To Explore The Clinical And Basic Research On The Pharmacological Mechanism Of Lianxia Ningxin Decoction In The Treatment Of Coronary Heart Disease With Phlegm-heat Syndrome By Means Of Network Pharmacology

LianXia NingXin(LXNX)formula,a Chinese prescription,which targets the phlegm-heat syndrome of coronary heart disease(CHD).This formula does not only have good effects on CHD and chest pain which is the main symptom of CHD,but also on the co-morbid diseases and symptoms related to CHD phlegm-heat symptom.The drug mechanism of LXNX formula is characterized as "multi-component,multi-target and multi-pathway".Thus,it will be ex-tremely difficult for us to identify LXNX formula’s drug targets one by one from thousands of proteins through traditional method.Network pharmacology researches on the drug action refer to the effect of a complex net-work,which is consisted of the whole organic proteins and their interactions,to the drug targets.This advantage of network pharmacology is suitable for analyzing the multi-target drug.There-fore,using network pharmacology to analyze herbal prescriptions could result in rapid progress for TCM pharmacology.The researches in this thesis investigated the underlying molecular mechanisms of LXNX formula to treat CHD phlegm-heat syndrome for better understanding the LXNX formula actions and promoting the application of this formula.The thesis included four researches:Research One:Literature reviewIn this sector,the current state,research principle,the databases and tools to which com-monly were used,and the representive research cases of the mechanism researches on herb formula based on network pharmacology were reviewed,for providing the theory foundation of next researches.ofResearch Two:The prediction and verification the target which LXNX formula treated sympathetic remodeling after coronary heart disease based on the scale-free network analysis.Objective:Sympathetic remodeling is one of significant and lethal co-morbid diseases re-lated to CHD phlegm-heat syndrome,and can lead to sudden cardiac death and malignant ar-rhythmias.LXNX formula can prevent the sympathetic remodeling after CHD based on previ-ous researches.Therefore,further predicted and verified research into the exact pharmacologi-cal mechanism of action for LXNX formula to treat sympathetic remodeling post CHD are needed.Methods:the data curated from pharmacological databases and literatures was organized into an herbs-ingredients-targets relationship dataset for LXNX formula.The most important drug targets of LXNX formula were identified after analyzed the node degrees of each drug target.Based on literature review and the degree of nodes,the drug target of LXNX formula to treat sympathetic remodeling was predicted by evaluating both its importance in LXNX for-mula and its role in the formation of sympathetic remodeling.The pre-clinical research was performed to verify the prediction result.The Sprague-Dawley(SD)rats were randomly divided into the following four groups:sham surgery(sham),MI,LXNX,and a confirmed target inhib-itor groups.Coronary artery ligation was not performed for rats in the sham group;for the MI,LXNX,and confirmed target inhibitor groups,coronary artery ligation was performed.Rats in the sham and MI groups were administered normal saline by gavage daily;those in the con-firmed target inhibitor group were given the inhibitor by gavage daily;rats in the LXNX for-mula group were administered LXNX formula by gavage daily.Heart rate variability(HRV)was measured in all rats after 30 days of intervention.Further testing was done on the peri-infarct zone of the rats via tyrosine hydroxylase(TH)immunohistochemical staining method,and COX-2 protein and mRNA expression levels were measured using western blotting(WB)and quantitative reverse transcription-polymerase chain reaction(qRT-PCR).Results:COX-2 is the potential drug target of LXNX formula to treat sympathetic remod-eling based on the results of scale-free network analysis and literature research.Preclinical re-search indicated that Both LXNX formula and celecoxib(a confirmed COX-2 inhibitor)signif-icantly reduced distribution of sympathetic remodeling around the peri-infarct zone;the levels of COX-2 protein and gene expression in the myocardium of rats post-MI were significantly decreased by LXNX formula and celecoxib.Both LXNX formula and celecoxib were shown to decrease the low-frequency(LF)component of HRV,a positive marker for cardiac sympathetic tone,and increase total power(TP)of HRV,which represents overall heart rate variability pos-itively.Conclusion:As LXNX formula has the same effects as celecoxib on the rats after MI,this study indicates that COX-2is one of the key drug targets of LXNX formula through which it inhibits adverse sympathetic nerve sprouting and electric remodeling.Research Three:Identification of co-morbid diseases and symptoms related to LXNX for-mula to treat CHD phlegm-heat syndrome.Obj ective:The co-morbid diseases and symptoms which can be treated by LXNX formula while CHD phlegm-heat syndrome was treated can reflect the drug mechanism of LXNX for-mula through network pharmacology analysis.Therefore,this research would identify the co-morbid diseases and symptoms of LXNX formula by which CHD phlegm-heat syndrome was treated based on the case reports of Professor Li Ping and previous research results in order to laying good foundation to next research.Methods:The digital outpatient case history was collected.Using the method of the case-control study,the co-morbid disease and symptom difference between CHD phlegm-heat syn-drome and non-phlegm heat syndrome were observed,and the co-morbid symptom improve-ment of LXNX formula treatment group and non-LXNX formula treatment group was com-pared.Results:The clinical study showed that the co-morbid symptoms(i.e.insomnia,palpitation,bitter taste and anxiety)were more significantly appeared in the CHD phlegm-heat group than non-phlegm-heat group.The co-morbid symptoms(i.e.palpitation and dizzle)were more sig-nificantly improved in the LXNX formula treatment group than non-LXNX formula treatment group.Conclusions:Combined with the previous results,co-morbid diseases related to LXNX formula were heart sympathetic nerve remodeling,arrhythmia,insomnia,anxiety and auto-nomic nerve system.LXNX formula can relieve CHD co-morbid symptoms significantly(i.e.chest pain,shortness of breath,palpitation,thirst,bitter taste in mouth,dizziness,chest tightness,and dream-disturbed sleep).Research Four:The mechanism research on LXNX formula to treat CHD phlegh-heat syn-drome:a disease model analysis.Objectives:The different seed gene lists associated with the herbs of LXNX formula,the CHD co-morbid diseases and symptoms which were relieved by the LXNX formula(co-morbid diseases and symptoms)were curated manually from biomedical databases and published bio-medical literatures.Module enrichment analysis was used to identify CHD-related disease mod-ules in the protein-protein interaction(PPI)network which were also associated to the targets of LXNX formula(LXNX formula’s CHD modules).The molecular characteristics of LXNX formula’s CHD modules were investigated via functional enrichment analysis in terms of gene ontology and pathways.We performed shortest path analysis to explore the interactions be-tween the drug targets of LXNX formula and CHD related disease phenotypes(e.g.co-morbid diseases and symptoms).Results:In the disease modular analysis,we identified two significant CHD related dis-ease modules(i.e.M146 and M203),which were targeted by the herbs of LXNX formula.Pathway and GO term functional analysis results indicated that G-protein coupled receptor signaling pathways(GPCR)of M146 and cellular protein metabolic process of M203 are im-portant functional pathways for the respective module.This is further confirmed by the shortest path analysis between the drug targets of LXNX formula and the aforementioned disease mod-ules.In addition,corticotropin releasing hormone(CRH)and natriuretic peptide precursor A(NPPA)are the only two LXNX formula target proteins with the low shortest path length(on average shorter than 3)to their respective CHD module and co-morbid disease and symptom gene groups.Conclusions:G-protein coupled receptor signaling pathway and cellular protein metabolic process are the key LXNX formula’s pathways to treat CHD disease phenotypes,in which CRH and NPPA are the two key drug targets of LXNX formula.Further evidences from Chinese herb pharmacological databases indicate that Pinellia ternate(Banxia)has relatively strong adjustive functions on the two key targets.Due to COX-2,CRH and NPPA are inflammatory cytokines,the mechanism of LXNX formula to treat CHD phlegm-heat syndrome may be related to in-flammatory response.