Study On The Molecular Network Mechanism Of Qilong Capsule In The Treatment Of Ischemic Stroke (Qi Deficiency And Blood Stasis Syndrome)

Background:Ischemic stroke(IS)is a major disease of global concern.According to a report in the Lancet in March 2019,stroke has become the second leading cause of death in the world,with IS accounting for the majority(84.4%).In China,the prevention and treatment of IS is facing greater challenges.China Stroke Prevention and treatment report 2019 points out that the incidence of IS in China is on the rise.Traditional Chinese medicine has certain advantages in the prevention and treatment of IS,among which the method of replenishing qi and activating blood circulation,as the basic treatment method of IS,has been widely used and developed in clinic,and it is the main line of treatment in every period of IS.Qilong capsule,derived from the classic prescription Buyang Huanwu decoction,is a representative proprietary Chinese medicine in the method of replenishing qi and activating blood circulation.A prospective,multicenter,non-randomized controlled study conducted by the research group proved that Qilong capsule can significantly improve the degree of neurological impairment in patients with acute IS qi deficiency and blood stasis syndrome,reduce the content of P-selectin in acute patients,and play a role in inhibiting platelet activation.However,the advantages and molecular mechanism of Qilong capsule in the treatment of IS are not clear.In recent years,the method of network pharmacology has been widely used to explore the mechanism of traditional Chinese medicine compound prescription,which provides an idea and feasible scheme for revealing the molecular mechanism of the synergistic effect of traditional Chinese medicine compound prescription on complex diseases from the whole level.Objective:To evaluate the advantages of Qilong capsule in the treatment of IS,and from the point of view of TCM holistic view and the combination of disease and syndrome,to explore the molecular network mechanism of Qilong capsule in the treatment of IS with qi deficiency and blood stasis syndrome,so as to provide a basis for its clinical accurate location.Research methods and results:Evaluating the advantages of effect:According to the standard of PRISMA2009 report,six RCT studies were included,and qualitative systematic evaluation and quantitative Meta analysis were carried out.The results showed that Qilong capsule combined with conventional western medicine was superior to routine western medicine in improving neurological impairment,reducing the recurrence rate of IS,improving activities of daily living and improving hemorheology in patients with IS,and the difference was statistically significant.Exploring the mechanism of molecular network:1.Data acquisition:We searched OMIM database and GeneCards database,obtained 2623 IS genes,searched SymMap database and HPO database,obtained 398 genes related to qi deficiency and blood stasis syndrome,collected 12 active components of Qilong capsule from Chinese Pharmacopoeia and national drug standards in 2020,searched TCMSP database and GeneCards database,combined with SEA platform prediction and literature collection,obtained 210 drug targets of Qilong capsule.Using UniProt database to standardize the gene data collected,and finally got three data sets for follow-up analysis:IS disease gene set,IS related qi deficiency and blood stasis syndrome gene set,Qilong capsule drug target set.2.Network construction and module partition:The above three target(gene)sets were uploaded to STRING database respectively,the gene interaction data were downloaded,and the network visualization was carried out by Cytoscape software.Three molecular networks were constructed:IS disease molecular network,IS related qi deficiency and blood stasis syndrome molecular network,Qilong capsule drug molecular network.Three molecular networks are divided by MCODE algorithm,MCL algorithm and DCU algorithm respectively,and the network structure entropy is calculated,and the algorithm with the minimum entropy is selected to divide the module.Finally,the MCL algorithm was used to divide the IS molecular network,and the MCODE algorithm was used to divide the molecular network of qi deficiency and blood stasis syndrome,and 18 syndrome modules are obtained.The molecular network of Qilong capsule was divided by MCODE algorithm,and 6 drug modules were obtained.The biological function enrichment analysis of the module was carried out by using the g:Profiler platform.The enrichment threshold was P<0.05.The enrichment results of GO biological process and Reactom pathway showed that the function of the module was related to IS and had biological significance.3.Identification and analysis of disease and syndrome combination module:The hypergeometric distribution function was used to identify the modules in which the genes of qi deficiency and blood stasis syndrome were significantly enriched in the 129 modules of IS disease.Taking P<0.05as the threshold of significant enrichment,eight disease syndrome combination modules of IS with qi deficiency and blood stasis syndrome were obtained.The core target genes of the disease-syndrome combination module were screened through two steps:first,25 nodes were screened by at least two of the median betweenness values of 0.00028548,closeness values of 0.47262702,and Degree values of 61;then,10 core target genes were identified by the cytoHubba plug-in in Cytoscape software:IL1B,FN1,INS,EGF,MAPK1,MMP2,VEGFA,CASP3,MAPK8,MMP9.The biological process of GO was analyzed by g:profiler platform.The results showed that the most significant enrichment results were apoptosis,oxidative stress and leukocyte differentiation,all of which were related to the pathological mechanism of IS.4.Identify and analyze the correlation module between drugs and syndromes:The eight disease-syndrome binding modules identified in part 3.2.3 were intersected with the molecular network of Qilong capsule respectively,and two drug-syndrome association modules were obtained:Module1 and Module2,contained 98 targets.According to the molecular function of the international authoritative protein knowledge base UniProt,the targets of 98 modules related to Qilong capsule and IS qi deficiency and blood stasis syndrome were classified into 11 categories according to the pharmacological effects of qi-invigorating and blood-activating drugs in the treatment of IS qi deficiency and blood stasis syndrome.On the whole,Qilong capsule has the characteristics of multi-target and multi-component synergistic effect in the treatment of IS qi deficiency and blood stasis syndrome;in addition,the number of targets of astragaloside IV,the main active component of Astragalus membranaceus,is the largest(19),and the number of targets of astragaloside IV and lumbrokinase is the largest in terms of anti-thrombosis.The core targets of Qilong capsule in the treatment of IS qi deficiency and blood stasis syndrome were screened by two steps(method is the same as 3.2.3),and the core targets of Qilong capsule in treating IS qi deficiency and blood stasis syndrome were obtained:SELP,MMP3,FGF2,JUN,FOS,HMOX1,PPARG,VCAM1,EGFR,PLG.GO biological process and Reactom pathway enrichment analysis were carried out on the correlation module between medicine and syndrome(same as 3.2.2).The results showed that Qilong capsule was involved in 30 biological processes in the treatment of IS with qi deficiency and blood stasis syndrome,including biological regulation,cell process,response to stimulation and anatomical development,including apoptosis(apoptoticprocess),regulation of programmed cell death(regulationofprogrammedcelldeath),inflammatory reaction(inflammatoryresponse)and so on.Participate in 9 signal pathways,including cell signal transduction,immune regulation,extracellular matrix and metabolism,including estrogen receptor signal pathway,EGFR signal pathway,PI3K/AKT signal pathway and so on.Conclusion:1.Qilong capsule may have certain advantages in improving neurological impairment,reducing the recurrence rate of IS,improving activities of daily living and improving hemorheology in patients with IS.2.The potential core target genes of IS qi deficiency and blood stasis syndrome may be:the molecular network mechanism of MMP9 and MAPK1,Qilong capsule in the treatment of IS qi deficiency and blood stasis syndrome may be related to anti-inflammation,anti-thrombosis,angiogenesis,anti-apoptosis and antioxidant stress,mainly through astragaloside,lumbrokinase and other active components to interfere with SELP,FGF2 and other targets,and regulate estrogen receptor signal pathway,PI3K/AKT signal pathway and so on.Innovation:1.The advantages of Qilong capsule in clinical treatment of IS were evaluated by systematic evaluation and Meta analysis,and from the point of view of the combination of TCM holistic view and disease syndrome,the molecular network mechanism of Qilong capsule in the treatment of IS with qi deficiency and blood stasis syndrome was explored by network pharmacology,which provided an objective basis for its clinical accurate localization and made its clinical location go deep into the molecular level.2.Through the computer prediction combined with the topological analysis of the module,it is concluded that the target PLG of astragaloside Ⅳ,the main active component of Qilong capsule,may be a new potential core target for the treatment of IS with qi deficiency and blood stasis syndrome,which is related to angiogenesis.