Protective Effect Of Different Compatibility Proportion Of Aconiti Lateralis Radix Praeparata And Ginseng Radix Et Rhizoma In Neonatal Rats Cardiomyocytes

Purpose: 1. We established the injury model of primary neonatal rats cardiac muscle cell sugar anoxia/ reoxygenation sugar, to survey the effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma of medicated serum of spontaneous imp frequency, cell viability, superoxde dismutase(SOD), malondialdehyde(MDA), Nitric oxide(NO) and lactate dehydrogenase(LDH), Bcl-2, Bax, Caspases-3 in neonatal rat hypoxia injury cardiomyocytes. In order to evaluate protective Effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma hypoxia injury in neonatal rats cardiomyocytes. 2. In order to evaluate the inducing toxicity effect of Aconiti Lateralis Radix Praeparata and different compatibility proportion of Panax ginseng in neonatal rats cardiomyocytes. 3. In order to evaluate the mechanism of protective Effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma hypoxia injury in neonatal rats cardiomyocytes.Method: 1. Primary culture of neonatal rat cardiomyocytes The new-born rats within 24 h, were placed in superclean bench, 75% alcohol disinfection. Then, Neonatal rats’ hearts were remove under sterile conditions, and cut into small pieces, digested in collagenase and trypsin treatment Differential adhesionseparation method was used to remove cardiac fibroblasts in order to obtain high purity cultured cardiomyocytes in this study. myocardial cells. 2. Model of cell myocardial damage Normal myocardial cells were cultured for 6days, proliferated to reached the suitabledensity. Cells that steady spontaneous imp frequency were selected for the use ofexperimental myocardial cells. The cardiomyocytes into the hypoxic box, slowly and continuously feeding the mixed gas(nitrogen content 95%) for 2h, taken out and aspirating balanced salt buffer, complete medium was added and incubated into CO2 incubator, the analog in vivo ischemia-reperfusion process, incubated 24 h. 3. Preparation of different compatibility proportion of serum drug Preparation of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma(0.5:1、1:1、2 :1、4:1、1:0、1:0.25、1:0.5、1:1、1:2) decoction. Wistar male rats(100 rats) were randomly divided into 10 groups. different compatibility proportion of decoction were given the rats, ig, 1times/d, for one week. After the last medicine administration, blood from the femoral artery, were placed under room temperature condition then centrifugatiing, obtaining serum. Serum were filtratd with 0.22 um membrane, in 56 ℃ water bath for sterilization, saving in-20 ℃. 4. Detection index and method 4.1 spontaneous imp frequency of cardiomyocytes Cardiomyocytes were seeded in 96-we Il culture plates at a density, were pretreated with different drugs depend on experimental groups, then incubated at 37°C for suitable time. After that, myocardial cells were placed under an inverted microscope, randomly selected 10 different horizons within 10 primary cardiomyocytes were observed, recorded cardiomyocytes imp frequency(times/min), the test was repeated six times, calculate the average myocardial imp frequency(times/min). 4.2 Assessment of myocardial cell viability(MTT assay) Cardiomyocytes were seeded in 96-we Il culture plates at a density, were pretreated with different drugs depend on experimental groups, then incubated at 37°C for suitable time. After that, the myocardial cells were washed with phosphate-buffered saline(PBS) three times and MTT dye was added to each well for the last 4h of treatment. The reaction stopped with the addition of dimethyl sulfoxide(DMSO) and the optical density was determined at 490 nm on a multi-well plate reader. All groups were assayed in six wells and the mean for each group was calculated. 4.3 Assessment of cardiomyocyte apoptosis with flow cytometryMyocardial cells all the experimental groups were digested by EDTA-free trypsin, stained with to Annexin V-FITC/Propidium Iodide, incubated at room temperature and protected from light for 5-15 min, detected by flow cytometry.(excitation wavelength 488 nm, emission wave length 530 nm). 4.4 Detection the level of intracellular SOD,MDA, LDH,NO in myocardial cells Myocardial cells were seeded in six wells, administrated by different experimental treatment. Detection were done in accordance with the instruction of SOD, MDA, LDH,NO detection kit after treatment, each set of OD values measured in amicroplate reader and the contents of SOD, MDA, LDH, NO in every sample was calculated according to these values. Each experiment was repeated six times. 4.5 Detection myocardial cells Caspase-3 activity Myocardial cells were seeded in six wells, administrated by different experimental treatment. Detection were done in accordance with the instruction of Caspase-3 detection kit after treatment, each set of OD values measured in fluorospectrophotometer. 4.6 Analysis of the Bcl-2, Bax, ERK1/2, p-ERK1/2 protein expression of myocardial cells. Myocardial cells were solubilized after complication of the treatment by using the Western or IP cell lysates, protein quantitation was performed by using BCA method. before the extrats went for immunoblotting analysis. Total proteins were separated on a SDS-PAGE gel, and transferred to a NC membrane after electrophoresis, incubated with primary and secondary antibody in turn after the membrane was blocked, signal was developed by using ECL substrate, acquired image by Integrated chemiluminescence image analysis system, the grayvalues of the lanes were measured and quantization as the expression of target proteins. Actin was used as loading control in this assay. 4.7 Analysis of myocardial cells Bcl-2, Bax and Caspases-3 m RNA expression. The total RNA of all cardiomyocytes were extracted with Trizol, purity and concentration of the extracted RNA was measured on a UV spectrophotometer. Then c DNA was synthesized by reverse transcription, and fluorescence quanititavie dectection of the target gene was performed afterwards. The primer of Bcl-2,Bax and Caspases-3 gene was designed according to Gene Bank sequence imformation, by means of Sequence Detection software version 1.2.35. Statistical analysis The data was analyzed by SPSS13.0. measurement data was presented as the means 土 SEM. Test of normality and homogeneity of variance was done in each group. Level of significance a=0.05Result: 1. Protective Effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma Hypoxia Injury in Neonatal Rats Cardiomyocytes 1.1 Comparison of cardiomyocytes imp frequency among various groups In 0~72h, Compared with NC group, spontaneous imp frequency decreased obviously in model group and blood serum group(P<0.05). Compared with BS group, spontaneous imp frequency increased obviously(P<0.05) in 6~48h in medication administration groups, besides FR(0.5:1) and FR(4:1) groups, and time-effect dependent, Result show:There was a protective effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma hypoxia injury in neonatal rats cardiomyocytes. Compared with other medicated serum groups, spontaneous imp frequency increased obviously(P<0.05) in medicated serum Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma(2:1)group in 48 h. In 24 h and 48 h, compared with FR(2:1) group, there was a significant difference(P < 0.05)in FR(0.5:1) and FR(4:1) groups, in 12 h, there was a significant difference(P<0.05)in FR(0.5:1) group. Further show, there was a significant protective effect in FR(2:1) group hypoxia injury in neonatal rats cardiomyocytes. 1.2 Comparison of cardiomyocytes viability among various groups In 0~72h, Compared with NC group, cardiomyocytes viability decreased obviously in model group and blood serum group(P<0.05). Compared with BS group, cardiomyocytes viability increased obviously(P< 0.05) in 6 ~ 72 h in medication administration groups, besides FR(0.5:1) group, and time-effect dependent. Result show:There was a protective effect in medicated serum groups hypoxia injury in neonatal rats cardiomyocytes. Compared with other medicated serum groups, cardiomyocytes viability increased obviously(P<0.05) in medicated serum FR(2:1) group in 48 h. In 24 h and 72 h, compared with FR(2:1) group, therewas a significant difference(P<0.05)in FR(0.5:1) and FR(4:1) groups. Further show, there was a significant protective effect in FR(2:1) group hypoxia injury in neonatal rats cardiomyocytes. 1.3 Comparison of cardiomyocytes apoptosis rate among various groups In 0~72h, Compared with NC group, cardiomyocytes apoptosis rate increased obviously in model group and blood serum group(P<0.05). Compared with BS group, cardiomyocytes apoptosis rate decreased obviously(P<0.05) in 6~72h in medication administration groups, besides FR(0.5:1) group in 6h, Compared with other medicated serum groups, cardiomyocytes apoptosis rate decreased obviously(P<0.05) in medicated serum FR(2:1) group in 48 h. 1.4 Comparison of SOD,MDA,LDH,NO among various groups Compared with BS group, the content of MDA, the secretory volume of NO and the release rate of LDH decreased obviously, the activity of SOD increased obviously(P<0.05)in medication administration groups. Compared with other medicated serum groups, the activity of SOD increased obviously, the content of MDA and the release rate of LDH decreased obviously(P<0.05)in medicated serum FR(2:1) group. Compared with FR(2:1) group, the secretory volume of NO(P<0.05)in FR(0.5:1) and FR(4:1) groups. 1.5 Comparison of the activity of Caspases-3 and Caspases-3m RNA among various groups Compared with BS group, the expression of Caspases-3 m RNA and the activity of Caspases-3decreased obviously(P < 0.05)in medication administration groups, Compared with other medicated serum groups, the activity of Caspases-3, the expression of Caspases-3 m RNA decreased obviously(P<0.05)in FR(2:1) group. 1.6 Comparison of the expression of Bcl-2, Bax protein and Bcl-2 m RNA among various group Compared with BS group, the expression of Bcl-2 protein and Bcl-2 m RNA increased gradually, the expression of Bax protein decreased obviously(P < 0.05)in medication administration groups, Compared with other medicated serum groups, There was a significant difference about above indexes changes in FR(2:1) group. Result show :in All medication administration groups, there was a significant protective effect in FR(2:1) group.1.7 The protective effects of different concentrations of medicine serum myocardial cells injury Compared with BS group, there was a protective effect in different concentrations of medicine serum myocardial cells injury. Compared with 5% concentrations of medicine serum group, there was a significant difference in spontaneous imp frequency, cell viability, the activity of SOD, the release rate of LDH, the expression of Bcl-2m RNA and Bcl-2, Bax protein in 10% and 15%concentrations of medicine serum group, but, there was no difference in 10% and 15%concentrations of medicine serum group. 2. The Inducing Toxicity Effect of Aconiti Lateralis Radix Praeparata and Different Compatibility Proportion of Panax Ginseng in Neonatal Rats Cardiomyocytes 2.1 Comparison of cardiomyocytes imp frequency among various groups In 0~4 h, Compared with BS group, spontaneous imp frequency increased obviously in FR(1:0) group(P < 0.05). Compared with FR(1:0) group, spontaneous imp frequency decreased obviously(P<0.05) in other medication administration groups. There were a time dependent manner within 0~4 h, in FR(1:0.25) group, Compared with FR(1:0.5) group, there was a significant difference in FR(1:0.25) group, but there was no difference in FR(1:1)、FR(1:2) and BS groups(P>0.05). Result show:compared with BS group, there was no significant difference in FR(1:0.5) group(P>0.05). 2.2 Comparison of cardiomyocytes viability among various groups In 0~4 h, Compared with BS group, cardiomyocytes viability decreased obviously in FR(1:0) group(P<0.05). Compared with FR(1:0) group, cardiomyocytes viability increased obviously(P<0.05) in other medication administration groups within 2~4 h. There were a time dependent manner within 0 ~ 4 h, in FR(1:0.25) group, Compared with FR(1:0.5) group, there was no difference in FR(1:1)、FR(1:2) and BS groups(P>0.05), there was a significant difference in FR(1:0.25) group, within 3~4 h(P<0.05). Result show:compared with BS group, there was no significant difference in FR(1:0.5) group(P>0.05). 2.3 Comparison of SOD,MDA,LDH,NO among various groups Compared with BS group, the content of MDA, the secretory volume of NO and the release rate of LDH increased obviously, the activity of SOD decreased obviously,(P<0.05)in FR(1:0) groups. Compared with FR(1:0) group, the activity of SOD increased obviously, the content of MDA, the secretory volume of NO and the release rate of LDH decreased obviously(P<0.05)in other medication administration groups, Compared with FR(1:0.5) group, there was no difference about above index changes in BS group(P>0.05), but there was a significant difference in FR(1:0.25) group(P<0.05). Result show: compared with BS group, there was no significant difference in FR(1:0.5) group(P>0.05). 2.4 The inducing toxicity effect of different concentrations of medicine serum in Neonatal Rats Cardiomyocytes Compared with BS group, there was a myocardial cells injury effect in different concentrations of medicine serum groups(FR1:0). Compared with different concentrations of medicine serum groups(FR1:0), there was a significant difference in spontaneous imp frequency, cell viability, the activity of SOD, the content of MDA, the release rate of LDH, the secretory volume of NO(P<0.05) in different concentrations of medicine serum groups(FR1:0.5), but, there was no difference in different concentrations of medicine serum(15%、10%、5%)groups(FR1:0.5)(P>0.05). 2.5 Comparison of the activity of Caspases-3 and Bcl-2 and Caspases-3m RNA among various groups Compared with BS group, the expression of Caspases-3 m RNA and the activity of Caspases-3 increased obviously, the expression of Bcl-2 decreased obviously(P<0.05)in FR(1:0) group. Compared with FR(1:0) group, the activity of Caspases-3, the expression of Caspases-3 m RNA decreased obviously, the expression of Bcl-2 increased obviously(P<0.05)in other medication administration groups, Compared with FR(1:0.5) group, there was no difference about above index changes in BS group(P>0.05), but there was a significant difference in FR1:0.25 group(P<0.05). Result show: compared with BS group, there was no significant difference in FR1:0.5 group(P>0.05). 2.6 Comparison of the expression of Bcl-2, Bax protein among various groups Compared with BS group, the expression of Bcl-2 protein decreased obviously, the expression of Bax protein increased obviously(P<0.05)in FR1:0 group, Compared with FR1:0 group, the expression of Bcl-2 protein increased obviously, the expression of Bax proteidecreased obviously(P<0.05)in other medication administration groups, Compared with FR(1:0.5) group, there was no difference about above index changes in BS group(P>0.05), but there was a significant difference in FR(1:0.25) group(P<0.05). Result show: compared with BS group, there was no significant difference in FR(1:0.5) group(P>0.05). 3. The mechanism of protective Effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma hypoxia injury in neonatal rats cardiomyocytes. Compared with BS group, the expression of Bcl-2, ERK1/2, p-ERK1/2 protein and Bcl-2 m RNA increased obviously, the expression of Bax protein and Bax、Caspases-3 m RNA decreased obviously(P<0.05)in FR(2:1) group(P<0.05). Compared with BS group, there was no significant difference about above index changes in FR(2:1)+B group(P>0.05).CONCLUSION: 1. There was a significant protective effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma hypoxia injury in neonatal rats cardiomyocytes, the optimization compatibility proportion in Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma(2:1); there was a significant difference between 5% and 10%, 15% concentrations of medicine serum groups, but there was no difference in 10% and 15%concentrations of medicine serum group(FR2:1). 2. There was a significant inducing toxicity effect of Aconiti Lateralis Radix Praeparata and different compatibility proportion of Panax ginseng in neonatal rats cardiomyocytes, the optimization compatibility proportion in FR(1:0.)5;There was no difference in different concentrations of medicine serum(15%、10%、5%)groups(FR1:0.5). 3. ERK1/2 signaling pathway may involve protective effect of different compatibility proportion of Aconiti Lateralis Radix Praeparata and Ginseng Radix et Rhizoma hypoxia injury in neonatal rats cardiomyocytes.