Efficacy Evaluation Of Qi Stagnation And Blood Stasis Syndrome Prescriptions And Research On The Whole Transcriptome Mechanism

The traditional Chinese medicine(TCM)compound preparation is a kind of formulae that should be formed based on the TCM theory,which could be originated from the ancient classics,or targeting the syndrome,or targeting the combination of disease and syndrome.Among them,the TCM compound preparations for syndrome is named syndrome drug,which is a sort of drug development model mostly featured with TCM characteristics.However,there is still a lack of TCM compound preparations in the market,partly because it is not clear whether the new syndrome drugs for treatment of syndromes are in accordance with the theory of TCM,and it remains obscure where the clinical advantages of this kind of syndrome drugs highlight compared with drugs targeting the combination of disease and syndrome.Therefore,supported by the research project of Chinese medicine industry special research on the evaluation method of new drug efficacy of TCM and based on review of current situation and methodology,this study took qi stagnation and blood stasis(QS&BS)syndrome as an example,and used theory research,network pharmacology research,clinical trial,and whole transcriptome mechanism research to figure out the key issues of the development of new drugs for syndromes.This study also provided a methodological reference for the clinical research model of new drugs for syndromes in "Technical Guidelines for Clinical Research of New Drugs in Syndromes".ObjectiveThe aims of this study were to provide the TCM theoretical basis for the development of new syndrome drugs for QS&BS syndrome,and to evaluate the effectiveness and safety of Xuefu Zhuyu Capsule as a new drug for QS&BS syndrome,and to explore Xuefu Zhuyu Capsule intervention mechanism for QS&BS syndrome.Methods1.Theoretical study on the intervention model of TCM based on ancient literature.Based on the representative Chinese medicine classic books involved in the book "History of Chinese Medicine" and the "Siku Quanshu Head Summary" and "Chinese Medicine" and the related contemporary literatures,we listed the theory of TCM intervention according to the times,and summarized the classification and theory of TCM intervention mode and intervention level.The comparative research method(the qualitative and comprehensive comparison method between the different intervention levels and the comparison between syndromes and prescriptions)was used to explore the theoretical basis of new syndrome drugs,including the difference,characteristics,advantages and scope of application compared with the drug targeting combination of disease and syndrome.2.Network pharmacology study of Xuefu Zhuyu Decoction for QS&BS syndrome.The composition of Xuefu Zhuyu Decoction was searched using the Chinese Medicine System Pharmacology Database,and the components were screened according to the absorption,distribution,metabolism,and excretion parameters,and the potential targets were predicted.The gene expression databases were used to search for the previously differential expression profiles of "QS&BS",and combined with the sequencing results of patients with QS&BS syndrome,the target of QS&BS syndrome was constructed Targets for QS&BS syndrome-related diseases were searched based on drug treatment target database,drug database,and disease gene association database.The Cytoscape software was used to build a molecular network for the regulation of QS&BS syndrome.3.Prospective multi-center clinical trial of Xuefu Zhuyu Capsule in the treatment of QS&BS syndrome.A multicenter central stratified randomized,placebo-controlled,double-blinded,clinical trial was designed and conducted to determine the cause and effects relationship between Xuefu Zhuyu Capsule and efficacy of QS&BS syndrome.According to the Diagnostic Scale of QS&BS Syndrome,subjects who were diagnosed with QS&BS syndrome were included.The central randomized system was used to realize stratified randomization and concealing of randomization.They were randomly divided into treatment group and control group according to 1:1 ratio,and double-blinded design was adopted,with subjects,researchers,outcome evaluators and statisticians being blinded by two levels.The treatment group was treated by Xuefu Zhuyu Capsule with 6 capsules/time,2 times/day,taken by warm boiled water,while the control group was Xuefu Zhuyu Capsule Placebo(blank)with 6 capsules/time,2 times/day,taken by warm boiled water.The leading-in period was 1 week and the treatment course was 7 weeks.During the leading-in period,other Chinese medicines for treating QS&BS syndrome were asked to stop.Effectiveness outcomes included the primary outcome of syndrome efficacy judged by the evaluation scale of QS&BS syndrome based on patient report outcome at 0,2,4,6,and 7 weeks and the secondary outcome of symptomatic efficacy performed at 0 and 7 weeks using the Single Symptoms and Signs Scale.Safety outcomes included blood routine,urine routine,stool routine,liver and kidney function,coagulation function,measured at 0 and 7 weeks,and adverse events observed and recorded throughout the trial.The sample size estimate determined that the sample content was 120 and the center-to-center ratio was 2:1:1.Statistical analysis was performed using R(V3.5.3)software.The continuous variables were measured in the form of mean�standard deviation or median and quartile,while the categorical variables were performed in the form of quantity and percentage.A two-sided test and P<0.05 was considered statistically significant.4.Transcriptome mechanism study of Xuefu Zhuyu Capsule for QS&BS syndrome.Five patients were selected separately from the treatment group and the control group in the clinical trial,high-throughput sequencing of the paired samples before and after the self-treatment was performed to detect differential expression profiles of the whole transcriptome and bioinformatics analysis was used to figure out the mechanism.Prospective nested case-control design was used by selecting five subjects with good efficacy and poor efficacy from the cohort of the treatment group and high-throughput sequencing of the pre-treatment samples,in order to screen the differential expression profiles of the whole transcriptome.Prospective design of prospective biomarkers design was also used by PCR testing all of the subjects in the cohort based on the screening differential RNAs,looking for biomarkers of drug sensitivity.Results1.Literature research indicated that Chinese medicine intervention can target on basic elements such as TCM constitution,diseases,etiology,pathogenesis,disease position,symptoms,Western medicine pathology and indicators,and intervention modes mainly include TCM constitution,disease,disease+symptom,disease+cause/pathogenesis+symptoms,disease+constitution+symptoms,pathogenesis+symptoms,pathogenesis+disease position,cause+pathogenesis+symptoms.The intervention levels mainly include yin and yang,qi and blood,viscera,triple focus,meridian,Weiqi Yingxue,Liujing and local.According to the scope of indications of prescriptions,the prescriptions can be divided into prescriptions with wide indications,prescriptions for intervention of visceral diseases,prescriptions strongly targeting disease,and prescriptions strongly targeting symptoms.The new drug of syndrome can be used for the conditioning of TCM constitution,pathogenesis and the same mechanisms at different stages of different diseases.The intervention level can be targeted at yin and yang,qi and blood,viscera,meridians,and prescriptions with a wide range of indications should be considered in the choice of prescriptions.Therefore,this QS&BS syndrome on the pathogenesis model and blood level can be used as the target indication for the development of new drugs for syndromes.Syndrome new drugs are different from the new drugs targeting combination of disease and syndrome,and the advantages embody in complicated diseases,complicated clinical manifestations,patients with multiple diseases,geriatric diseases and complex situation after long-term treatment.2.The network pharmacology study predicted that Xuefu Zhuyu Decoction might interact with the targets of both QS&BS syndrome and associated diseases.A total of 418 compounds was screened for Xuefu Zhuyu Decocotion,and 338 targets were predicted,the function was enriched in Cytokine Signaling in Immune Signal path et al.A total of 783 differentially expressed genes of QS&BS syndrome was selected from patients with QS&BS syndrome,and their functions were enriched in signaling pathways such as Signalling by NGF.As for QS&BS syndrome related diseases,coronary atherosclerotic heart disease targeted 912 genes,gastroenteritis disease targeted 228 genes,osteoarthritis disease targeted 76 genes,dysmenorrhea disease targeted 13 genes.QS&BS syndrome "formulae-disease-syndrome" regulation molecular network was build,where Xuefu Zhuyu decoction targeted genes and QS&BS syndrome intersected a total of 7 genes,and 84 intersections with coronary heart disease targets,29 intersections of gastritis target,and 4 intersections of dysmenorrhea target,and 5 intersections of osteoarthritis target.3.Clinical trial showed that Xuefu Zhuyu Capsule had better effects in the treatment of QS&BS syndrome than placebo.In terms of the main outcome,the difference between the two groups was statistically significant compared with the baseline QS&BS test scores at 7 weeks after treatment,and the median reduction of QS&BS syndrome PRO score in treatment group was at least 5.5 after 7 weeks,which was better than the placebo group.In terms of secondary outcomes,Xuefu Zhuyu Capsule improved the pain(p=0.015),chest tightness(p=0.001),palpitation(p=0.004),bulging(p=0.000),bloating(p=0.000),sigh(p=0.021),insomnia(p=0.027),dizziness(p=0.003),numbness(p=0.000),dark complexion(p=0.037),purple lips(p=0.037)which were superior to placebo,and there was no statistically significant difference between the two groups in improving irritation(p=0.432)and lump(p=0.246).As for the safety outcomes,except for stool routine,Kappa test indicates homogeneity for blood routine,liver and kidney function,coagulation function,urine routine,urine biochemistry,electrocardiogram results before and after treatment.A total of 13 patients had 25 adverse events,and there was no statistically significant difference between the two groups.4.The whole transcriptome mechanism study completed high-throughput sequencing of 25 samples of 5 samples and real time PCR of 30 samples,figured out the sensitivity biomarker of Xuefu Zhuyu Capsule.There were 58 differential expressed mRNAs,17 differential expressed miRNAs,691 differential expressed IncRNA,161 differential expressed circRNAs in paired samples of before and after treatment.There were 71 differential expressed mRNAs,16 differential expressed miRNAs,616 differential expressed lncRNA,34 differential expressed circRNAs in paired samples of before and after intervention in control group.There were 107 differential expressed mRNAs,45 differential expressed miRNAs,721 differential expressed IncRNA,242 differential expressed circRNAs screened in effective samples and noneffective samples.The functions of the differential genes are mainly enriched in signaling pathways associated with QS&BS syndrome and QS&BS syndrome-related diseases.The sensitivity analysis suggested that the PCR quantitative results of FZD8 and circRNA 13799 are statistically significant between the effective group and the noneffective group.The 75%stratification results of FZD8 could predict the effect of Xuefu Zhuyu Capsule on the treatment of QS&BS syndrome.The area under the ROC curve was 0.765,and the prediction had certain accuracy.Conclusions?New syndrome drug has the theoretical basis of TCM in terms of TCM intervention,intervention level and prescription indication.?Xuefu Zhuyu Capsule could effectively improve the syndrome of QS&BS,and can be used as a new syndrome drug for QS&BS syndrome treatment.?Xuefu Zhuyu Capsule can play a role in the treatment of QS&BS syndrome by interfering with the target genes of QS&BS syndrome and related disease target genes.FZD8 can be used as a predictor biomarker of the sensitivity of Xuefu Zhuyu Capsule in intervention of QS&BS syndrome.