To Investigate The Mechanism Of Qili Qiangxin Capsule Against Chronic Heart Failure Based On Network Pharmacology

Purpose: Qiliqiangxin Capsule(QLQX),composed of 11 kinds of Chinese herbal medicines,has been used in the treatment of chronic heart failure(CHF)in China,providing an effective treatment for CHF.QLQX has the characteristics of "multi-component,multi-target,multi-channel" treatment of traditional Chinese medicine,so it is difficult to study all the drug targets of QLQX one by one.Network pharmacology breaks through the concept of "one drug,one target" dominated by traditional pharmacology research,and coincides with the philosophical basis of systematic and holistic view of traditional Chinese medicine.In order to deeply understand the pharmacological mechanism of QLQX on chronic heart failure,we designed this study by combining network pharmacology with experiment.Material and method: 1.The target and mechanism of QLQX in the treatment of CHF were analyzed based on network pharmacology: QLQX is produced by Shijiazhuang Yiling Pharmaceutical Company Limited,and its herbal components include Astragalus mem branaceus,ginseng,aconite,Salvia miltiorrhiza,Tinglizi,Alisma orientalis,polygonatum odoratum,Ramulus Cinnamomi,safflower,cortex Periplocae and Pericarpium Citri Reticulatae.This study firstly used TCMSP,CNKI,Google Scholar and Pub Med to search the chemical components and targets of all Chinese herbal medicines in QLQX one by one,construct a QLQX component-target interaction network,and analyze the target using the cytoscape software module.In the network topology parameters,Rich Fun software is used to analyze the enrichment of the target in the organization,and Metascape online database is used to analyze the biological processes and molecular functions involved in the targets.Secondly,the online database was used to search the targets of Chronic heart failure,and the Venn map was used to obtain the targets of QLQX in the treatment of Chronic heart failure.The high expression of these targets in different cardiac cells was analyzed using the HUMAN PROTEIN ATLAS database,and to establish target-high expression cell type interaction network of QLQX-anti-Chronic heart failure.The biological processes of cardiomyocytes,fibroblasts,endothelial cells,immune cells and smooth muscle cells were analyzed by Metascape database to explain the regulation of QLQX on different cell types.Third,in order to study the potential biological mechanism of QLQX against Chronic heart failure at the level of protein-protein interaction,Go and KEGG enrichment analysis was performed on the core protein interaction network.2.Effect and mechanism of QLQX on cardiac remodeling and cardiac function in CHF mice: In order to verify the results of network pharmacology,we established a mouse model of chronic heart failure by AngiotensinⅡ(AngⅡ)intervene for 4 weeks.Sixty C57 mice were randomly divided into Control group,AngⅡgroup,AngⅡ+QLQX group and AngⅡ+Captopril group,15 mice in each group.The general state and blood pressure of mice were observed.High resolution small animal ultrasound was used to detect the cardiac structure and function of mice in each group,such as left ventricular systolic diameter(LVIDs),left ventricular diastolic diameter(LVIDd),left ventricular ejection fraction(EF%),fractional shortening(FS%).The serum and heart of mice in each group were extracted,and the heart was weighed to calculate the cardiac hypertrophy index,namely the ratio of heart weight to body weight(HW/BW)and the ratio of heart weight to tibia length(HW/HL).HE staining was used to observe the general morphology of the heart,WGA staining was used to observe the hypertrophy of myocardial cells,Masson and Sirius red staining was used to observe the cardiac fibrosis,and immunofluorescence staining was used to observe the inflammatory reaction in the heart.The expressions of BNP,IL-6,IL-1 β and TNF-a in serum were detected by ELISA to evaluate the severity of heart failure and the level of inflammation.RT-PCR and Western blot were used to detect the m RNA and protein expressions related to myocardial hypertrophy,fibrosis and inflammatory response pathway.Results: 1.The target and mechanism of QLQX in the treatment of CHF were analyzed based on network pharmacology: The results of the study showed that pharmacological studies yielded 700 targets for CHF and 239 targets for QLQX.QLQX targets are significantly enriched in human cardiomyocytes,endothelial cells,smooth muscle cells and immune cells,which may be involved in inflammatory reaction,negative regulation of cell adhesion,migration,apoptosis,and reactive oxygen species metabolism,indicating the potential therapeutic effect of QLQX on CHF.The target of QLQX was combined with the target of Chronic heart failure to obtain 99 overlapping targets,which have different high expression in different cells in the heart: Immunocytes(52),endothelial cells(51),cardiomyocytes(34),smooth muscle cells(33)and fibroblasts(31).We analyzed the biological process of these targets in cardiomyocytes,fibroblasts,endothelial cells,smooth muscle cells and immune cells.A protein-protein interaction network with 99 nodes and 1747 edges was obtained by topological screening.The average node degree value was 38,in which the node represented the target,the edge represented the association between two targets,and the degree value represented the intensity of the association.The top ten targets of correlation strength were Interleukin-6(IL-6),Albumin(ALB),Threonine kinase 1(AKT1),Vascular endothelial growth factor(VEGFA),Tumor necrosis factor(TNF),Nitric oxide synthase 3(NOS3),Epidermal growth factor(EGF),Mitogen activated protein kinase 8(MAPK8),Chemokine-8(CXCL8)and Endothelin 1(EDN1).It is suggested that they play an important role in QLQX anti CHF.By analyzing KEGG signaling pathway of nodes in the network,we found that the top 15 signaling pathways related to CHF were AGE-RAGE,IL-17,blood flow shear stress and atherosclerosis,HIF-1,TNF,NF-kappa B,c GMP-PKG,JAK-STAT,MAPK,AMPK,NOD like receptor,PI3K-Akt,TGF-β,Toll,and Cytokines and cytokine receptor signaling pathways.It is suggested that QLQX may play a role in the treatment of CHF through the above pathways.2.Effect and mechanism of QLQX on cardiac remodeling and cardiac function in CHF mice: 2.1 Blood pressure of mice: at the beginning of the experiment,there was no significant difference in systolic blood pressure and diastolic blood pressure of mice in each group.The systolic blood pressure of AngⅡgroup was significantly higher than that of Control group one week after pump embedding.After drug treatment,the systolic blood pressure of AngⅡ +QLQX group and AngⅡ+Captopril group was significantly lower than that of AngⅡgroup,and there was no significant difference between AngⅡ+QLQX group and AngⅡ+Captopril group.The trend of diastolic and systolic blood pressure in each group was consistent.It was suggested that QLQX could reduce the increase of blood pressure induced by AngⅡin mice.2.2 Cardiac function of mice: the cardiac function of mice in AngⅡgroup was significantly lower than that in Control group,which could be reflected by the decrease of EF and FS.After drug treatment,the EF value of AngⅡ+QLQX group was significantly higher than that of AngⅡgroup,and the EF value of AngⅡ+Captopril group was also significantly higher than that of AngⅡgroup.The EF value of AngⅡ+QLQX group was not significantly different from that of AngⅡ+Captopril group.At the same time,the trend of FS value and EF value was consistent.The level of BNP in serum of mice in each group also confirmed the results of ultrasound.It is suggested that QLQX can improve the cardiac function damage induced by AngⅡin mice.2.3 Myocardial hypertrophy in mice: the gross picture of the heart,HE staining and WGA staining showed that after Ang Ⅱ intervention,the heart of mice in Ang Ⅱ group was significantly larger than that in Control group,and QLQX or Captopril could alleviate the myocardial hypertrophy induced by AngⅡ.Cardiac hypertrophy index(HW/ BW,HW/TL)and cardiac hypertrophy index(ANP,BNP,MYH7 m RNA)also supported the above results.Western blot showed that QLQX alleviated AngⅡinduced cardiac hypertrophy by regulating ERK1 / 2 and AKT / STAT3.2.4 Myocardial fibrosis in mice: Masson and Sirius red staining showed that the degree of myocardial fibrosis in AngⅡgroup was significantly higher than that in Control group,and QLQX or Captopril could alleviate the myocardial fibrosis induced by Ang Ⅱ.The myocardial fibrosis indexes(TGF-β,SMA,CollagenⅠ,Collagen Ⅲ)detected by RT-PCR also supported the above results.Western blot showed that QLQX alleviated AngⅡinduced myocardial fibrosis by regulating TGF-β/Smad3 pathway.2.5 Serum and heart inflammation in mice: immunofluorescence staining showed that after AngⅡintervention,IL-6 in heart from the AngⅡgroup was significantly higher than that in the Control group,and QLQX or Captopril could alleviate the heart inflammation induced by AngⅡ.The inflammatory markers in heart(IL-6,IL-1 β,TNF-a)detected by RT-PCR and serum(IL-6,IL-1β,TNF-a)detected by ELISA also supported the above results.Western blot showed that QLQX alleviated Ang Ⅱ induced cardiac inflammation by regulating RAGE/NF-κ B pathway.Conclusion:1.In this study,we used the research mode of network pharmacology to visualize QLQX components,component targets,disease targets and distributed cell types,and analyzed the interaction relationship between different levels,so as to study the action characteristics of QLQX multi targets and the important signal pathways in biological networks.The top ten targets were IL-6,ALB,AKT1,VEGFA,TNF,NOS3,EGF,MAPK8 and CXCL.The important signaling pathways include AGE-RAGE,NF-κB,MAPK,Akt and TGF-β.2.Chronic heart failure mouse model was prepared and animal experiments were carried out in vivo.Through gross observation,histopathology and molecular biology methods,it was confirmed that QLQX improved cardiac remodeling and cardiac function damage induced by AngⅡin CHF mice,mainly through three aspects: myocardial hypertrophy pathway ERK1/ 2,AKT/STAT3,myocardial fibrosis pathway TGF-β/Smad3,inflammation pathway RAGE/ NF-κ B.The therapeutic characteristics of the prescription were "multi-component,multi-target and multi-channel".