Study On The Mechanism Of Qishen Granule Inhibiting Cardiomyocyte Apoptosis Based On Drug Target Prediction

ObjectiveHeart failure is the end stage of various cardiovascular diseases.Ventricular remodeling induced by cells apoptosis is one of the basic mechanisms of heart failure.P53/Mdn2 plays a critical role in regulating apoptosis in various cardiovascular diseases.Targeting on it has been becoming the hotpots in clinical.QiShenKeLi(QSKL),developed by our team,possesses the effect of treating heart failure(HF)through the therapeutic principles of supplementing qi,activating blood and detoxication.However,the underlying hasn’t been defined yet.In the present study,we firstly investigated the effects and targets of QSKL on inhibiting cell apoptosis in vitro.And then,we filtered the components-targets by network pharmacology to find the apoptosis-related target proteins,and we also gained the corresponding pharmaceutical components by reverse docking,finally experiments in vitro were taken to validate the results.All of these will provide new treatment drugs as well as strategy for prevention of HF.Method1.An apoptosis model induced by OGD/R was established and cells were divided into 4 groups:control group,model group,QSKL group and Quercetin group.Drugs were added at the same time of establishing model.After corresponding treatment,cell viability,release of LDH,ROS generation in cell,concentration of Ca2+,mitochondrial membrane potential and cell apoptosis were detected.Besides,real-time quantitative PCR(Q-PCR)was used to detect the mRNA expressions of Fas and FasL and western blot was used to detect proteins related to apoptosis,including p-Akt,p53/Mdm2,Bcl-2,Bax,caspase-8 and cleaved-caspase-3.2.To investigate effect of QSKL on inhibiting apoptosis by PI3K/Akt signaling pathway,cells were divided into 4 groups:control group,model group,QSKL group and LY294002 group.LY294002 was added 1 hour before establishing model.CCK-8 was used to detect cell viability and western blot was used to detect the expressions of p-Akt and cleaved-caspase-3.3.Network pharmacology was applied to filter the whole components and relative targets of QSKL.Less components and targets were filtered according to oral bioavailability(OB)and drug likeness(DL).Apoptosis-related targets were selected from the results and corresponding components were obtained by reverse docking.4.A model of apoptosis induced by H2O2 was established and cells were divided into 4 groups:control group,model group,Luteolin group and Quercetin group.Drugs were added 6 hours before establishing model.Indicators detected including cell viability,ROS level and apoptotic rate.Q-PCR and Western Blot were used to detect the mRNA expression of Mdm2 and the proteins of Akt,p53,Bcl-2,Bax,caspase-8 and cleaved-caspase-3 respectively.Result1.The regulation of QSKL on apoptosis and apoptosis related proteinsIn the process of determining the condition of apoptosis model,cell survival rates at three time points,of which oxygen-glucose deprivation 4,6,8 hours,and reoxygenation for 12 hours,were 73.5±3.4%,65.3±3.1%and 43.3±1.1%.Oxygen-glucose dep rivation 8 hours,and reoxygenation for 12 hours were determined to be the ideal condition to induce apoptosis.In the process of determining the non-toxic concentration of QSKL,2000μg/ml and 1500μg/ml QSKL inhibited cell growth significantly(p<0.01,p<0.05),while 1000μg/ml、800μg/ml、600μg/ml、400μg/ml and 200μg/ml QSKL had no effect on cell viability.To observe the effect of QSKL on cell viability,cell survival rate in model group was 54.3±6.6%.Compared with model group,QSKL in 400μg/ml、600μg/ml、800μg/ml and 1000μg/ml up-regulated cell viability(p<0.01)and cell survival rates were 73.7±4.6%,84.2±2.9%,87±1.4%,74.5±3.7%.Compared with control group,cell morphology in model group had significant changes:cells showed as shrinking and intercellular gap became larger.Aggregation of particles occurred in cytoplasm and some cells floated off.In addition,LDH release increased,ROS generated in cells and concentration of Ca2+ both elevated,mitochondrial membrane potential and red/green fluorescence ratio decreased,apoptotic rate was 23.9±5.8%(p<0.01).Compared with model group,application of QSKL could protect cells,improve cell morphology and reduce LDH release,intracellular ROS generation and Ca2+ concentration(p<0.01).In addition,QSKL increased mitochondrial membrane potential,which thus elevating the ratio of red/green fluorescence(p<0.01).And the apoptotic rate dropped(8.9±2.2%),which had a significant difference compared with the model group(p<0.01).Q-PCR results showed that,compared with control group,the mRNA expressions of Fas and FasL increased significantly.In the detection of western blot,Bcl-2 decreased slightly in model group,but had no difference compared with control group,while Bcl-2/Bax ratio was significantly lower(p<0.01),caspase-8 and cleaved-caspase-3 increased significantly(p<0.01),Bax,p53 and Mdm2 increased(p<0.05).Compared with the model group,QSKL significantly reduced the mRNA expression of FasL(p<0.01),increased Bcl-2/Bax ratio(p<0.01),reduced the expressions of p53,caspase-8 and cleaved-caspase-3(p<0.01),and increased the expression of Mdm2(p<0.05).Quercetin,as the positive control group,had the similar results.2.The regulation of QSKL on PI3K/Akt signaling pathway.Compared with control group,cell viability in model group decreased significantly(p<0.01).QSKL could protect cells and elevate cell viability(p<0.01).After application of LY294002,the protective effect of QSKL on cell viability was blocked,and had no significant difference compared with the model group(p>0.05).In western blot assay,compared with control group,cleaved-caspase-3 protein expression increased(p<0.01),p-Akt expressed at lower level in model group(p<0.05).Compared with model group,QSKL significantly reduced cleaved-caspase-3 expression(p<0.01),and increased the expression of p-Akt(p<0.05).After application of LY294002,the protective effect of QSKL was inhibited,and there were no difference on the expressions of cleaved-caspase-3 and p-Akt compared with model group(p>0.05).3.Filtering of components and targetsWe gained 572 components and 656 targets with network pharmacology filtering the whole formula.According to the OB value≥30%and DL value≥0.2,173 ingredients and 283 targets were filtered and the former six compounds of which corresponded to most targets were quercetin,kaempferol,luteolin,β-sitosterol,tanshinone ⅡA and naringenin.Filtered the targets relating to apoptosis,the target of caspase-3,caspase-8,Bcl-2,Bax,p53 and Mdm2 were gained.By using reverse docking,the unique molecule Luteolin was found to target on Mdm2.4.The regulation of Luteolin on apoptosis and apoptosis-related proteinsIn the model of apoptosis induced by H2O2,cells viabilities were decreased with the increasing concentrations of H2O2.Compared with control group,cells viabilities with 10μM and 200μM H2O2 decreased obviously(p<0.01).Cell survival rate with 100μM H2O2 was 52.48±0.0416%and 100μM H2O2 was selected to induce apoptosis.There was no influence on cell viabilities incubated with 5μM,10μM,20μM Luteolin for 6 hours.Luteolin could inhibit the reduction of cell viability in dose-dependent manner.Cell viability in 10μM group was higher than 5μM group(p<0.01),but had no significant difference with 20μM group(p>0.05).Compared with control group,cell morphology changed significantly in model group:cells showed as shrinking,structural disorder,some cells became round and brighten,even floated off.ROS generated in cells increased obviously(P<0.01)and apoptotic rate was 67.47±3.05%.Compared with model group,Luteolin improved cell morphology back to the normal state.ROS generated in cells decreased significantly(P<0.01)and apoptotic rate decreased to 9.8 ± 0.85%.Q-PCR results suggested that compared with control group,the mRNA expression of Mdm2 increased in model group(P<0.05).Proteins expressions of Bcl-2 and Akt decreased in model group,but had no difference compared with control group(p>0.05).However,the levels of p53,caspase-8 and cleaved-caspase-3 increased significantly(P<0.01),as well as Bax(P<0.05).Compared with model group,Luteolin could increase the expressions of Mdm2 and Bcl-2(P<0.05),significantly elevated level of Akt(P<0.01).Besides,Luteolin also inhibited the expressions of p53 and cleaved-caspase-3(P<0.05),as well as Bax and caspase-8(P<0.05).Quercetin had the similar results as Luteolin.Conclusion1.The mechanisms of QSKL in inhibiting apoptosis include the following aspects:QSKL upregulates the expression of p-Akt,regulates the ratio of p53/Mdm2,which increases the expression of Mdm2 and decreases p53.Besides,QSKL can decrease the mRNA expression of FasL,restore the balance of Bcl-2/Bax,as well as other apoptosis-regulated proteins,such as caspase-8 and cleaved-caspase-3.This study indicates that QSKL inhibits apoptosis and prevents HF may through regulating p-Akt-p53/Mdm2 signaling pathway.2.Application of network pharmacology in drug-target prediction can obtain relavant molecules and targets rapidly and selectively from the complex traditional Chinese medicine.Combined with experiments can provide a powerful way in the research of Chinese medicine prescription.Through the network pharmacology prediction,we found that Luteolin from honeysuckle is the unique molecule corresponded to Mdm2.In the further study,we observed that Luteolin not only scavenges free radicals,but also regulates apoptosis-related proteins.It had the effects of up-regulating Akt,increasing Mdm2 expression and inhibiting the expression of p53.All of these contribute to protect cells from the injury induced by H2O2.This experiment provides new ideas and targets for the further study of QSKL on the prevention and treatment of heart failure.