| BackgroundWith the progress of precision medicine research,human diseases show an interrelated view:disease states are different by different clinical phenotypes,but different diseases may be caused by the dysfunction of the same target.Studying the relationship between diseases plays an important role in new drug development and drug repositioning.At present,cardiovascular death still accounts for the first total cause of death of urban and rural residents in China,and the social burden is heavy.Among them,ischemic cardiomyopathy(ICM)is the main cause of heart failure,with about 18 million people dying every year,accounting for 31%of all deaths,and traditional Chinese and Western medicine lack targeted drug treatment.While in the disease association,stable angina pectoris(SAP)and ICM belong to the category of stable coronary heart disease(SCAD),and there is a relationship between disease progression in pathological mechanisms,so it is of important clinical value to explore effective drugs for ischemic cardiomyopathy by studying the relationship between diseasesObjectiveBased on the paired disease progression module to explore the intrinsic correlation mechanism of SAP,ICM and chronic heart failure(CHF),screening the core prescription of ICM,through the preclinical efficacy and effect mechanism of prescription pharmacodynamic experiments and proteomics,then using RT-qPCR technology to verify the relationship between disease,explore the feasibility of broad-spectrum antiischemic drug development path based on the same target and pathway.Method1 Exploration of the relationships between diseasesBy integrating the data of multiple databases,paired disease progression module(PDPM)is constructed to identify the relationship between SAP,ICM and CHF.Finally,the three diseases were used to explore the relationship through functional enrichment analysis and literature validation in disease progression.2 Screening of core prescriptions for ischemic cardiomyopathyBased on the whole genome sequencing data of a previously known effective drug(Danhong Injection)in the treatment of SAP,using gene co-expression network and module pharmacology method found the effective treatment of ICM effective target,based on the target module screening potential treatment of ischemic cardiomyopathy.3 Experimental study of prescription pharmacodynamicsForty-six quarantine-eligible Beagle dogs were randomly divided into two groups,six in the normal group and 40 in the model control group.Model Control animals were ligated with a left anterior descending coronary artery,replicating a model of ischemic cardiomyopathy.Thirty-six animals were divided into model group,tongmai granule group,low,medium and high dose group and xiaoxintong group,with six animals in each group.Each drug administration group was administered at 5 mL/kg gavage,and the normal and model groups once a day for 8 weeks.Beagle dog weight was monitored weekly during drug administration.ECG tests and cardiac function-related indicators were performed before and at the 6 and 8 weeks of administration,including heart rate,cardiac output(CO),left ventricular end diastolic volume(LVEDV),left ventricular end diastolic pressure(LVEDP),left ventricular end systolic volume(LVESV),Left Ventricular Internal Diastolic Dimension(LVIDD),Left ventricular Posterior Wall Dimensions(LVPWD),Left ventricular Internal Dimension Systole(LVIDS),Left ventricular posterior wall systolic(LVPWS),ejection fraction(EF),Serum brain natriuretic peptide(BNP)and copeptin(CPP).The heart of each group was euthanized the next day after the last drug administration,and the organ coefficients were weighed and calculated.HE staining and Masson staining were performed to observe the myocardial cells,myocardial interstitial tissue structure and myocardial fibrosis level under light microscope.4 Target validation of efficacy mechanism and inter-disease relationshipThe day after the last administration,7 groups of 42 Beagle dogs with left ventricular myocardial tissue were removed,and DIA proteomics technology was used to screen for differential proteins and perform functional enrichment analysis of the differential proteins to explore the molecular mechanism of prescription efficacy.Based on the sample size of myocardial tissue,five dogs were selected from each group for RT-qPCR verification of the middle target by the disease progression module and the pathway interaction between different proteins with efficacy.Results1 Disease relationship between SAP,ICM and CHF:A total of 16 pairs of PDPMs(K>0.3777)were between SAP,ICM and CHF,including 6 pairs of SAP-ICM and 5 pairs of ICM-CHF and SAP-CHF.For the two phases of SAP-ICM and ICM-CHF,A total of 27 unique genes in the SAP-ICM stage,Where ICM modules 5 and 7 are unique modules(including ADAMTS9 and NPPA genes),Twenty-seven genes involved in inflammation,angiogenesis,fibrosis,apoptosis,and calcium homeostasis,It affects AGE-RAGE signaling,PI3K-Akt signaling,and HIF-1 signaling;A total of four unique genes in the ICM-CHF stage,The ICM modules 6 and 9 are unique to the modules(including the STAT1,ADORA1,and APLN genes),all of these involve inflammation.A total of 11 overlapping genes in SAP-ICM-CHF,Where modules 1 and 8 in the ICM are common modules,five of the genes involved(AGT,REN,CDH5,PGF,FLT1),overlapping 11 genes mainly involved in blood pressure,inflammation,angiogenesis,and electrolyte function,affects renin-angiotensin system,Rap1 signaling pathway,Chagas disease,and Pathways in cancer.2 Core prescription screening of ischemic cardiomyopathy:Zsummary<0 was used as the standard to screen the difference modules of Danhong injection in the treatment of stable angina pectoris.One difference module was screened out from the data of all the case groups treated by Danhong injection,among which related genes of 7 modules matched with 3 TCM related chemical components.Two difference modules were screened from the effective case group of Danhong injection,among which 3 genes in DHsub-Black module matched 2 chemical components related to Traditional Chinese medicine,and 10 genes in DHsub-Brown module matched 8 chemical components.3 Prescription pharmacodynamics experimental study①Effect of on ST segment:At the 8th week of administration,the tongmai granule group,low,medium and high dose group and xiaoxintong group were significantly reduced compared with the model control group(P<0.05).② Effect of on cardiac function:for 6 weeks,compared with the model control group,CO in medium and high doses and xiaoxintong groups were significantly increased(P<0.05),EF in low,medium and high doses(P<0.05),LVPWS and LVPWD in low doses(P<0.01),LVESV and LVIDD(P<0.05).Compared with the tongmai granule group,the CO was significantly increased in the medium dose group(P<0.05).At the eighth week of administration,EF increased in medium and high dose and xiaoxintong groups(P<0.05),LVESV and LVIDS were decreased(P<0.05),and LVPWS in medium and high dose groups(P<0.05).Compared with tongmai granule group,the heart rate was significantly reduced in the xiaoxintong group(P<0.01).③ Effect of on BNP and CPP:Compared with model control group,BNP and CPP in Tongmai granule group,Geshi Tongmai granule low-dose,medium-dose and high-dose groups and Xiaotong Tong granule group were significantly decreased(P<0.01).Compared with Tongmai Granule group,CPP of Geshi Tongmai granule low-dose group was significantly increased(P<0.05).④ For myocardial histopathology:Compared with model control group,the cardiac organ coefficients of tongmai granule group,Geshi Tongmai granule medium-dose and high-dose groups and Xiaotong Tong group were significantly decreased(P<0.05).HE staining and Masson staining showed that compared with the model control group,the range and degree of cardiac lesions in the geshitongmai granule high-dose group and xiaotongxin group were significantly reduced.Pathological grading results showed that compared with model control group,the degree of myocardial inflammatory infiltration and myocardial tissue fibrosis in geshitongmai granule high-dose group and Xiaotongxin group were significantly reduced(P<0.05).4.Study on therapeutic mechanism:Taking the low-dose group as the control,there were 5 overlapping differential proteins in the normal group,model group,Tongmai Granule group and Xiaotong Tong group,respectively COL14A1,ATG9A,MYOM1,PDIA5 and OAT.Four overlapping differential proteins were found in the medium-dose group as control,which were KRT2,PPP1R13B,HNRNPUL1 and ANKFY1.Nine overlapping differential proteins were found in the high-dose group as control,which were PAFAH1B2,RO60,OXSR1,PPP1R12C,ME3,MRPS18B,PAFAH1B3,COPS6 and F1PYE8.There was an overlapping differential protein TGFB1I1 compared with each dose group.The construction of PPI network showed that OAT,PPP1R12C and TGFB1I1 were overlapped differential proteins in low-dose group and high-dose group,respectively.Compared with model group,low dose group differences in protein mainly affects human cytomegalovirus infection and human papillomavirus(HPV)infection pathways,dosage group differences in protein main influencing oxytocin signaling pathways,high dose group differences in protein main influence vascular smooth muscle contraction channel,including vascular smooth muscle contraction pathway in high dose group for comparison,Common pathway of differential protein enrichment in normal group,model group,Tongmai granule group and Xiaotong Tong group.Compared with the low-dose group,the difference proteins in the medium-dose group and the high-dose group were mainly involved in the adhesion patch pathway,while the difference proteins in the high-dose group were involved in the spliceosomal pathway.5.Target verification of PDPMs:Rt-qPCR was used to verify the target of PDPMs,and it was found that compared with the model group,the expression levels of AGT and NPPA genes in geshitongmai low-dose group,high-dose group and Xiaotong group showed an upward trend,and the expression levels of AGT genes in Geshitongmai medium-dose group also showed an upward trend,with statistical significance(P<0.05).Compared with the normal group,The expression levels of AGT and NPPA genes in geshitongmai low-dose group,medium-dose group,high-dose group and Xiaotong group showed an upward trend,with statistical significance(P<0.05).The expression levels of ADORA1 gene in model group and low-dose group showed an upward trend,with statistical significance(P<0.05).Genes(AGT,NPPA,ADORA1)have been identified to be involved in the renin-angiotensin-aldosterone system,and these three genes and the differential protein PPP1R12C affect the vascular smooth muscle contraction pathway and oxytocin signaling pathway.Four pathways of differential protein enrichment overlapped with SAP-ICM stage in the low-dose group VS model group,accounting for 66.67%(4/6)of the total pathways in SAP-ICM stage,including malaria signaling pathway,flassin signaling pathway,Kaposi sarcoma-associated herpes virus infection pathway,and human papillomavirus infection pathway.In the low-dose vs model group,two pathways of differential protein enrichment overlapped with SAP-ICM-CHF,and 50%(2/4)were Rapl signaling pathway and cancer signaling pathway respectively.Compared with model group,the overlap rate of differentially overlapped proteins in each dose group was 11.11%(3/27)with SAP-ICM stage genes(ICAM1,FN1,TIMP1),and 16.67%(1/6)with ICM-CHF stage genes(STAT1).The overlap rate with SAP-ICM-CHF stage was 36.36%(4/11),and the overlapping genes were AGT,FLT1,CRP and CDH5.Conclusion1 By using PDPM method,we found that inflammation is a factor involved in genes at different stages of the three diseases,among which the SAP-ICM-CHF gene is also involved in angiogenesis,blood pressure and electrolytes,mainly affecting the renin-angiotensin-aldosterone system.In addition to inflammation and angiogenesis,the genes in SAP-ICM stage are also involved in fibrosis,apoptosis,calcium homeostasis and other functions,mainly affecting the age-rage signaling pathway.2 Geshitongmai granules can significantly reduce ST segment in ecg of Beagle dogs with ischemic cardiomyopathy and improve cardiac function and myocardial tissue fibrosis degree;At the same time,the low dose was the effective dose,the high dose had the best effect,and the middle dose had the same effect as the positive drug Tongmai granule.3 Differential proteins in the low-dose group of Geshitongmai granules mainly affected the pathways of human cytomegalovirus infection and human papillomavirus infection;The differential proteins in the medium dose group mainly affected the oxytocin signaling pathway.Differential proteins in the high-dose group mainly affect the vascular smooth muscle contraction pathway,which is also the common pathway for the enrichment of differential proteins in the high-dose group as the control.Compared with the low-dose group,the difference proteins in the medium-dose group and high-dose group were mainly involved in the adhesion patch pathway,while the difference proteins in the high-dose group were involved in the spliceosomal pathway.4 Through the gene and pathway coverage and RT-qPCR verification,Geshitongmai granules can act on multiple targets and pathways at various stages of SAP,ICM and CHF,and have effects on three diseases with ischemic pathologic nature.It is suggested that it is feasible to explore the development path of broad spectrum anti-ischemia drugs based on the same target and pathway,and provide a kind of thinking for the scientific connotation of"treating different diseases with the same treatment" in TCM. |
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