| The treatment of stable angina pectoris(SAP)of coronary heart disease(CHD)is of great significance to the poor prognosis of the patient's disease,and blood stasis is one of its basic pathogenesis.Many clinical literatures show that,blood circulation drugs has a good clinical effect to SAP,but there are still insufficient.The disadvantage of Chinese Herbal Medicine is the components and mechanisms are unclear.The Chinese patent medicines aim at limited symptoms.And active ingredients of a single Chinese herbal medicine cannot fully display the feature of traditional Chinese medicine(TCM).The development of modernization of TCM requires that the study of prescriptions should clarify the role of the components,the target points,and the role links.And the"Effective Components Combination Formula" can solve all the problem.And it shows great value.At the same time,the new formulations can better exert the multi-components multi-channel,multi-target advantages of TCM.The biomolecular network is a method of system biology research.Basing on the use of biomolecule networks,it is possible to construct a co-expression network at the gene level,then from the change of gene molecules to signal pathways,at last to fully understand the mechanism of drug action.This study intends to through a RCT to verify the efficacy and safety of Dangshen Tongluo Jiaonang(DSTLJN).And then use network pharmacology to predict the target of DSTLJN.Some patients' blood samples were subjected to high-throughput sequencing,to build an IncRNA-mRNA interaction network.And then GO and KEGG analyses were performed to explore the mechanism of drug action.This study will provide a basis for the clinical application and mechanism study of the "Effective Components Combination Formula".1.Literature review:In this part of the study,the author reviewed 58 clinical literatures and 30 documents,to analyze the clinical research progress of SAP and development of "Effective Components Combination Formula".2.Clinical researchOBJECTIVE:Using RCT to to evaluate the clinical curative effect and safety of DSTLJN.METHODS:The patients in this study were from outpatients from March 2017 to December 2017 in Guang'anmen Hospital.A total of 40 patients who were randomly entered the treatment group(TG)or control group(CG).Interventions:The two groups maintained the basic treatment of Western medicine,and the TG took DSTLJN for treatment,1 capsule/time,3 times a day,orally;the CG took DSTLJN simulator for treatment,1 capsule/time,3 times a day,orally.Both groups were treat for 5 weeks.Observations include general information.Efficacy indicators include:(1)total angina pectoris efficacy,angina pectoris episodes,duration,and angina score;(2)nitroglycerin consumption,nitroglycerin stoppage rate;(3)ECG efficacy;(4)efficacy and symptoms of TCM syndromes.Safety indicators include vital signs,blood,urine,stool routines,liver function,and kidney function.The test results were statistically analyzed using SPSS 19.0 statistical software.RESULTS:Baseline:TG consisted of 20 patients,including 12 male and 8 female.The age range was 56-70,and the average age was 62.15±4.66.There were 20 patients in CG,including 13 male and 7 female.The age range was 54-69,and the average age was 61.2±4.57.There was no significant difference between the two groups in baseline(p>0.05).Efficacy:(1)The total effective rate of angina pectoris:the total effective rate in the treatment group was 90%,and the total effective rate in the control group was 60%.(2)The number of episodes of angina pectoris:the number of pain in the treatment group after treatment was significantly less than that in the control group(p<0.05).(3)Angina pectoris scores:there was a statistically significant difference in angina pectoris scores before and after treatment between the two groups(p<0.05).(4)Nitroglycerin consumption(mg):there was a difference in the amount of nitroglycerin between the two groups before and after treatment.Statistical significance(p<0.05).(5)Efficacy of electrocardiogram:the total effective rate in the treatment group was 80%,and the total effective rate of the electrocardiogram in the control group was 70%;(6)Efficacy of TCM syndromes:the total effective rate of TCM syndromes in the treatment group was 90%,the total effective rate of TCM syndromes in the control group was 50%,the difference between the two groups was statistically significant(p<0.01),and the treatment group had better efficacy in the clinical symptoms of chest tightness,chest pain,and fatigue.(7)Safety indicators:both groups changed within normal range.3.Experimental study:3.1 Network Pharmacology StudyOBJECTIVE:To use network pharmacology to predict the possible targets of Dangshen Tongluo Capsules,which are assigned to the Huoxuehuayu Group,and to predict the target genes of Danshen Tongluo Capsules by GO and KEGG analysis of the target.A common analysis was performed with the next high-throughput sequencing results.METHODS:Using OMIM and TTD database to obtain "CHD" target set.Using TCMSP database and PharmMapper database to obtain target set "Yao".Using Uniprot database to unified name.Using Venn diagrams to get the intersection targets of "CHD" and "Yao".Using GeneMANIA network software and MAS 3.0 for analysis the target gene function,target pathway,and target interaction network.RESULTS:A set of "CHD" targets was obtained from the OMIM and TTD database search and contained 204 target genes.The "Yao" target set predicted from the TCMSP and PharmMapper databases contained 286 eight-point genes.After the two trgets were collected and combined,three targets for the prediction of DSTLJN were obtained,which were IL10,F2,and AR.GeneMANIA's analysis showed a 67.64%physical correlation,but only 13.5%of the co-expression features.MAS 3.0 found that 20 GO functions were involved and 12 were involved in the KEGG pathway.The top five GO functions were physiological process(G0:0065007),cellular process(GO:0009987),biological regulation(GO:0050789),physical process(G0:0007582)and binding(GO:0005488),accounting for 50.8%of all features.The KEGG pathway is mainly involved in the actin cytoskeleton regulation,complement pathway,epidermal growth factor receptor signaling pathway,and the interaction of cytokines and their receptors.3.2 Molecular Biology ExperimentsOBJECTIVE:Through high-throughput sequencing technology with blood samples,construct the co-expression network of IncRNA-mRNA,to explore the mechanism of DSTLJN by analyzing GO function and KEGG pathway of target genes that have a co_expression relationship.METHODS:Collection of blood samples.Screen and determine the differential genes using high-throughput sequencing after quality inspection.The results were compared with the reference genes using hist2,bowtie2,eXpress software and FPKM algorithm.Expression level tests were performed and sample correlation tests were performed at the same time.The DESeq software was used to calculate the difference times,and change genes of |log2FoldChange|>1 and p value<0.05 were defined as the differential genes for this experiment.The differential lncRNA and mRNA were calculated using Pearson's correlation coefficient,and Pearson's correlation coefficient not less than 0.8 with p value<0.05 was defined as a co-expression relationship.GO and KEGG enrichment analysis:according to the differential gene with the co-expression relationship in the previous step,the number of differential genes included in each GO entry and KEGG pathway was counted,and each GO entry and KEGG pathway were calculated using the hypergeometric distribution test method.The Enrichment Score of the differential gene enrichment;the GO function was analyzed using topGO,and the top 10 items mainly including BP,CC,and MF were described in detail;KEGG selected coronary artery disease-related pathways,inflammation-related pathways,and enriched with more gene pathways for analysis.RESULTS:Differential genetic screening resulted in a total of 181 differential lncRNA,of which 98 were up-regulated genes and 83 were down-regulated genes.A total of 312 differential mRNAs were screened,of which 172 were up-regulated genes and 140 were down-regulated genes.There were 333 pairs of lncRNA and mRNA co-expression relationships,of which 226 pairs were positively correlated with 108 pairs being negatively correlated.GO analysis showed that gene function mainly focused on oxygen transport and maintenance of oxygen homeostasis,cell membrane trafficking,and myoblast differentiation and filopodia formation and immune processes.Analysis of its signal pathways revealed that there are 5 signaling pathways with significant genetic differences,including cGMP-PKG signaling pathway(has 04022)and toll-like receptor signaling pathway(has 04620)are closely related to coronary heart disease pathways.The three signaling pathways associated with the two pathways are MAPK signaling pathway(has 04010),P13K-AKT signaling pathway(has 04151),and apoptotic signaling pathway(has 04210).There are up-regulation of CREB and down-regulation of PDE3 in cGMP-PKG signaling pathway,down-regulation of p38 in toll-like receptor signaling pathway,down-regulation of DAXX and p38 in MAPK signaling pathway,up-regulation of CREB,down-regulation of Bim,and apoptosis in P13K-AKT signaling pathway.There are DAXX and p38 downregulation in the signal pathway.4.CONCLUSIONS:The "Effective Components Combination Formula" DSTLJN has good clinical effect on coronary heart disease patients with SAP and blood stasis syndrome.It can effectively improve the clinical symptoms of patients,significantly reduce the amount and frequency of nitroglycerin,safe and no bad responses.Based on biomolecular network molecular biological studies found that co-expressed differentially expressed genes are mainly concentrated in the MAPK signaling pathway,cGMP-PKG signaling pathway and Toll-like receptor signaling pathway,proving that the blood circulation "Effective Components Combination Formula" drug DSTLJN has cardioprotective function and relieve-inflammation function.Its cardioprotective function may be achieved through DAXX/ASK1/p38 and cAMP/PKA/CREB signaling processes.The similar changes in regulation and positive feedback changes exist in cAMP/PKA/CREB signaling process of the cGMP/PKG signaling pathway and the DAXX/ASK1/p38 signaling process of the MAPK signaling pathway.Maybe this reflects the idea of compatibility of TCM in the side.Network pharmacology prediction results shows that the predicted gene can inhibit the toll-like receptor signaling pathway through indirect action. |
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