Study On The Mechanism Of Wenxin Decoction Treating Unstable Angina With Yang Deficiency And Blood Stasis Syndrome Based On MiR-126-5p/Bcl-212 Apoptosis Pathway

1.BACKGROUNDCoronary heart disease(CHD)is a global disease,and one of the main causes of death,its accounts for 48%of all cardiovascular mortality or approximately one of every seven deaths,which becoming a serious public health problem and leading to a serious financial burden.Unstable angina(UA)is a typical acute coronary syndrome of CHD.In patients presenting with refractory or severe angina,an interventional strategy is preferable.In addition to invasive interventional therapy,Western medicines for the treatment of unstable angina mainly including anti-plate,anti-coagulation,regulation of blood lipids and lowering blood pressure,the purpose is to stabilize the plaque.But there is considerable debate regarding the use of invasive strategy versus conservative strategy.Therefore,it is necessary to find a supplementary replacement therapy as a conservative treatment strategies to support interventional therapy for UA.Traditional Chinese medicine(TCM)treatment of coronary heart disease has been for thousands of years,its clinical efficacy is remarkable.In recent years,there have been many studies on the treatment of UA with TCM.The results show that the combination of TCM and coronary heart disease secondary prevention drugs can achieve better clinical efficacy than using western medicines alone.With the supplementary of TCM,its not only can reduce the morbidity and mortality of UA,but also improve the patients’ life quality,control or delay the progression of coronary heart disease,reduce the complications of CHD and keep patients in a stable condition for a long time.The concept of coronary heart disease is similar to "chest impediment"/"chest pain" in traditional Chinese medicine and its basic pathogenesis is deficient root with overdo superficial.Clinical epidemiology shows that "Yang deficiency and blood stasis" is a common syndrome type in CHD,so that"strengthening Yang and activating blood" becomes one of the main treatments of coronary heart disease,and achieves famous clinical effect.Wenxin Decoction is the specific treatment TCM decoction for Yang deficiency and blood stasis syndromeMiRNA is a type of microRNA that does not have the ability to encode a protein.It specifically regulates target gene expression at the post-transcriptional level by specifically binding to the 3’ end of the target mRNA.Studies have shown that miRNA regulate the development of unstable angina,involving the regulation of cardiomyocyte apoptosis,endothelial cell injury,cardiac angiogenesis and other mechanisms.MiRNAs specifically expressed in cardiomyocytes are closely related to cardiovascular processes such as cardiac hypertrophy,myocardial remodeling,heart failure,myocardial ischemia-reperfusion,and myocardial infarction.MiRNAs are generally considered to be high stability,but miRNA can decay very rapid relying on neuronal activity in some cells.Study reported that the expression of miRNA can be up and down regulated with the disease progress and treatment intervention.As a potential target for early diagnosis and treatment of diseases.miRNA has become a research hotspot.2.PURPOSES2.1.Study on the clinical effectiveness of Strengthening Yang and Activatins Blood Therapy(WenXin Decoction)in the treatment of UA patients2.2.Study on the effect of Strengthening Yang and Activating Blood Therapy(WenXin Decoction)on the expression of miR-126-5p in with UA patients2.3.Study on the effect of Strengthening Yang and Activating Blood Therapy(WenXin Decoction)on H2O2-induced apoptosis of hypoxic cardiomyocytes and expression of miR-126-5p/Bcl-212/Caspase-9/3 network genes3.METHODS3.1 Part Ⅰ Clinical observation of Strengthening Yang and Activating Blood Therapy(WenXin Decoction)in the treatment of UAa.)Case inclusion and treatment:80 patients with UA yang deficiency and blood stasis syndrome confirmed by coronary angiography were randomly divided into the experimental group(n=40)and the control group(n=40).The cases were from Guang’anmen Hospital.The control group was treated with western medicine;the experimental group was treated with Strengthening Yang and Activating Blood Therapy on the basis of Western medicine treatment,1 dose/day,twice in the morning and evening,for 4 weeks.b)Observation indexes:’TCM syndrome score(using Chinese medieine yang deficiency blood stasis syndrome score scale);quality of life evaluation:Seattle Angina Questionnaire(SAQ);blood lipid test(high density lipoprotein(HDL)-C),low density lipoprotein(LDL-C),triglyceride(TG),cholesterol(TC),free fatty acid(FFA);inflammatory index(C-reactive protein(CRP));safety index(liver Renal function test).3.2 Part Ⅱ Study on the effect of Strengthening Yang and Activating Blood Therapy(WenXin Decoction)on expression of miR-126-5p in patients with Unstable Anginaa)14 patients with UA yang deficiency and blood stasis syndrome confirmed by coronary angiography were included,treated by Strengthening Yang and Activating Blood Therapy came from the Guang’anmen Hospital.b)3 mL of peripheral vein being taken before and after treatment,extract total RNA respectively for qRT-PCR detectionc)qRT-PCR detection:detected the expression miR-126-5p and mRNAs involved in Bcl-212/Caspse-9/Caspse-3 signal pathway.3.3 Part Ⅲ Study on the effect of Wenxin Deeoction on H2O2-induced Hypoxia Cardiomyocyte Apoptosis and Gene Expression of miR-126-5p/Bcl-212/Caspase-9/3 Regulation Pathwaya)Cell culture:Cardiomyocytes(H9c2 cells,ATCC)b)Screening of the optimal concentration of WenXin Decoctionc)Establishment of myocardial injury model(H2O2 induced)d)Screening of the maximum non-toxic concentration of WenXin Decoctione)Transfectionf)Experiment groupsA,normal group(Control)B,injury group(Model)C,injury+Wenxin Decoction group(Model+wenxin)g)Cell proliferation assay:CCK-8 assayh)qRT-PCR detection:detecting the expression of miRNA and its target gene mRNAs at various time points and concentrations.i)Protein detection:(western blot.WB)j)Detection of cell cycle and apoptosis4.RESULTS4.1 Part Ⅰ Clinical observation of Strengthening Yang and Activating Blood Therapy(WenXin Decoction)in the treatment of UA4.1.1 After treatment with Wenxin Decoction,the Seattle angina pectoris questionnaire(SAQ)scores of the two groups of UA patients were significantly improved,and the TCM syndrome scores decreased significantly,the difference was statistically significant(P<0.05);4.1.2 Wenxin Decoction significantly reduced the triglyceride(TG)level in UA patients,and the difference between the groups was statistically significant (P<0.05).4.1.3 There were no significant differences in the indexes of liver and kidney function between the two groups of UA patients before and after treatment(P>0.05).4.2 Part Ⅱ Study on the effect of Strengthening Yang and Activating Blood Therapy(WenXin Decoction)on expression of miR-126-5p in patients with UA patients4.2.1 Strengthening Yang and Activating Blood Therapy(WenXin Decoction)can up-regulate miR-126-5p expression in UA patientsAfter treatment with WenXin Decoction,the expression of miR-126-5p in UA patients was significantly up-regulated(P<0.05).4.2.2 Strengthening Yang and Activating Blood Therapy(WenXin Decoction)regulates miR-126-5p/Bcl-212/Caspase-9/Caspase-3 singnal pathway Genes in UA PatientsAfter treatment with WenXin Decoction,the expression of Bcl-212 was significantly up-regulated(P<0.05).The downstream target genes caspse-9/caspse-3 showed a trend of decreased though without a significant difference.It is suggested that WenXin Decoction can increase the expression of miR-126-5p and Bcl-212,decrease the expression of caspse-9/caspse-3,which may be a potential mechanism of WenXin Decoction anti-apoptosis.4.3 Part Ⅲ Study on the effect of Strengthening Yang and Activating Blood Therapy(WenXin Decoction)on H2O2-imduced Hypoxia Cardiomyocyte Apoptosis and Gene Expression of miR-126-5p/Bcl-212/Caspase-9/3 Regulation Pathway4.3.1 Strengthening Yang and Activating Blood Therapy up-regulates the expression of miR-126-5p in hypoxic cardiomyocytes induced by H2O2.Hypoxia cardiomyocyte model was induced by H2O2 to explore the relationship between the regulation of miR-126-5p expression and cardiomyocyte apoptosis by Strengthening Yang and Activating Blood Therapy.The results of qRT-PCR showed that the expression of miR-126-5p was significantly down-regulated in the injury model group compared with the normal cell group(P<0.05),but in the injury+WenXin Decoction group was significantly higher than that in the injured model group(P<0.05).4.3.2 Strengthening Yang and Activating Blood Therapy has a regulatory effect on proteins expression in Bcl-212/Caspase-9/Caspase-3 apoptotic signal pathway.The WB detection results showed that compared with the normal group,the expression of Bcl-212 protein in the injury group was decreased(P<0.05),while in the injury+WenXin Decoction group was significantly up-regulated compared with the injury group(P<0.05);Compared with the normal cell group,the expression of Caspase-9/3 proteins were increased in the injury group(P<0.05);while in the injury+WenXin Decoction group was lower than that in the injury group(P<0.05).4.3.3 Strengthening Yang and Activating Blood Therapy reduces H2O2-induced apoptosis of hypoxic cardiomyocytes.The results of flow cytometry showed that the apoptosis rate of myocardial cells in the injury group was significantly up-regulated compared with the normal group(P<0.05).The apoptosis rate of the injury+Wenxin decoction group was significantly lower than that of the injury group(P<0.05).Above results suggest that the effect of Strengthening Yang and Activating Blood Therapy on regulation of miR-126-5p expression is closely related to cardiomyocyte apoptosis.5.CONCLUSIONS5.1 Strengthening Yang and Activating Blood Therapy(WenAXin Decoction)for the treatment of UA yang deficiency and blood stasis syndrome has clinical effectiveness and safety.5.2 Wenxin Decoction has the effect of regulating genes expression in UA patients.5.3 Wenxin Decoction has anti-apoptotic effect,and its mechanism may be related to up-regulating miR-126-5p expression and targeting regulation of Bcl-212/Caspase-9/3 network pathway.