"TCM-network-effects" Integration Approach And Its Application In Mechanism Research Of Yixinshu Capsules And Other Chinese Medicine

BackgroundTraditional Chinese medicine prescriptions(TCMP)are made from different traditional Chinese medicines refer based on the guidance of Chinese medicine theory.TCMP have been widely used because of their good clinical curative effect and less side effects,and have become the main forms and means of clinical treatment of traditional Chinese medicine.However,due to the complex composition of TCMP,it restricts the in-depth study of its mechanism of action,affecting its clinical application.In order to solve the problem of vague clinical orientation and unclear mechanisms of TCMP,this study proposes an integrated pattern of "Traditional Chinese medicine(TCM)-network-effects" through the study of Yixinshu capsule(YXS)the representative TCMP,which systematically analyzed the molecular network relatedness with the mechanism of action and clinical application characteristics of YXS,and extended this pattern to study other representative TCMP,Xiaoerfupi granules(XEFP).In this study,we took YXS as an example to propose and validate the feasibility of research pattern of "TCM-network-effects".YXS is a preparation developed on the basis of "Sheng Mai San" from "Neiwai shangbian huolun",and is one of the representative drugs for TCMP.YXS consists of seven common Chinese herbal medicines,Panax ginseng,'Fructus Schisandra chinensis,Astragalus membranaceus,Radix Salviae miltiorrhizae,Radix Ophiopogonis,Rhizoma Chuanxiong,and Fructus Crataegi.YXS has been clinically applied to cardiovascular diseases such as chronic heart failure(HF),cardiac neurosis,coronary heart disease and angina pectoris.However,most studies are limited to clinical observations of YXS for treatments of heart failure,and the molecular mechanism of YXS intervention in heart failure has not yet been studied.This restricts the clinical positioning and precise application of YXS.Therefore,it is necessary to carry out researches on the role of YXS intervention in heart failure.In addition,this pattern of "TCM-network-effects" was extended to be applied to the study XEFP.XEFP is a representative TCMP derived "Shen-Ling-Bai-Zhu-San".XEFP is mainly composed of Rhizoma Atractylodis Macrocephalae(Bai zhu),Pericarpium Citri Reticulatae(CHEN PI),Sclerotium Poriae Cocos(Fu ling),Semen Nelumbinis Nuciferae(Lian zi),Codonopsis pilosula(Dang shen),Hawthorn Fruit(Shan zha).XEFP has the effects of strengthening spleen-stomach and improving digestive function,and clinically used to treat children with spleen and stomach qi deficiency,indigestion.However,most of the studies are limited to XEFP interventions in clinical observations of anorexia in children,and the molecular mechanisms of XEFP intervention in functional dyspepsia(FD)have not been studied.Therefore,it is necessary to extend the research pattern of "TCM-network-effects" to the analysis of the role of XEFP in FD.Therefore,in order to solve the problem of the unclear clinical positioning and the unclear mechanism of action of YXS and XEFP,we proposes a pattern of "TCM-network-effects",which uses the network pharmacology analysis of the chemical constituents of TCM,combined with proteomics,establishing a network of TCM and biological effects,selecting appropriate pharmacological models,revealing the interaction between TCM components,targets and the biological significance of each node,clarifying their complex mechanisms of action,and guiding clinical orientation.The research contentPart I Analytical study on effect of Yixinshu capsule on heart failure based on integration pattern of“TCM-network-effects"Firstly,based on the network pharmacology strategy,the chemical composition,molecular targets and disease network of YXS were analyzed,and the effect of YXS intervention on heart failure was investigated through in vivo and in vitro heart failure models.On this basis,through the detection of hippocampal tissue in rats with heart failure,we investigated the effects of YXS hippocampal tissue dysfunction induced by heart failure.1 Based on BATMAN-TCM database,334 chemical components related to YXS and corresponding 1874 molecular targets were extracted.Combining the analysis of the DAVID database and the STRING database,the YXS network of chemical components,molecular targets,and diseases was obtained.The enrichment results revealed that YXS may be associated with atherosclerosis,heart failure,depression,and anxiety disorders.Through the three dimensions of TCM theory,YXS clinical experience and bioinformatics analysis,we focused on the efficacy and mechanism of YXS intervention in heart failure.2 Rats of heart failure model were established by ligation of the left anterior descending coronary artery,and were detected by echocardiography and biochemical indicators.It was found that continuous intervention of YXS for 6 weeks could significantly reduce-ventricular enlargement,increase left ventricular ejection and significantly reduce the expression levels of BNP and NT-pro BNP in rats with heart failure.The hiPS-CMs(human iPS cell-derived cardiomyocytes)of heart failure model induced by endothelin-1(ET-1)were detected BNP levels.The results show that YXS can improve BNP expression levels in hiPS-CMs heart failure model.The above results indicate that YXS can improve cardiac function in heart failure rats.3 Proteomics methods were used to quantitatively compare the proteomic characteristics of hiPS-CMs models induced by ET-1 under different intervention conditions,enrich biological signaling pathways and key interacting proteins,and select the potential molecular targets of action of YXS intervention in heart failure.The potential molecular targets of action are:NPPA(natriuretic peptide precursor A),TNNI3(troponin I type 3,cardiac),GAL-3(galectin-3),HSP 70(Heat shock protein 70),FABP3(heart-type fatty acid-binding protein 3),ACLY(ATP-citrate synthase),and CKAP5(Cytoskeleton-associated protein 5).The ELISA method was used to verify the changes in the in vitro and in vivo models of heart failure.Combined with the network pharmacology analysis,a map of the molecular target network of YXS interaction and its corresponding effector molecule was constructed,suggesting that YXS may play a role in the intervention of heart failure through molecular networks(BNP,TNNI3,GAL-3,FABP3,and CKAP5).4 Based on the results of network pharmacology,evans blue was used to detect the permeability of the blood-brain barrier in hippocampus,and Western Blot was used to detect the expression of proteins ZO-1 and AQP4 related to permeability of blood-brain barrier.It was found that YXS can ameliorate the abnormal blood-brain barrier of the hippocampus induced by the heart failure.ELISA was used to detecte the expression levels of IL-1 beta,TNF-a,and IL-6 in hippocampal tissue.The results indicated that YXS can improve the expression of hippocampal inflammatory factors induecd by heart failure.Using network pharmacology to further analyze YXS chemical constituents and molecular targets,we enriched the peroxisome proliferator-activated receptor(PPAR)signaling pathway.Then,using the Western Blot method,we verified that YXS may reduce hippocampus through intervention of PPARy signaling pathway.Part II Analytical study on effect of xiaoerfupi granules on functional dyspepsia based on integration pattern of "TCM-network-effects"Based on the "TCM-network-effects" integration pattern,this study applied this integration pattern to the research of XEFP.1 Based on the BATMAN-TCM database,113 XEFP-related chemical components and corresponding 456 molecular targets were extracted.Combining the analysis of the DAVID database and the STRING database,the network relationship of XEFP chemical components,molecular targets,and diseases was obtained.It was found that XEFP may be associated with diseases such as functional dyspepsia(FD).Through the three dimensions of TCM theory,XEFP clinical experience and bioinformatics analysis,this study focused on the efficacy and mechanism of XEFP intervention in functional dyspepsia.2 A rat model of functional dyspepsia was established by iodoethylamine and overnight fasting.After drug intervention for 3 weeks,the gastric emptying function,salivary amylase,gastrin,and nitric oxide synthase levels in the serum were measured.The results showed that XEFP can significantly improve the symptoms of FD.3 Based on the study of pharmacodynamics,a proteomic study of gastric tissue was conducted and a total of 4045 proteins were identified.The use of network pharmacology to analyze XEFP chemical components and molecular targets enriches two key targets of HTR3A and c-FOS.Combined with Western Blot verification,it was found that XEFP may interfere with functional dyspepsia by regulating HTR3A and c-FOS.Part ? Screening the key transcription factor target and its effect of Danhong injection against ischemic strokeDuring my PhD period,I also conducted some other research work.Based on the "Chinese medicine-network-effects" pattern,the preliminary study in our research lab has obtained the efficacy and proteomic data of Danhong injection(DHI)anti-ischemic stroke.The purpose of this study is to primary screen the key transcription factors on ischemic stroke with DHI intervention through molecular biology experiments.1 Western Blot and EMSA experiments were used to screen out the key transcription factors SATB1 and POU3F1 for anti-ischemic stroke.Combined with literature information,this study focused on the transcription factor SATB1.2 Bioinformatics predictions suggest that there may be interacting targets of SATB1 with Bcl-2 and IL-4;Bcl-2 and IL-4 were detected by real-time fluorescence PCR,and the resulsts suggest that SATB1 may regulate Bcl-2 and IL-4 to exert the effects of anti-ischemic stroke.To sum up,this study was to investigate the representative TCMP,Yixin Capsule(YXS)and XEFP)based on the integrated pattern of "TCM-network-effects",and the analysis of the association between molecular groups and molecular networks,systematically analyzed theirs mechanisms of action and clinical application characteristics.The innovation points of the study:1 Based on the research pattern of "TCM-network-effects" integration,through research on the mechanism of YXS intervention in heart failure,it was revealed that YXS may interfere with heart failure through molecular target networks(BNP,TNNI3,GAL-3,FABP3,and CKAP5).2 It was found that YXS can improve the effects of hippocampal abnormalitiesinduced by heart failure and reveal that it may exert its effect through the effects on PPARy signaling pathways.3 The role of XEFP in the intervention of functional dyspepsia was clarified and it was found that XEFP may exert functional dyspepsia through HTR3A and c-FOS targets.