| Objective(A)Experimental ResearchTo explore the effect of Aralosides on myocardium cell H/R injury,and whether its mechanism is related to the PI3K/Akt/GSK3β signal transduction pathway and the mitochondrial ATP sensitive potassium channel by establishing the H9c2 rat myocardial cells with hypoxia/reoxygenation injury model of simulating MIRI process at the cellular level.(B)Clinical ResearchTo observe the efficacy and safety of Yangxinshi tablets in the treatment of angina pectoris in the real world,and analyze the factors that may affect the clinical effect by clinical epidemiological observational research methods,.Methods(A)Experimental researchExperiment one:Exploration of H9c2 myocardial cell culture and model time,drug concentration.We use CCK-8 method to detect cell survival rate by setting different hypoxia/reoxygenation time so as to determine the optimum time of hypoxia/reoxygenation injury,on this basis,to explore the suitable concentration of As protecting myocardial cells from H/R injury;Experiment two:Primary exploration of Aralosides protective effect on H9c2 myocardial cells injury induced by hypoxia/reoxygenation.After establishing H/R injury model of rat H9c2 cells pretreated with As,the survival rate of each group was detected by CCK-8 assay,AnnexinV-FITC/PI was used to observe the apoptosis of myocardial cells in each group,the activity of LDH in the cell culture medium and the content of MDA,the activity of SOD were detected by microplate and WST-1 method,the relative content of apoptosis related factors Caspase-3,Bax,Bcl-2mRNA were detected by Reverse Transcription Polymerase Chain Reaction,Western blot was used to detect the expression of apoptosis related protein such as Caspase-3,Bax,Bcl-2,Caspase-9,CytC;Experiment three:Research of protective effect of Aralosides on hypoxia/reoxygenation injury model of H9c2 myocardial cell based on PI3K/Akt pathway.To establish H/R injury model of rat H9c2 myocardial cells pretreated with As and PI3K/Akt signaling pathway specific inhibitor LY294002,the survival rate of each group was detected by CCK-8 assay,and the apoptosis rate was determined by AnnexinV-FITC/PI method,LDH leakage in cell culture medium、the content of MDA and activity of SOD were detected in each group,the expression of Caspase-3 and phosphorylation of PI3K/Akt/GSK3β signaling pathway related protein Akt and GSK3β in each group were detected by Western blot;Experiment four:Research of the protective effect of Aralosides on H/R injury of H9c2 myocardial cell based on mitochondrial ATP sensitive potassium channel(mitoKATP).To establish H/R injury model of H9c2 myocardial cells pretreated with As and mitoKATP inhibitor 5-hydroxy decanoic acid(5-HD)and activator diazoxide(DZ),cell viability was determined by CCK8 assay,Western blot was used to detect the expression of Caspase-3 in each group of cell,the content of Cytochrome C in mitochondria and cytoplasm was determined by Western blot after the mitochondrial and cytosolic proteins were isolated.(B)Clinical ResearchMulti stage sampling was used to collect stable angina pectoris of coronary heart disease patients used Yangxinshi tablets with gender,age and duration of illness by medical information,we observe total score of TCM syndrome,angina population after administration of the attacks,the Seattle Angina Questionnaire scores and vital signs,to observe the efficacy and safety of Yangxinshi tablets in the treatment of stable angina pectoris through follow-up,logistic regression analysis was used to analyze the factors influencing the curative effect of Yangxin tablets.Results(A)Experimental research sectionExperiment one:we select hypoxia 12h/reoxygenation 2h as the hypoxia/reoxygenation model time of H9c2 myocardial cell,0.1mg/L is chosen as the drug concentration of Aralosides in this experiment;Experiment two:the results showed that the survival rate of H9c2 myocardial cells in Aralosides group pretreated with As was significantly higher than Model after H/R injury(P<0.01),apoptotic cell in Aralosides group were less than Model,compared with model group,the leakage of LDH in Aralosides group decreased significantly(P<0.01),the content of MDA decreased(P<0.05),the vitality of SOD increased(P<0.05),the results of PCR showed that the relative content of Caspase-3mRNA in model group increased significantly compared with Control(P<0.01),the relative content of apoptosis related factor BaxmRNA also increased(P<0.01),the relative content of anti apoptosis factor Bcl-2mRNA decreased significantly(P<0.01),it showed that H/R damage can promote the apoptosis of H9c2 cells,however,Caspase-3mRNA in Aralosides group pretreated by As after H/R injury decreased compared with the Model(P<0.05),the relative content of BaxmRNA decreased,but the difference was not statistically significant(P>0.05),as an anti apoptosis related factor,Bcl-2mRNA increased significantly(P<0.01);Western blot showed that the expression of myocardial apoptosis related proteins such as Caspase-3,Bax,Caspase-9,CytC in model group increased compared with Control after H/R injury(P<0.05),the content of Bcl-2 decreased(P<0.05),compared with the Model group,the content of apoptosis related proteins including Caspase-3,Bax,Caspase-9,CytC in Aralosides group pretreated with As decreased(P<0.05),the expression of Bcl-2 increased(P<0.05);Experiment three:the results showed that the survival rate of myocardial cell in Aralosides+LY294002 group reduced significantly than Aralosides(P<0.01),the results showed that apoptosis rate of myocardial cell in the model group increased compared with Control(P<0.05),cell in Aralosides group were given H/R injury pretreated with As,its apoptosis rate decreased compared to Model(P<0.05),compared with Aralosides group,the apoptosis rate of Aralosides+LY294002 group increased(P<0.05),indicating that once LY294002 block PI3K/Akt signal specificity,the effect of Aralosides reducing apoptosis caused by H/R injury was significantly inhibited;compared with Aralosides,LDH leakage in As+LY294002 group were significantly higher(P<0.01),the content of MDA increased(P<0.05),the activity of SOD decreased significantly(P<0.01);the results of Western blot showed the expression of apoptosis related protein Caspase-3 of Aralosides+LY294002 group increased significantly compared with Aralosides after adding specific inhibitor of PI3K/Akt pathway(P<0.01),the phosphorylation level of Akt,GSK3 protein in Aralosides group increased compared with Model(P<0.05),and the phosphorylation of Akt,GSK3βprotein in Aralosides+LY294002 group were obviously lower than Aralosides after joining the LY294002(P<0.01);Experiment four:the results showed that survival rate of myocardial cell in 5-HD group were given Mitochondrial ATP sensitive potassium channel inhibitor(mitoKATP)5-hydroxy decreased significantly compared with Control(P<0.01),the survival rate of myocardial cell in Aralosides+5-HD group decreased compared with Aralosides(P<0.01),the results of Western blot showed that the expression of Caspase-3 protein in Aralosides+5-HD group increased significantly compared with Aralosides(P<0.01),isolated cells mitochondria and cytoplasm,Western blot was used to detect the expression of CytC in mitochondrial and cytoplasmic,the relative expression of CytC of Model decreased in mitochondrial,and increased in the cytoplasm,the difference was statistically significant compared with Control(P<0.05),the expression of CytC in Aralosides group increased in mitochondrial,decreased in cytoplasmic,the difference was statistically significant compared with Model(P<0.05),showed that the content of CytC released from mitochondria to cytosol increased after H/R,the content of CytC released from mitochondria to cytosol decreased after pretreated with As,the expression of CytC in 5-hydroxy decanoic(5-HD+H/R)group with the mitoKATP blocker decreased in mitochondria,increased in cytoplasm,the difference was statistically significant compared with Control(P<0.05),the expression of CytC increased in mitochondria and decreased in cytoplasm(P<0.05)with mitoKATP Activator diazoxide(DZ)treatment,these results suggest that mitoKATP is related to the release of CytC from mitochondria to cytoplasm,and the CytC expression level of Aralia+5-hydroxy decanoic group decreased in mitochondria and increased in cytoplasm compared with Aralosides,the difference was statistically significant(P<0.01),it showed that the effect of Aralosides to reduce the release of CytC from mitochondria to cytoplasm was significantly inhibited in the closing of the channel specificity.(B)Clinical ResearchThe results showed that the clinical symptoms curative effect of Yangxinshi tablets on patients with coronary heart disease,angina symptoms and TCM syndromes have some improvement,TCM syndrome score decreased(P<0.01),at the same time,patients with angina pain score and the average duration of four visits were gradually improved(P<0.01),the Seattle angina questionnaire in five dimensions including patient level of physical activity limitation,angina,stable angina pectoris,full treatment Cognitive satisfaction and disease score of four visits were gradually improved(P<0.01);the safety aspect,there were two cases of adverse events in this study,were upper respiratory tract infection,does not belong to the drug induced adverse reaction,serious to a lesser extent,does not affect the use of monitoring drugs,patients with stable vital signs,the safety of clinical use;multivariate statistical analysis results showed that patients with Yangxinshi tablets after treatment.The influence factors of TCM syndrome total score are:duration,complications,alcohol and total dose;There are some factors that influence the improvement of VAS score of angina pectoris:the first use of Yangxin tablet and the total dose.The factors that affect the average duration of angina pectoris were:gender,duration,total dose.Multivariate statistical analysis results show the Seattle angina questionnaire dimensions effect:gender,duration,total dose,BMI,combined diseases will affect the physical limitation of the treatment effect;gender,course of disease,BMI,whether the first use of Yangxinshi tablets are the influence factors of stable angina curative effect;age,BMI,total dose,the course of disease,is the main factor affecting the angina pectoris;gender,age,course of disease,drinking,whether the first use of the total dose of Yangxinshi tablets,are the main factors influencing treatment satisfaction;gender,duration of disease,BMI,the initial use of Yangxin tablets are the major factors that affect the cognitive level of disease.Conclusion1.Aralosides pretreatment has protective effect on H9c2 rat myocardial cells from H/R injury,it can reduce H/R injury by decrease apoptosis,necrosis and oxidative stress injury,the mechanism may be partly related to the activation of PI3K/Akt/GSK3β signaling pathway,and the opening of mitochondrial ATP sensitive potassium channel.2.Yangxin tablet is safe and effective in the treatment of stable angina pectoris of coronary heart disease,the factors such as age,sex,duration of disease,dosage of prescription and merger of other diseases may affect the clinical efficacy of Yangxin tablets. |
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