Clinical And Genomic Study Of Shenshuguanxin Formula Combined With Western Medicine In The Treatment Of Intermediate Coronary Lesions(Qi Deficiency And Phlegm Stasis)

The first part:Clinical study on the treatment of intermediate coronary lesions with Shenzhuguanxin formula combined with Western MedicineObjectiveIn this study,taking the patients with intermediate coronary lesions in Lingnan area as the research object,taking the method of Supplementing Qi,promoting blood circulation and resolving phlegm as the starting point,the clinical research and safety study of the treatment of critical coronary disease with the combination of Shenshuguanxin formula and Western medicine were carried out.MethodsIn this clinical study,the patients with intermediate coronary lesions were taken as the study object.The experimental group was given western medicine combined with Shenshuguanxin formula,and the control group was given western medicine combined with placebo.After six months of continuous treatment,the efficacy and safety of Shenshuguanxin formula in the treatment of intermediate coronary lesions were evaluated.The main outcome measures were:coronary artery CT related indexes(myocardial transmural perfusion gradient,left ventricular CT value and aortic root CT value),blood lipid(low density lipoprotein,total cholesterol,triglyceride,high density lipoprotein),hsCRP,TCM syndrome score,Seattle Angina scale score.ResultsThere were 60 patients with critical coronary lesions completed the trial,30 in the experimental group and 30 in the control group.There was no significant difference between the two groups in age,gender,weight,course of disease,personal history,family history,clinical medication and distribution of the number of patients.1.The results of clinical study showed that there was no significant difference in the degree of coronary artery stenosis and calcification score between the two groups before and after the intervention(P>0.05);As for the myocardial microcirculation indexes(myocardial perfusion gradient,CT value of left ventricle and CT value of aortic root)before and after the intervention),there was no significant difference between the two groups(P>0.05).2.There was no significant difference in LDLC level between the test group and the control group before treatment(P>0.05).After 6 months of treatment,the level of LDLC in the experimental group was reduced compared with that before treatment,and the difference was statistically significant(LDLC before treatment:2.84±0.93 mmol/L VS after treatment:2.39±0.65 mmol/L,P=0.006);after 6 months of treatment,the level of LDLC in the experimental group was lower than that in the control group,and the difference was statistically significant(LDLC in the experimental group:2.39±0.65 mmol/L VS control group:2.77±0.74,P=0.040).There was no significant difference in TC level between the two groups before treatment(P>0.05),and the TC level of the test group and the control group decreased after 6 months of drug treatment compared with that before treatment,and the difference was statistically significant(TC of the test group:before treatment,4.42+0.96 mmol/L after VS treatment,3.73±0.52 mmol/L after VS treatment,P<0.001,TC of the control group:before treatment,4.70+1.24 mmol/L after VS treatment,4.21±0.73mmol/L,P=0.042).Comparing the TC level of the test group and the control group after 6 months of treatment,it was found that the TC level of the test group was lower than that of the control group,and the difference was statistically significant(TC 3.73±0.52 VS in the test group and TC 4.21±0.73 in the control group,P=0.006),indicating that on the basis of conventional western medicine treatment,Shenshuguanxin formula could more effectively reduce the TC level of patients with coronary critical lesions.However,there was no significant difference in the levels of triglyceride(TG)and high density lipoprotein(HDLC)between the two groups before and after 6 months of treatment(P>0.05),and there was no significant difference in the levels between the two groups after treatment.3.There was no significant difference in the level of hsCRP between the two groups before treatment,and there was no significant statistical difference in the level of hsCRP between and within the two groups after 6 months of treatment(P>0.05).4.In terms of the efficacy of TCM syndromes,after 3 months of treatment,according to the scores of TCM syndromes of patients,the total effective rate was 66.7%in the experimental group and 20%in the control group.After 6 months of treatment,the total effective rate was 76.6%in the experimental group and 36.37%in the control group according to the TCM syndrome score of the patients.Comparing the efficacy of TCM syndromes between the experimental group and the control group after 3 months and 6 months of treatment,the experimental group has more advantages in the efficacy of TCM syndromes,and has statistical significance(P<0.05).5.For the score of Seattle Angina Scale,the scores of Seattle Angina Scale(SAQ)before treatment were compared between the test group and the control group,and there was no significant difference in SAQ scores between the two groups before treatment.The SAQ scores(PL,AS,AF,TS,DP)of the test group and the control group after 3 and 6 months of treatment were compared separately.It was found that the SAQ scores of the test group after 3 and 6 months of treatment were higher than those of the control group,and the difference was statistically significant(P<0.05).6.In terms of safety,the changes of liver function(glutamic oxalacetic aminotransferase,alanine aminotransferase)level and renal function(urea nitrogen,creatinine)of patients in the experimental group and the control group before and after treatment were compared,suggesting that there was no significant change in liver function and renal function indexes in the experimental group and the control group before and after treatment(P>0.05),and Shenshu Guanxin prescription has certain safety.The second part:literature analysis of genomics of cardiovascular disease Objective(1)To collate and summarize the genomic literature of common cardiovascular diseases.(2)To sort out the genomic literature of cardiovascular diseases related to traditional Chinese medicine.MethodsThis paper adopts bibliometric analysis method.Firstly,the key words were determined,mainly including coronary atherosclerotic heart disease,myocardial infarction,myocardial ischemia,hypertension,heart failure and atrial fibrillation.Gene chip,genomics,gene expression profile,transcriptome,lncRNA,miRNA and mRNA were determined as the key words,mainly through the retrieval of China national knowledge Internet,CNKI,Chinese VIP information,Wanfang Data and PubMed database.The basic data of the eligible documents were extracted and counted by Excel and SPSS.ResultsThrough the preliminary screening of the titles and abstracts of the reading literature,864 articles on the application of genomics in the research of cardiovascular diseases were obtained.The full text of the reading was downloaded and 116 articles were finally included in the literature meeting the inclusion criteria.There are 8 articles on the application of genomics technology to coronary heart disease,22 articles on myocardial infarction,16 articles on myocardial ischemia,6 articles on heart failure,36 articles on hypertension,14 articles on atrial fibrillation,6 articles on heart failure,2 articles on cardiomyopathy,3 articles on rheumatic heart valve disease and 9 articles on pulmonary hypertension.In the included literature,59 studies explored the differential gene expression profile(mRNA)of diseases,32 studies explored the differential miRNA of diseases,8 studies explored the lncRNA expression of diseases,6 studies explored the circRNA expression of diseases,1 study selected the joint analysis of lncRNA+mRNA+miRNA,4 studies selected the joint analysis of lncRNA+mRNA,3 studies selected the joint analysis of miRNA+mRNA,1 study selected the joint analysis of lncRNA+mRNA In one study,lncRNA+circRNA was selected for association analysis,and DNA was used as the research index.Of the 116 eligible studies,41 involved traditional Chinese medicine.There are 8 TCM related genomic studies involved in myocardial ischemia,including 4 genomic studies related to acupuncture intervention in myocardial ischemia and 4 genomic studies related to TCM compound intervention in myocardial infarction.There are 23 researches related to traditional Chinese medicine,8 of which involve acupuncture treatment,11 involve intervention effect of traditional Chinese medicine on hypertension,4 involve syndrome type of traditional Chinese medicine,including 1 case of syndrome type related to hyperactivity of liver Yang,and 3 involve syndrome type related to blood stasis.One of the related studies of heart failure involves the related group analysis of the therapeutic effect of traditional Chinese medicine on heart failure.In the included TCM literature,21 studies supplemented the RT-PCR verification of the selected differential genes,only one of which provided the number of samples for verification,and the rest did not provide the number of samples for verification.In 22 studies,GO enrichment analysis of differential genes and KEGG pathway analysis were carried out.From the perspective of pathway,molecular function,biological process,cell composition and other aspects,the intervention of traditional Chinese medicine on cardiovascular disease was discussed in depth.ConclusionThrough the research of cardiovascular disease genomics,we can find that gene chip technology has been widely used in cardiovascular system diseases,which promotes the research of cardiovascular diseases.Analysis of the current TCM related genomics research found that at present,it is mainly focused on the genomics related research of TCM intervention mechanism,while the TCM syndrome type related genomics research is relatively less.It is concluded that the quality of TCM related genomics research needs to be improved.The third part:A genomic study of Shenzhuguanxin formula compared with placebo combined with western medicine in the treatment of intermediate coronary lesionsObjectiveTo explore the mechanism of Shenshuguanxin formula in improving lipid metabolism in patients with coronary critical lesions from the genomics level,to screen out molecular markers for Shenshuguanxin formula in treating coronary critical lesions,and to provide theoretical basis at the gene level.MethodsCombined with clinical studies,according to the matching principles of gender,age,drug use and so on,4 patients in the operation coronary heart prescription group and 7 patients in the placebo group were finally selected,and the whole blood of patients after 6 months of intervention was drawn for lncRNA chip-related research.The expression of lncRNA,circRNA and mRNA in whole blood of case group and control group were detected by gene chip detection technology,and the differences of lncRNA,mRNA and circRNA between the two groups were screened by bioinformatics method.The obtained differential genes were analyzed by gene function enrichment analysis(Gene Ontology,GO Gene Ontology)and gene pathway enrichment analysis(KEGG:Kyoto Encyclopedia of Genes and Genomes,Kyoto Encyclopedia of Genes and Genomes,Kyoto Encyclopedia of Genes and Genomes).The interaction between differential lncRNAs and differential miRNAs was screened through the SarBase database,and then integrated with the miRNA-mRNA interaction.The lncRNA-mRNA-miRNA ceRNA network was established using Cytoscape software.The lncRNA-mRNA relationship pairs were further screened by lncRNA CIS(cis)target gene analysis,lncRNA trans(trans)target gene analysis and LncRNA transcription factor association analysis to explore the function of lncRNA.Results1.Through gene chip detection technology and bioinformatics analysis method,we finally found that there were 35 differential lncRNAs(30 up-regulated genes and 5 down-regulated genes),85 differential mRNAs(55 up-regulated genes and 30 down-regulated genes)and 20 differential circRNAs(17 up-regulated genes and 3 down-regulated genes)in case group compared with control group.The functions of the top 10 mRNAs,lncRNAs and circRNAs with the largest difference multiples were retrieved and analyzed,and SLC22A1 in the differential mRNAs was found to be closely related to lipid regulation.ARHGAP1,TCF12,CEMIP and COLGALT1 in differential circRNAs are closely related to lipid metabolism.2.According to the results of the chip experiment,the differential genes were analyzed by protein interaction network,and finally 10 core genes were obtained:CACNA2D2,CACNA2D3,DNA JC6,FGF12,SGSM2,CACNA1G,LRP6,KIF25,OXTR,UPB1.Searching the core gene functions finally found that LRP6,SGSM2,OXTR were closely related to atherosclerosis and lipid metabolism.3.GO items of total differential mRNA were enriched with 113 molecular functions(MF),107 cellular components(Cellular Component),and 367 biological processes(Biological Process).Among them,GO items closely related to coronary critical lesions mainly include:cholesterol metabolism,action potential of cardiomyocytes during contraction,Wnt pathway,arterial blood pressure regulation and other items.KEGG pathway with Shenshuguanxin formula to reduce blood lipids in coronary critical lesions mainly includes:PI3K/Akt/mTOR signaling pathway,I3K-Akt signaling pathway,Rapl signaling pathway,Hippo signaling pathway.4.The genes included in the ceRNA network of LncRNA-miRNA-mRNA of Shenshuguanxin formula compared with placebo mainly include:lncRNA(DPY19L2P2,HCG15,ANKRD36BP2),miRNA(miR-24,miR-27,miR-363,miR-125),mRNA(CNNM3,PEG10,MCOLN2,ULK3).Among them,miR-24 and miR-27 have been confirmed as important target markers in lipogenesis,inflammation,lipid metabolism,oxidative stress and other aspects of the body.5.In this study,21 different circRNA were screened out.Go analysis and KEGG enrichment analysis were carried out for the host gene of the differentially expressed circRNA to obtain the biological process that the host gene of the differentially expressed circRNA might affect.Finally,the go enrichment project of the host gene of circRNA contains 64 molecular functions(MF),84 cellular components and 123 biological processes.Through go analysis,it was found that the total circRNA host gene was mainly enriched in DNA repair,lipophilic positive regulation,protein kinase C activity positive regulation,histone H3-K9 acetylation regulation,CTD phosphatase activity,L-aspartate transmembrane transport activity and other biological processes.Through KEGG analysis of host gene of different circRNA,we can get the possible pathway of host gene of different circRNA.According to the results of KEGG,we found that the host gene of different circRNA was mainly enriched in the following pathways:O-glycan biosynthesis,lysine degradation,p53 signal pathway,cell cycle,ubiquitin mediated protein hydrolysis,cell aging,human papillomavirus infection,etc.6.KEGG pathway analysis of LncRNA co-expressed differential genes was mainly enriched in the following pathways:p53 signaling pathway,focal adhesion,TGF-beta signaling pathway,Ras signaling pathway,PI3K-Akt signaling pathway,AMPK signaling pathway,cGMP-PKG signaling pathway,DNA repair,etc.According to the correlation between lncRNA and gene expression,the cis-regulated genes of lncRNA mainly include:the relationship between lncRNA ITFG1-AS1 and ITFG1.Analysis of target genes of lncRNA trans(trans)action mainly found that four mRNAs(RAD54L2,CNNM3,KIF18B,GPANK1)and three lncRNAs(NR₁34460.1,NR₁45490.1,XR₀01753092.2)formed lncRNA-mRNA target interaction network maps.7.In our study,the mRNA(ALDH1A2,CD300A,CEP72,DDAH2,HDAC5,SYT14)and lncRNA(LOC105374150,DNAJC9-AS1,LOC105372209)were tested by q-PCR in 11 samples.Among them,three primers,ALDH1A2,SYT14 and LOC105372209,were designed for high CT value or non-single peak of dissolution curve,and the pre experiment failed.The results of Q-PCR showed that the difference genes CEP72,HDAC5,DNAJC9-AS1 and LOC105374150 in Shenshuguanxin formula group were up-regulated and the difference ddah2 was down regulated,which was statistically significant.The Q-PCR experiment further confirmed that the treatment of coronary critical lesions by Shenshuguanxin formula may be closely related to genes CEP72,HDAC5,DDAH2,lncRNA DNAJC9-AS1,LOC105374150.ConclusionIn the genomics study of Shenshuguanxin formula in the treatment of coronary critical lesions,there were differences in lncRNA,mRNA and circRNA between Shenshuguanxin formula and placebo group in the treatment of coronary critical lesions.The improvement of lipid metabolism in patients with coronary borderline lesions by Shenshuguanxin formula is closely related to the following molecular markers:miR24,miR27 and mRNA:SLC22A1,ABCB1,SGSM2,LRP6,OXTR,circRNA:ARHGAP1,CEMIP,COLGALT1 and lncRNA:DPY19L2P2,HCG15,ANKRD36BP2.Through GO analysis and KEGG enrichment analysis,we found that the mechanism of Shenshuguanxin formula in treating patients with coronary critical lesions of qi deficiency and phlegm stasis type may be closely related to cholesterol metabolism,myocardial contraction,regulation of endothelial cell proliferation,and mTOR signaling pathway,PI3K-Akt signaling pathway,metabolic pathway,Ras signaling pathway,Rapl signaling pathway,cAMP signaling pathway,Hippo signaling pathway.Among them,the pathways related to Shenshuguanxinfang in reducing blood lipids in coronary critical lesions mainly include:PI3K/Akt/mTOR pathway,Rapl signaling pathway.