| Qilong capsule(Chinese medicine Z20000097,medical insurance class B)is legislated to replenish qi and activate blood circulation,with equal emphasis on replenishing qi and activating blood circulation,mainly for ischemic stroke with qi deficiency and blood stasis syndrome.However,the clinical effectiveness of Qilong capsule in real-world clinical practice has not been evaluated,and the therapeutic mechanism is not clear.The compound prescription of traditional Chinese medicine has the characteristics of complex components and diverse target pathways.In order to solve the clinical problem that the clinical effectiveness of Qilong capsule is not clear in the real world,and its mechanism is not clear,this study proved the clinical effectiveness of Qilong capsule in real clinical practice through a prospective cohort study,and screened out the key genes,signal pathways and biological processes regulated by Qilong capsule by using RNA-Seq and RT-qPCR.This study provides real-world evidence for Qilong capsule to accurately locate ischemic stroke patients with qi deficiency and blood stasis syndrome,and reveals the therapeutic mechanism of"multi-component,multi-target,multi-pathway" of Qilong capsule from the transcriptome level,and provides an objective biological basis for accurately locating ischemic stroke of qi deficiency and blood stasis syndrome in clinical practice.ObjectiveA prospective cohort study was conducted to evaluate the clinical effectiveness of Qilong capsule in the treatment of ischemic stroke with qi deficiency and blood stasis syndrome in real world clinical practice.RNA sequencing and RT-qPCR were used to screen the key genes,signal pathways and biological processes regulated by Qilong capsule and explain its therapeutic mechanism.Methods1.Prospective cohort study of Qilong capsule in the treatment of ischemic stroke with qi deficiency and blood stasis syndrome:the participants were ischemic stroke patients with qi deficiency and blood stasis syndrome.The patients were divided into exposure group and non-exposure group according to whether they took Qilong capsule or not.All included patients were given conventional treatment.The total sample size was not less than 2249 cases.The primary outcomes were mRS score,NIHSS score and BI score,while the secondary outcomes included TCM syndrome(qi deficiency syndrome,blood stasis syndrome),psychological index(SDS,SAS),blood lipid index,blood coagulation index and homocysteine.The patients were followed up at the 12th and 24th week after treatment to evaluate the effectiveness.Interpolation method was used to deal with missing data,propensity score matching method(PSM)was used to deal with confounding factors,and clinical effectiveness was evaluated by difference test,aligned rank transformation analysis of variance(ART ANOVA)and generalized linear mixed model(GLMM).The research results are reported in accordance with the STROBE-cohort study standard.2.Study on differential expression of mRNA after intervention with Qilong capsule based on RNA sequencing:(1)The experimental blood samples were taken from patients in the previous clinical trial at baseline and 12 weeks after treatment with Qilong capsule.According to the screening principle of patients with the largest reduction in NIHSS score and mRS score and the largest increase in BI score after 12 weeks of treatment.A total of 40 blood samples were collected from 20 patients with 2.5ml Paxgene Blood RNA tube,before and after treatment.(2)Blood samples were collected from 20 patients before treatment as the untreated group(20 biological samples repeated)and 12 weeks after treatment with Qilong capsule as the treatment group(20 biological samples repeated).In addition to the main analysis group,the subgroups were set up according to the clinical characteristics of the patients,including sex,age(≤60 years old and>60 years old),initial or recurrence.(3)The experimental methods include total RNA extraction,total RNA quality control,library construction and sequencing,sequencing data filtering and quality control,differential gene detection and differential gene enrichment analysis.(4)DEGseq method was used to screen differential genes.In order to improve the accuracy of screening,we defined genes with log2FoldChange≥1 and Q-value≤0.001 as significantly differentially expressed genes.(5)The GO function and KEGG Pathway enrichment analysis of the differential genes were carried out.The calculated P-value was corrected by Bonferroni and FDR respectively,and the threshold was P-value or Q-value(corrected P-value)<0.05.The GO term and Pathway satisfying this condition were defined as GO term and Pathway which were significantly enriched in the candidate genes.The main signal transduction pathways and biological functions of differential genes can be determined by GO function and KEGG Pathway significant enrichment analysis.3.Study on key genes and Regulation Mechanism of Qilong capsule after intervention based on RT-qPCR:(1)AREG,CAV2,PEDS1-UBE2V1,MYL9,ITGA2B,CXCL8,LY96,GNG10 and CXCL10 with significant difference and high gene expression were selected in the sequencing results.β-actin was used as the internal reference gene.(2)The RNA sequencing part of the same batch of patients’ blood samples were selected for the verification experiment.A total of 10 patients(3 females and 7 males;5 patients aged<60 years old,5 patients aged>60 years old;6 cases of initial episodes,4 cases of recurrence),20 blood samples for RNA verification(before treatment,after treatment).(3)The experimental methods include total RNA quality detection,mRNA reverse transcription,real-time fluorescence quantitative PCR amplification(sequence and primer design,PCR reaction system).(4)The relative changes of gene expression in real-time quantitative PCR were analyzed by 2-delta delta Ct method.The three-repeat average Ct and the average internal reference Ct of 10 samples in the untreated group and the treatment group were calculated as the mean of the two groups,and then compared with the untreated group(2-⊿⊿Ct is set to 1),the expression ratio 2-⊿⊿Ct of each target gene in the treatment group and the untreated group was calculated.If the ratio is more than 1,the gene expression is up-regulated after intervention.If the ratio is less than 1,the expression level is down-regulated.To determine whether the verified target gene is consistent with the upward and downward trend of transcriptome sequencing.At the same time,combined with the results of transcriptome sequencing and enrichment,the main signal pathways and biological processes regulated by key genes were explained.Results1.A prospective cohort study of Qilong capsule in the treatment of ischemic stroke with qi deficiency and blood stasis syndrome:(1)From November 3,2016 to January 16,2019,a total of 2468 patients were screened.At baseline enrollment,97 cases were excluded due to non-compliance with the enrollment criteria.A total of 2371 patients entered the study cohort.A total of 69 cases were eliminated and lost to follow-up at the 12th and 24th week of follow-up.Finally,2302 qualified cases were included in the analysis(1260 cases in the exposure group,1042 cases in the non-exposed group).(2)Comparison of the difference between the two groups of 2302 cases with full data set before and after treatment:①at the 24th week after treatment,Qilong capsule combined with conventional treatment was significantly superior to conventional treatment alone in reducing mRS score,NIHSS score,Qi deficiency syndrome score,blood stasis syndrome score,SDS score,SAS score and increasing BI score(P<0.05).②at the 12th week after treatment,Qilong capsule combined with conventional treatment was significantly better than conventional treatment alone in reducing the content of total cholesterol(P<0.05).(3)Comparison of the difference between groups before and after treatment with PSM data set:①at the 24th week after treatment,Qilong capsule combined with conventional treatment was superior to conventional treatment alone in reducing mRS score,NIHSS score,Qi deficiency syndrome score,blood stasis syndrome score,SAS score and increasing BI score(P<0.05).②at the 12th week after treatment,Qilong capsule combined with conventional treatment was superior to conventional treatment alone in reducing the content of triglyceride(P<0.05).(4)Alignment rank transformation variance model analysis of 600 cases data set:the main outcome index analysis of the three measurements showed that the P value of mRS and BI in the grouping was less than 0.05,indicating that there was a statistically significant difference in the data between mRS and BI,while the P value of the interaction between grouping and measurement times was greater than 0.05,indicating that there was no interaction between measurement times and grouping.When the P value of NIHSS is less than 0.05,it shows that the difference of NIHSS data in each measurement time is statistically significant,while the P value of grouping and measurement interaction is more than 0.05,indicating that there is no interaction between measurement times and grouping.(5)Generalized linear mixed model analysis:the mRS score,NIHSS score and BI score were modeled on the full data set of 2302 cases and the data set of 600,and the results showed that:①in terms of mRS score,the estimation of odds ratio of mRS in exposed group decreased by 99.83%with one time,and that of mRS in non-exposed group decreased by 95.8%with one time.The decrease rate of mRS in exposure group was faster than that in non-exposure group,and the difference was significant(P<0.05).The results of the full data set of 2302 cases showed that the estimated odds ratio of mRS in the exposure group decreased by 99.82%with one time,and the estimated odds ratio of mRS in the non-exposure group decreased by 97%with one time.The decrease rate of mRS in the exposure group was faster than that in the non-exposure group,and the difference was significant.②in terms of NIHSS scores,the results of the data set of 600 cases showed that the estimated odds ratio of NIHSS in the exposure group decreased by 99.98%with one time,and the estimated odds ratio of NIHSS in the non-exposure group decreased by 99.77%with one time.The decrease rate of NIHSS in the exposure group was faster than that in the non-exposure group,and the difference was significant(P<0.05).The results of the full data set of 2302 cases showed that the estimated odds ratio of NIHSS in the exposed group decreased by 99.99%with one time,and the estimated odds ratio of NIHSS in the non-exposed group decreased by 99.97%with one time.The decline rate of NIHSS in the exposed group was faster than that in the non-exposed group,and the difference was significant(P<0.05).③in terms of BI scores,the results of the data set of 600 cases showed that the BI scores of patients in the exposure group increased faster than those in the non-exposure group,and the difference was significant(P<0.05).2.Study on differentially expressed mRNA after the intervention of Qilong Capsule based on RNA sequencing:(1)The average total RNA concentration of 40 samples was 282.08 ng/μL,and the average total Rin value was 5.61 μg;except for the two samples whose RIN values are 6.6 and 5.1,the rest are all higher than 7.The 28S/18S is between 1.3 and 2.9,indicating that the purity and completeness of 40 total RNAs are high,which meets the requirements of subsequent library construction and sequencing.(2)Using the DNBSEQ platform for sequencing,40 samples produced a total of 1869.27Mb original data.After the original data were filtered,a total of 1619.21Mb(86.62%)of the original data were retained as high-quality reads(clean reads),indicating that the sequencing data results are good,most of the high-quality reads are retained,and the clean reads differences between samples are small,which can be used for follow-up analysis.(3)The results of significant difference gene detection showed that 39 significant difference genes were detected in the main analysis group,40 significant difference genes were detected in the male subgroup,151 significant difference genes were detected in the female subgroup,61 significant difference genes were detected in the age≤60 years old subgroup,84 significant difference genes were detected in the age>60 years old subgroup,and 52 significant difference genes were detected in the first onset subgroup,102 significant difference genes were detected in the recurrence subgroup.(4)Enrichment analysis showed that Qilong capsule improved inflammation and atherosclerosis and repaired neurological defects by regulating signal pathways and biological functions in the following five aspects.Such as:①immune response(LY96,CXCL8,CXCL10,etc.participate in the regulation of immune response,LY96,HP,HLA-DRB5,etc.participate in the process of immune system).②inflammatory response related signal pathways(LY96,CXCL8,CXCL10,etc.participate in the regulation of inflammatory response,AREG,ITGA2B,FN1,etc.participate in the regulation of PI3K-AKT signal pathway,ITGA2B,FN1,GP1BB,etc.participate in the regulation of ECM-receptor interaction,AREG and CAV2 participate in the positive regulation of MAPK cascade,GNG10,CXCL10,CXCL8,etc.participate in the regulation of chemokine signal pathway,PEDS1-UBE2V1,CXCL8,CXCL1,etc.participate in the regulation of IL-17 signal pathway.LY96,CXCL8,CCL4L1,etc.are involved in the regulation of NF-κB signal pathway,LY96,CXCL8,CAV1,etc.are involved in the regulation of Toll-like receptor signal pathway,CXCL8,CXCL1,CCL4L1,etc.are involved in the interaction of cytokine receptors,FN1,ARG1,CAV1,etc.are involved in TGF-β signal pathway,DAAM2,CAV1,BAMBI,etc.are involved in the regulation of Wnt signal pathway.③regulation of atherosclerosis(CAV2,ITGA2B,GSTT1,etc.participate in the regulation of fluid shear stress and atherosclerosis),④regulation of cell-related functions(AREG,CAV2,IGFBP2,etc.participate in the regulation of cell proliferation and differentiation,CXCL8,E2F1,KIR2DL1,etc.participate in cell senescence,HBA1,HBB,HP,etc.participate in apoptosis and clearance,CAV2,ITGA2B,CAV1,etc.participate in the regulation of cell adhesion,ITGA2B,MYL9,FN1,etc.participate in the regulation of leukocyte migration.⑤ blood pressure regulation(AREG,KLK1,HBB,etc.participate in blood pressure regulation).3.Study on key genes and Regulation Mechanism of Qilong capsule after intervention based on RT-qPCR:(1)The concentration of 20 samples is between 200-700 ng/μl and the purity is between 2.0 and 2.3.The concentration and purity meet the requirements of the next reverse transcription and amplification experiment,and the RNA quality of all samples detected by electrophoresis is good.(2)After PCR amplification,the amplification efficiency of the target gene was basically the same as that of the internal reference,and the melting curve was a single peak,indicating that there was only one product with good specificity.The effect of PCR amplification have good quality.(3)RT-qPCR results showed that the expression ratio 2-⊿⊿ct of AREG,CAV2,ITGA2B,PEDS1-UBE2V1 and MYL9 in the treatment group was more than 1,which was up-regulated compared with the untreated group after intervention,while the expression ratio 2-⊿⊿Ct of LY96 and GNG10 in the treatment group was less than 1,which was down-regulated compared with the untreated group in the after intervention.(4)The expression levels of genes AREG,CAV2,PEDS1-UBE2V1,MYL9,ITGA2B,LY96 and GNG10 were consistent with the upward and downward trends of the corresponding genes of RNA sequencing,on the one hand,it indicated that AREG,CAV2,PEDS1-UBE2V1,MYL9,ITGA2B,LY96,GNG10 may be the key genes regulated by Qilong capsule;on the other hand,it showed that the results of RT-qPCR verification were consistent with those of RNA sequencing,and the sequencing results were reliable.According to the results of transcriptome sequencing and enrichment analysis,it can be found that Qilong capsule participates in immune response,inflammation-related signal pathway,atherosclerosis,cell-related function,blood pressure regulation and other key signal pathways and biological process regulation through the above key genes.Conclusion1.On the basis of conventional treatment,Qilong capsule can significantly reduce mRS score,NIHSS score,qi deficiency syndrome score,blood stasis syndrome score,SDS score,SAS score,total cholesterol content,triglyceride content and increase BI score,improve neurological impairment,qi deficiency and blood stasis,depression and anxiety,reduce blood lipids and improve patients’ activities of daily life,and have good safety.In real-world clinical practice,Qilong capsule is clinically accurately positioned for patients with ischemic stroke of Qi deficiency and blood stasis syndrome and has significant effectiveness.2.Qilong Capsule in the treatment of ischemic stroke patients with Qi-deficiency and blood-stasis syndrome mainly regulates the expression of key genes such as AREG,CAV2,PEDS1-UBE2V1,MYL9,ITGA2B,LY96,GNG10,and participates in immune response and inflammatory response-related signal pathways(inflammation response regulation,PI3K-AKT signaling pathway,ECM-receptor interaction,positive regulation of MAPK cascade,chemokine signaling pathway,IL-17 signaling pathway,NF-κB signaling pathway,Toll-like receptor signaling pathway),atherosclerosis regulation,cell related functions(cell proliferation and differentiation,cell adhesion,leukocyte migration),blood pressure regulation and other key signaling pathways and biological processes.Through the above process to improve the inflammation and atherosclerosis of patients,promote nerve cell and tissue regeneration,restore nerve function and improve the ability of daily life.Among them,the monarch drug Huangqi is related to immune response and inflammatory response-related signal pathways and the regulation of atherosclerosis,and the monarch drug Dilong is related to the regulation of atherosclerosis,cell-related functions,and blood pressure regulation.The ministerial medicines Danshen,Danggui,Chishao,chuanxiong,Honghua,and Taoren are related to inflammatory response-related signal pathways,atherosclerosis regulation,cell-related functions,and blood pressure regulation.Qilong Capsule,under the action of the above-mentioned drug compatibility,uses the legislation to invigorate qi,activate blood,remove blood stasis and dredge collaterals,and jointly participate in the above five key signal pathways and biological processes.The therapeutic mechanism of Qilong capsule accurately located in patients with ischemic stroke with qi deficiency and blood stasis syndrome was explained from the transcriptional group level.Highlight of innovation1.Combining the propensity score matching method with the aligned rank transformation variance analysis and the generalized linear mixed model,the prospective cohort study confirmed the clinical effectiveness of Qilong capsule in the treatment of ischemic stroke with qi deficiency and blood stasis syndrome in real clinical practice.It provides real-world evidence for clinical accurate localization of qi deficiency and blood stasis syndrome.2.By using RNA sequencing technology and RT-qPCR verification technology,it was found that Qilong capsule participated in the key signal pathway and biological processes such as immune response,inflammation-related signal pathway,atherosclerosis,cell-related function and blood pressure regulation by regulating the expression of key genes such as AREG,CAV2,PEDS1-UBE2V1,MYL9,ITGA2B,LY96 and GNG10,and preliminarily explained the therapeutic mechanism of "multi-component,multi-target,multi-pathway" of Qilong capsule.It provides an objective biological basis for clinical accurate localization. |
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