| Objective:In recent years, a large number of study both at home and abroad have shown that oxidative stress related apoptosis is the common mechanism of cardiovascular damage caused by many different pathological factors. There are excessive oxygen free radicals generated in many pathological processes of cardiovascular diseases, such as atherosclerosis (AS), hypertension, ischemic heart disease, hyperlipidemia and so on, while the anti-oxidative defensive mechanism in these processes are inhibited. The damage of myocardial cells cause by oxidative stress is relevant to its intensity. It’s reversible in a certain stress intensity range. But the damage can become irreversible, which mainly includes myocyte apoptosis and necrosis, if beyond the certain range.The proto-oncogene Pim (Proviral integration site of murine) family in mammalian cells is serine/threonine kinase belongings to the calmodulin-dependent protein kinase, including Pim-1, Pim-2and Pim-3. Many experimental studies have confirmed the existence of a significant correlation between the upregulation of Pim kinase and inhibition of apoptosis. Pim-2expression continued to promote long-term cell resistance to various apoptosis signal stimulation. It’s confirmed that Pim-1protects myocardial injury via Akt pathway, the expression of Pim-2in Pim-1deficient mice increased4.6times;7days after myocardial infarction of Pim-1-deficient mice, Pim-2expression increased2.75times. But Pim-2expression and function in myocardial cells is still unknown, it is necessary to explore it’s function and mechanism of myocardial injury.Radix Notoginseng is a medicine with a variety of cardiovascular activity, it’s widely used in clinical in the prevention and treatment of cardiovascular disease. With the advance of modern chemistry and pharmacology, Panax notoginseng saponins (Panax notoginseng saponin, PNS) is identified to be the main active ingredient of the Radix Notoginseng, contains a variety of monomer saponin, such as ginsenoside Rbl, ginsenoside Rgl, the notoginsenoside R1. Focusing on notoginsenoside R1in this study, our purpose is to understand the protective effect of notoginsenoside R1in myocardial injury, and to investigate the cell signaling pathways and proteins involved in apoptosis regulation, and Pim-2effect in notoginsenoside R1protection.Method:1. Primary culture of neonatal rat cardiomyocytesNeonatal rats’ hearts were removed in surgical sterile conditions, digested in trypsin in4℃overnight, and then gradually digested the heart tissue with collagenase into isolated cells. Cells were inoculated into a culture flask. Differential adhesion separation method was used to remove cardiac fibroblasts in order to obtain pure myocardial cells in this study.2. Model of H2O2-induced oxidative stress in myocardial cellsNormal myocardial cells were cultured for2-3days, proliferated to reached the density of80%-95%. Cells that pulsed at the rate120～140times per minute were selected for the use of experimental myocardial cells. Different time points and concentrations were tested for H2O2intervention. Proper concentration of H2O2and time point to intervene was used in follow-up experiment.3. Notoginsenoside R1myocardial preconditioningCultured myocardial cells first2-3days, cell growth close to80%confluence, myocardial cells pretreated with different concentrations notoginsenoside R1cultured24h, toxicity tests conducted Notoginsenoside R1, select the appropriate high, medium and low dose groups related follow-up experiments.4. Assessment of cell viability (MTT assay)Cardiomyocytes were seeded in96-well culture plates at a density of1×104cells/well, were incubated at37℃for48h, and then pretreated with different drugs depend on experimental groups. After that, the cells were washed with phosphate-buffered saline (PBS) three times and MTT dye was added to each well for the last4h of treatment. The reaction stopped with the addition of dimethyl sulfoxide (DMSO) and the optical density was determined at490nm on a multi-well plate reader. All groups were assayed in six wells and the mean for each group was calculated.5. Analysis of cardiomyocyte apoptosis with flow cytometryMyocardial cells according to 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 for5～15min, analysed by flow cytometry (excitation wavelength488nm, emission wave length530nm).6. Detection the level of intracellular LDH、MDA、SOD in myocardial cellsMyocardial cells were seeded in six wells, grouped by different experimental treatment. Analysises were done in accordance with the instruction of LDH, MDA, SOD detection kit after treatment, each set of OD values measured at420nm in a microplate reader and the contents of LDH, MDA, SOD in every sample was calculated according to these values. Each experiment was repeated three times.7. Real-time quantitative PCR analysis of myocardial cells Pim-2mRNA expression.Cardiomyocytes were seeded to6-well plates2×107/well, cultured for2-3days. Cells were collected in optimal condition for the following experiment. Samples were divided as following groups:normal groups, control groups,0.5,1,2,3,6hours H2O2treated group,50μM H2O2was used for myocardial cells. The total RNA of cardiomyocytes was extracted with Trizol, purity and concentration of the extracted RNA was measured on a UV spectrophotometer. Then cDNA was synthesized by reverse transcription, and fluorescence quanititavie dectection of the target gene was performed afterwards. The primer of Pim-2gene was designed according to Gene Bank sequence imformation, by means of Primerpremier5.0software.7. SiRNA silencing myocardial cells’Pim-2expression.Cells were cultured in six wells and ninty-six wells for2or3days to reach80%～90%confluent, cells of optimal condition were used for further expriments. SiPORTTM NeoFXTM was incubated with DMEM and SiRNA diluent in the same volume for15min at room temperature. The mixture was added into the culture plates for transfection for next48～72h.After that cells were collected for Western blot analysis and flow cytometry analysis of cardiomyocyte apoptosis rates.8. Western blot analysis of the 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 PVDF 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 KODAK Image Station2000MM system, analysis the image with Image Tool3.0, the gray values of the lanes were measured and quantization as the expression of target proteins. Actin was used as loading control in this assay.10. Statistical analysis:The data was analyzed by SPSS13.0. measurement data was presented as the means±SEM. Test of normality and homogeneity of variance was done in each group. If datas accorded with normality and homogeneity of variance, statistical analysis was made by one-way ANOVA followed by LSD test, otherwise statistical analysis was made by rank sum test, and multiple comparison was made by Tamhane’s T2. Multiple enumeration data was analyzed by Kruskal-Wallis. Hemodynamics parameters in different time points was analyzed by repetitive measurement and analysis of variance. Level of significance a=0.05Result:1. Notoginsenoside Rl roles in H2O2-induced myocardial injury1.1The concentration-response relationship of myocardial cells injury to H2O2. MTT assay shows that after treating the cardiomyocytes with different concentrations of H2O23hours, viability of cells were evidently reduced. Cells pulse became slower or even stop when observed through the microscope, there are large pieces of cells shedding in the area that cells was relative crowded. Compared to the normal control,cell viability after H2O2intervention at various concentration were all reduced, the statistical analysis of the difference between each group were statistically significant (F=734.372,P<0.001), and cell viability were more apparently reduced with higher concentration of H2O2.1.2The different concentrations of notoginsenoside R1on myocardial cell viability. Myocardial cells were preconditioned with different concentrations of notoginsenoside for24hours. Comparisons of cell viability were made between groups treated with different concentrations of notoginsenoside R1and the normal control group. Statistical analysis showed that there are no statistically difference of cell viablility between groups treated with0.1×10-6mol/L,1×10-6mol/L,10x10-6mol/L notoginsenoside R1and groups of normal control (P>0.05), while the concentration of100×10-6mol/L groups’ viability were significantly lower than controls after treating myocardial cells for24hours (P<0.01).1.3Pretreatment of Notoginsenoside R1on myocardial cell viability in H2O2injury models. Myocardial cells were pretreated with low, medium and high concentrations (0.1×10-6mol/L,1×10-6mol/L,10×10-6mol/L) of notoginsenoside for24hours, then H2O2(SOμmol/L) was added for3hours, normal control group and model group were set up at the same time. Comparisons of myocardial cells viability were made between groups of different concentrations notoginsenoside R1in H2O2injury models. The statistical analysis showed that the cell viability difference between the groups was statistically significant ((F=519.758, P<0.001). Notoginsenoside R1pretreatment reduced the decline of myocardial cell viability in a dose-dependent manner after H2O2treatment.1.4Pretreatment of Notoginsenoside R1reduced the apoptosis rate of myocardial cell in the H2O2injury model. The apoptotic rates detected by flow cytometry were used to compare the effects of notoginsenoside R1of multiple concentrations in H2O2-induced models. After pretreatment by notoginsenoside R1, the apoptotic rates of cells were lower campared to the model group, especially in high concentration groups, with significantly improvement of apoptotic rates, After statistical analysis, differences of each groups were statistically significant (P <0.0001).1.5Activities of LDH, MDA and SOD in notoginsenoside R1pretreated myocardial cells of H2O2intervention. Compared with the normal control group, SOD activity of model groups were significantly reduced, while the activity of LDH, MDA was significantly increased (P<0.001). Notoginsenoside R1lowered SOD activity in a dose-dependent manner, while increased LDH and MDA activity. The difference among the groups was statistically significant (P<0.001).2Notoginsenoside R1effects on H2O2-induced myocardial apoptosis signaling pathways and apoptosis-related protein expression2.1Notoginsenoside R1effects on H2O2intervention myocardial cells MAPK signaling pathway2.1.1Western Blot detection of protein expression of p-ERK1/2, total ERK1/2in each group and analysis of the target bands by optical density (IOD). Compared with normal group, model group (H2O2) p-ERK1/2expressions were significantly increased. P-ERK1/2expression in groups of low, medium and high dose of notoginsenoside R1pretreatment were significantly reduced compared to the modele groups, especially in of the high-dose groups. There are significant differences of p-ERK1/2expression among the groups (P<0.01).2.1.2Western Blot detection of protein expression of P-JNK, total JNK in each group and analysis of the target bands by optical density (IOD). Compared with the normal group, model group (H2O2) P-JNK expression was significantly increased (P <0.001). There are no significant difference between groups of multiple concentrations of notoginsenoside Rl pretreatment and model groups (P>0.05).2.1.3Western Blot detection of protein expression of P-P38, P38in each group and analysis of the target bands by optical density (IOD). Compared with normal group, model group (H2O2) P-P38expression was significantly increased (P<0.001), notoginsenoside R1low. There are no significant difference between groups of multiple concentrations of notoginsenoside R1pretreatment and model groups (P>0.05). 2.2Changes of expression of Bax/Bcl-2apoptotic proteins of cardiomyocytes after pretreatment of notoginsenoside R1. Bax/Bcl-2expression was detected by Western Blotting analysis in each group; analyses of the target band optical density (IOD) were performed aferward. Normal groups have a certain amount of Bax, Bcl-2expression, Bax expression of model groups were significantly higher than the normal groups, notoginsenoside R1groups have a decreased Bax expression compared to model groups (P<0.01); Compared with the normal group, model groups have reduced Bcl-2expression. Notoginsenoside R1groups expression of BCL-2were gradually increased compared to model groups (P<0.01). The Bax/Bcl-2ratio of model groups was significantly higher to normal groups, notoginsenoside R1groups have reduced Bax/Bcl-2compared to model groups (P<0.01).3Pim-2expressions and its role in H2O2-induced myocardial injury3.1H2O2intervention significantly upregulated Pim-2mRNA expression in myocardial cells.50mM/L H2O2was addd to myocardial cells cultures for0.5,1,2,3,6h, the normal control group was setted up at the same time. Pim-2mRNA expression analysed by real-time quantitative PCR. It’s showed that Pim-2mRNA expression increased in groups after the intervention of H2O2.There are statistically significant between groups (P<0.001). Comparsions between each groups were statistically analysed too.Which0.5h and1h groups showed no significant difference compared to the control group (P>0.05); the remaining groups were significant different between each others (P<0.0001), especially in3h, Pim-2mRNA level increased to the most obvious level;6h groups showed reduced Pim-2mRNA expression, but the level was still higher than the normal control group.3.2H2O2intervention significantly increased Pim-2protein expression in myocardiocytes. Pim-2protein expressions were increased after H2O2intervention, indicated by Westernblotting analysis of Pim-2protein expression at different time points. The growth of Pim-2expressions were correlation with the growth of mRNA expression analysed by real-time fluorescence quantitative PCR. The difference between each groups were statistically significant (P<0.01).3.3Pim-2siRNA interference on apoptosis of myocardial cells. Samples of myocardial cells cultures were randomly divided into normal group, H2O2group, siRNA group (H2O2+siRNA), and negative control group (H2O2+NC). The results show model group compared with the normal group, the apoptosis rate of model group was increased significantly compared to the normal control group. The siRNA interference further increased the apoptosis rate (P<0.001), negative control group was significant different compared to the normal group (P<0.001), but no statistical difference when compared to the model group (P>0.05).3.4Impact of Pim-2siRNA interference on activity of LDH, MDA and SOD. SOD activity after Pim-2siRNA interference was significantly reduced compared with the model group, while LDH, MDA content compared with the model group was significantly increased, the differences between groups has statistical significance (P <0.001). Indexes of negative control group were not statistically significant (P>0.05) when compared with the model group.4Pim-2protein expression and regulation4.1Notoginsenoside R1increased Pim-2protein expression. Pim-2protein expression of myocardial cells was analysed by Western Blotting, the results showPim-2expression of the model group (H2O2) was significantly higher than the normal group, notoginsenoside R1of different concentration further increased the Pim-2expression, the most Pim-2expressoin was observed in the high dose notoginsenosdie R1group. Difference among the groups was statistically significant (P<0.01).4.2Pim-2siRNA interference on notoginsenoside Rl pre-treatment H2O2injured cardiac myocyte. Samples of myocardial cells were divided into normal group, model group (H2O2), notoginsenoside R1group (H2O2+NG Rl10μM), siRNA transfection group (H2O2+NG R1lOμM+siRNA), siRNA negative control group. Apoptosis rates were analysed by flow cytometry, the result showed that the model group was significantly higher compared with the normal group (P<0.001), apoptotic rates in oginsenoside R1group were decreased compared with the model group. There are no significant difference between siRNA group, siRNA negative control group and notoginsenoside R1group (P>0.05).4.3Pim-2siRNA interference on Bax/Bcl-2protein expressons. Samples of myocardial cells were divided into normal group, H2O2group, siRNA group (H2O2+siRNA), and negative control group. Western blotting was used to detect the expression in Bax, Bcl-2protein, the target bands were analysed with optical density (IOD). The expression of Bax in model group was significantly higher than normal control group. SiRNA group showed further increase compared with model group expression (P<0.01), there is no significant difference on Bax expression between negative control group and model group. Bcl-2expression of model group was decreased when compared to normal group, SiRNA group showed further decrease compared with model group expression (P<0.01), there is no significant difference on BCL-2expression between negative control group and model group. Bax/Bcl-2ratio was significantly increased when comparison was made through the model group and normal group (P<0.01), there was further increase in SiRNA group, but no significant difference between negative control group and normal group..Conclusion:1, H2O2can inhibit cardiac activity, and promote apoptosis of cardiac myocyte, resulting in the destruction of the oxidative balance system. Notoginsenoside R1can inhibit H2O2-induced myocardial injury, enhance myocardial cell viability, cell antioxidant activity, and protect cardiac myocyte from apoptosis.2. H2O2enhanced myocardial cells phosphorylation of ERK, JNK, P38proteins. Notoginsenoside R1reduced phosphorylation of JNK after H2O2intervention of myocardial cells, but did not have significant regulation on ERK and P38phosphrylation. Bax expression decreased, while Bcl-2expression increased simultanteously in H2O2intervention after pretreatment of notoginsenoside R1. It’s suggested that Notoginsenoside R1protect cardiomyocyte from apoptosis through it’s regulation of the ERK signaling pathway and apoptosis-related proteins.3. H2O2can induce expression of Pim-2kinase in myocardial cells. Pim-2siRNA interference can effectively silencens Pim-2gene, and blocked its protective effect on myocardial injury.4. Pim-2expression in the myocardial cells increased by Notoginsenoside R1in H2O2intervention. Notoginsenoside R1can still prevent cardiomyocyte apoptosis dispite of SiRNA interference of Pim-2, indicating that functions of notoginsenoside R1against myocardial cell apoptosis is not only regulated by Pim-2, but also through variety of apoptosis-related proteins such as Bax and Bcl-2. |
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