| ObjectiveThe prevalence of acute myocardial infarction is so high,and current treatments have drawbacks.Most patients still have heart failure,sudden cardiac death and other malignant events.Inflammato oxidative stress are important pathogenesis of myocardial infarction,but there are currently no targeted drugs.Apoptosis is an important form of myocardial cell necrosis,and intervention in apoptosis can prevent the progression of the disease.The mechanism of Chinese medicine in treating myocardial infarction remains to be revealed.The purpose of this research is to confirm the clinical efficacy of Yi Qi Huo Xue Recip(YQHX)in the treatment of myocardial infarction,and to explore its therapeutic mechanism through basic experiments.Methods1.Clinical research.Sixty patients who met the diagnostic criteria of Western medicine for acute myocardial infarction and TCM syndromes with Qi deficiency and blood stasis syndrome were selected and divided into standard western medicine treatment group(control group),YQHX+western medicine standard treatment group(treatment group).Observe Chinese medicine Syndrome score,the level of LVEF and E/Ea detected by cardiac color Doppler ultrasound,6 minutes walking distance,the expression of NT-ProBNP and H-CRP in patients'serum,and the levels of TNF-?,IL1?,IL-6,ROS and p-FOXO3a detected by ELISA in two groups.2.Basic experiment(1)The effect of YQHX on inflammatory factors in cardiomyocytes induced by H2O2.First,the oxidative stress model of cardiomyocytes was constructed,and CCK-8 was used to detect the optimal concentration of hydrogen peroxide to construct the model.Then CCK-8 was used to detect the maximum dose of YQHX that has no effect on cell viability.The effect of YQHX on the concentration of inflammatory factors TNF-?,IL-1? and IL-6 in the culture supernatant of cardiomyocytes was detected by ELISA.The experiment was divided into normal group,model group,model+YQHX low-dose group,model+YQHX middle-dose group,model+YQHX high-dose group.(2)Effect of YQHX on the level of oxidative stress in cardiomyocytes induced by H2O2.DHE detection and ROS kit were used to detect the effect of YQHX on ROS levels.CAT,MDA,and SOD kits were used to detect the effect of YQHX on the levels of CAT,MDA and SOD in cardiomyocytes.(3)Effect of YQHX on H2O2-induced cardiomyocyte apoptosis.Cell flow cytometry was used to detect the effect of YQHX on myocardial cell apoptosis.Western-blot was used to detect the effect of YQHX on the protein levels of cardiomyocyte apoptosis pathway.(4)The expression of Sirt4 and p-FOXO3a in cardiomyocytes induced by H2O2.Western-blot was used to detect the expression of Sirt4 and p-FOXO3a in the myocardial cells of neonatal rats induced by H2O2.The effect of YQHX on the expression of Sirt4,p-FOXO3a,and FOXO3a acetylation were also detected by WB.(5)The effect of YQHX on inflammatory factors through the Sirt4/FOXO3a signal pathway.First,the interference plasmid of Sirt4 were constructed,and RT-qPCR was used to detect knockdown effect of Sirt4.The experiment was divided into normal group,model group,model+YQHX high-dose group,model+YQHX high-dose+Sirt4 knockdown negative control group,model+YQHX high-dose+Sirt4 knockdown group.ELISA was used to detect the expression of inflammatory factors in each group.(6)The effect of YQHX on the level of oxidative stress through the Sirt4/FOXO3a signal pathway.The experimental groupings were the same as(5).DHE detection and ROS kit were used to detect ROS levels in each group.CAT,MDA and SOD kits were used to detect the levels of CAT,MDA and SOD in each group.(7)The effect of YQHX on cell apoptosis through Sirt4/FOXO3a signal pathway.The experimental groupings were the same as(5).Flow cytometry was used to detect the level of apoptosis in each group,and Western Blot was used to detect the expression of apoptosis-related proteins Bcl-2,Bax,BIM,and cleaved caspase3 in each group.Western Blot was used to detect the expression of Sirt4,p-FOXO3a,and FOXO3a acetylation.Results1.Clinical study.The results of the analysis of the efficacy of TCM syndromes showed that the treatment group has a higher treatment efficiency than the control group.The treatment group's overall syndrome score improvement was better than that of the control group.Chest pain,chest tightness,palpitations,and fatigue symptoms were better improved in the treatment group than the control group.There was no statistical difference in the symptoms of shortness of breath between the two groups.The result of echocardiography treatment group can significantly increase LVEF,but there is no difference in E/Ea between treatment group and control group.The treatment group can reduce NT-ProBNP,wall montion score index and increase the amplitude of endocardial movement during systolic period,which were better than the control group.In the two groups,H-CRP was no statistical difference.ELISA results summary:YQHX can significantly reduce the level of oxidative stress in patients with myocardial infarction,increase the phosphorylation level of FOXO3a,reduce the expression of IL-1?,and have no effect on TNF-? and IL-6.2.Basic experiments.(1)CCK-8 test results showed that 25uM hydrogen peroxide can significantly inhibit the activity of cardiomyocytes and increase the oxidative stress damage of cardiomyocytes,so 25uM hydrogen peroxide concentration was selected to induce rat cardiomyocyte damage.The 100ul/ml dose of YQHX was the maximum non-toxic dose,so the low dose was 25ul/ml,the middle dose was 50ul/ml,and the high dose is 100ul/ml for subsequent experiments.The expression of inflammatory factors in the model group was higher than that in the normal group,and the expression of inflammatory factors gradually decreased with the increase in the dose of YQHX.(2)The results of DHE and ROS detection showed that compared with the normal group,the ROS level of the model group increased,and the expression level of ROS gradually decreased with the increase of the dose of YQHX.The expression level of MDA in the model group increased,and the expression level of MDA decreased gradually with the increase of the dose of YQHX.The expression level of SOD and CAT in the model group decreased,and with the increase of the dose of YQHX,the expression level of SOD and CAT gradually increased.(3)The results of flow cytometry showed that compared with the normal group,the level of apoptosis in the model group was higher,and the level of apoptosis gradually decreased with the increase in the dose of YQHX.The expression level of Bax,BIM and cleaved caspas3 in model group increased,and the expression levels of them gradually decreased with the increase in the dose of YQHX.The expression level of Bcl-2 in the model group decreased,and with the increase of the dose of YQHX Bcl-2 level gradually increased.(4)WB results showed that compared with the normal group,the expression of Sirt4 and p-FOXO3a in the model group decreased,and the acetylation level of FOXO3a increased,with the increase in the dose of YQHX,Sirt4,p-FOXO3a level gradually increased,and the acetylation FOXO3a level decreased.(5)RT-qPCR results showed that plasmid interference can effectively reduce the expression of Sirt4.Compared with normal results of ELISA,the expression of inflammatory factors TNF-?,IL-1? and IL-6 in the model group increased.Compared with the model group,the expression of inflammatory factors in the model+high-dose group decreased.Compared with the model+high-dose group,the expression of inflammatory factors in the model+high-dose+Sirt4 knockdown negative control group remained unchanged,and the expression of inflammatory factors in the model+high-dose+Sirt4 knockdown group rose but was lower than the model group.(6)Compared with the normal results of DHE and ROS detection,the expression of ROS in the model group increased.Compared with the model group,the expression of ROS in the model+high-dose group decreased.Compared with the model+high-dose group,the ROS expression of the model+high-dose+Sirt4 knockdown negative control group remained unchanged,and the ROS expression of the model+high-dose+Sirt4 knockdown group recovered but was lower than the model group.CAT,SOD and MDA test results:Compared with the normal group,the expression of SOD and CAT in the model group decreased,and the MDA increased.Compared with the model group,the expression of SOD and CAT in the model+high-dose group increased,while MDA decreased.Compared with the model +high dose group,the SOD,CAT and MDA expressions of the model+high dose+Sirt4 knockdown group remained unchanged,while the SOD and CAT expression of the model+high dose+Sirt4 knockdown group decreased but were higher than the model group,and the MDA expression increased but lower than the model group.(7)Cell flow cytometry results:Compared with the normal group,the expression of apoptotic cells in the model group increased.Compared with the model group,the expression of apoptotic cells decreased in the model+high-dose group.Compared with the model+high-dose group,the expression of apoptotic cells in the model+high-dose+Sirt4 knockdown negative control group remained unchanged,and the expression of apoptotic cells in the model+high-dose+Sirt4 knockdown group rose but was lower than that of the model group.WB results showed that compared with the normal group,the expression of BAX,BIM,and cleaved caspas3 in the model group increased,and the expression of Bcl-2 decreased.Compared with the model group,the expression of Bax,BIM,and cleaved caspas3 in the model+high-dose group decreased,and the expression of Bcl-2 increased.Compared with the model+high dose group,the expression of Bax,BIM,cleaved caspas3,and Bcl-2 in the model+high dose+Sirt4 knockdown negative control group remained unchanged,while the expression of Bax,BIM,and cleaved caspas3 in the model+high dose+Sirt4 knockdown group rose but Lower than the model group,Bcl-2 expression decreased but higher than the model group.ConclusionsYQHX can inhibit inflammatory damage and oxidative stress in patients with myocardial infarction.Cell experiments show that QYHX protects cardiomyocytes from inflammatory factors and oxidative stress,and inhibits cell apoptosis through Sirt4/FOXO3a pathway,... |
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