membranes were incubated with peroxidase conjugated second antibody in blocking buffer for 2 h. The labeled proteins were detected with enhanced chemiluminescence. The results were analyzed by KODAK Image Station 4000MM exposure imaging system. The experiment was performed three times.10. Statistical MethodsSPSS 13.0 statistical soft was used. Statistical analysis Results were expressed as mean±SD. Statistical significance of two samples was determined using Independent-SamplesT Test, mutiple was determined using one-way ANOVA Statistical significance was determined using One-Way ANOVA, multiple comparisons using post-hoc analysis with Bonferroni correction. When the variance of arrhythmia, the use of robust estimation Welch, then the use of Dunnett’s T3 compared methods. The test criterion is at a value of a=0.05.Result1. The result of the preparation of BDMSThere was no rat die in the experiment, and the process of preparation of BDMS is smoothly.2. Neonatal cardiomyocyte culturesJust isolated cardiac cells were spherical, then begin to adherent to wells after 4h cultured, showing round, then into a spindle. Cells continue to expand in the wells, extending pseudopods, and become irregular; A small number of individual adherent cells were spontaneously beating in a different frequency and rhythm after 12h cultured;Two days later cells are into a single layer, after 3 to 4 days cells extend pseudopodia contacting with each other into a network, and gradually forme cell clusters or single cells layer, into a radial array, beating rhythmically; sat a rate of 30 to 120 times/min.3. The impact of different concentrations of Dox on cardiomyocytes viabilityThe cardiomyocytes viability of Dox groups decreased compared with the control group (P=0.000). Dox groups had significant reduction in cell viability. With the increase of the concentrations of Dox, the cardiomyocytes viability was decreased, 2uM Dox group was lower than 0.5uM Dox group and luM Dox group, the difference is significant (P=0.000).4uM Dox group was lower than 2uM Dox group, but the difference was not significant (P=0.262).4. The impact of Dox at different times on cardiomyocytes viability:The cardiomyocytes viability of different time groups decreased compared with the control group (P=0.000). Different time groups had significant reduction in cell viability. With the prolongation of the time, the cardiomyocytes viability was decreased,24h Dox group was lower than 6h Dox group and 12h Dox group, the difference is significant (P<0.05).48h Dox group was lower than 24h Dox group, but the difference was not significant (P=0.491).5. The impact of Dox on cardiomyocytes apoptosis.Hoechst 33258 Staining proved that the Dox group showed a morphological features of cardiomyocytes apoptosis:cell body deflate, cellular organ compact, nuclear body shrinkage, cell shrinkage, chromatin condensation, chromosome fragmentation, chromosome fragmentation.6. The impact of BDMS on cardiomyocytes viabilityThe cardiomyocytes viability of BDMS group was higher than the control group (P=0.008).7. The impact of BDMS on cardiomyocytes viability induced by Dox.Compared with the DOX group, the cardiomyocytes viability of BDMS group was increased (P=0.000).8. The impact of BDMS on cardiomyocytes apoptosis induced by Dox.Hoechst 33258 Staining proved that the cellular nucleus of the control group showed a diffuse homogeneous nuclear fluorescence, the Dox group showed a morphological features of cardiomyocytes apoptosis:cell body deflate, cellular organ compact, nuclear body shrinkage, cell shrinkage, chromatin condensation, chromosome fragmentation, chromosome fragmentation, the BDMS group showed slight morphological features of cardiomyocytes apoptosis, compared with the Dox group, the degree of apoptosis was obviously relieved.9. The result of Western-Blot analysisCompared with the control group, the protein level of Bcl-2 in BDMS group and DOX group was reduced significantly(P=0.000), the protein level of Bax and p53 was increased significantly(P=0.000). Compared with the DOX group, the protein level of Bcl-2 in BDMS group was increased significantly(P=0.001), the protein level of Bax and p53 was reduced significantly(P=0.000, P=0.022).Conclusions1. Within certain range of concentrations of DOX(≤2uM), DOX can inhibit cardiomyocytes viability significantly depending on the concentration.2. Within certain range of time of DOX(<24h), DOX can inhibit cardiomyocytes viability significantly depending on the time.3. DOX (2uM 24h))can inhibit cardiomyocytes apoptosis.4. BDMS can inhibit cardiomyocytes viability and apoptosis induced by DOX. 5. BDMS can inhibit cardiomyocytes apoptosis through promote the protein level of Bcl-2 and inhibit the protein level of p53 and Bax.