| Background:Heart failure is a serious terminal stage of various cardiac diseases,and it is also a difficult and hot point in domestic and international research because of its increasing morbidity,high mortality and high rehospitalization rate.The pathogenesis of heart failure involves many aspects,energy metabolic remodeling is a key factor in the occurrence and development of heart failure.This study based on YQHXLSF effect on energy metabolism regulation,use clinical trial evaluates the efficacy of YQHXLSF,use network pharmacology technology to forecast the targets of regulating energy metabolism in the treatment of heart failure and verify the mechanism of YQHXLSF regulating energy metabolism by animal experiments to research the mechanism of YQHXLSF in the treatment of heart failure,and provide a new research idea for the prevention and treatment of heart failure with Traditional Chinese Medicine(TCM).Objective:1 To evaluate the clinical effect of Yiqi Huoxue Lishui Fang(YQHXLSF)on chronic heart failure(CHF)patients with qi deficiency,blood stasis and water stasis syndrome,and its effect on left ventricular systolic function.2 To predict the mechanism of YQHXLSF in the treatment of chronic heart failure and the signaling pathway involved in regulating energy metabolism by network pharmacology.3 To investigate whether YQHXLSF can protect mitochondrial structure,improve mitochondrial respiratory function and regulate mitochondrial energy metabolism to treat heart failure by activating AMPK/PGC-1αsignaling pathway.Methods:1 Clinical trial(1)This clinical trial was a randomized controlled study,patients with CHF were randomly divided into control group and treatment group.(2)In the control group,patients will be treated with conventional western medicine;In the treatment group,patients will be treated with YQHXLSF on the basis of conventional western medicine.(3)After 4 weeks of treatment,observe the changes of TCM symptom score,TCM syndrome score,Lee’s heart failure score,NYHA heart function class,NT-proBNP and LVEF before and after treatment in the 2 groups.GLS was detected by using two dimensional speckle tracing to evaluate cardiac systolic function.2 Network pharmacology research(1)Components and targets of YQHXLSF were retrieved from the TCMSP database.Heart failure related genes were summarized from GeneCards and OMIM databases.Use Venn diagram to obtain the target of YQHXLSF in the treatment of chronic heart failure.(2)Use Cytoscape software to construct the network diagrams of the disease-drug-active component-action target.Use the string online database to establish the protein-protein interaction network in order to obtain core targets.(3)GO and KEGG pathway enrichment analyses were performed on the intersection targets of drugs and diseases using DAVID database.3 Animal experiment(1)Establish the heart failure after myocardial infarction in model of SD rats by ligating the anterior descending branch of left coronary artery.The study rats were divided up into 5 groups:①Sham;②Model;③TMZ;④YQHXLSF-M;⑤YQHXLSF-H.Distilled water,TMZ and YQHXLSF were given for intervention.(2)After 4 weeks of continuous intervention,record 24h urine volume of each group.Echocardiography was used to examine cardiac structure and function.Use HE stain and Masson stain to analyze myocardial histopathological changes and TUNEL stain to determine myocardial apoptosis.(3)Respective ELISA kits were used to examine the ROS content,the activity of mitochondrial respiratory chain complexes,mitochondrial membrane potential and ATP content.Use transmission electron microscope to observe the mitochondrial ultrastructure.(4)Use Western Blot analysis the expression levels of AMPK/PGC-1α signaling pathway-associated proteins.Use Real-time PCR analysis mtDNA copy number and the mRNA expression levels of AMPK/PGC-1α signaling pathway.Results:1 Clinical trial(1)After treatment,the scores of TCM symptom,TCM syndrome and Lee’s heart failure score in the 2 groups were decreased compared with that before treatment(P<0.01);The decrease degree of total score of shortness of breath,fatigue,chest tightness,facial edema,TCM syndrome and Lee’s heart failure score in the treatment group was better than that in the control group(P<0.05,P<0.01);The curative effect of TCM syndrome score,Lee’s heart failure score and the improvement of NYHA heart function class in treatment group was better than that in control group(P<0.05,P<0.01).(2)After treatment,LVEF in 2 groups was improved(P<0.05,P<0.01);The levels of NT-proBNP and GLS in the treatment group were improved compared with those before treatment(P<0.01);The improvement effect of LVEF,GLS and the decrease rate of NT-proBNP in the treatment group were better than those in the control group(P<0.05,P<0.01).(3)GLS has positively correlated with NT-proBNP,LVDd,LVDs,TCM syndrome score and NYHA heart function class(P<0.05,P<0.01),it also has negatively correlated with LVEF and LVFS(P<0.01).2 Network pharmacology research(1)116 active ingredients and 205 targets of YQHXLSF in the treatment of CHF were screened out.(2)Construct the network diagrams of the disease-drug-active component-action target and screen out the key active ingredients of YQHXLSF;Core target genes include AKT1,JUN,MAPK1,RELA,APP,CTNNB1,NFκB1,CCND1,IL-6.(3)Enrichment analysis obtained 158 pathways of YQHXLSF in the treatment of chronic heart failure,mainly focusing on energy metabolism,glucolipid metabolism,oxidative phosphorylation,mitochondria,inflammatory response,DNA replication and repair,translation,signal transduction,apoptosis and autophagy.The main pathways involved in energy regulation are PI3K/Akt signaling pathway,AMPK signaling pathway and cAMP signaling pathway.3 Animal experiment(1)24h urine volume:Compared with Sham group,24-hour urine volume in Model group was significantly decreased(P<0.01).Compared with Model group,24-hour urine volume in YQHXLSF-M and YQHXLSF-H group was significantly increased(P<0.01).(2)Echocardiography results:Compared with Sham group,LVDd and LVDs in Model group were increased(P<0.01),LVPWTd,LVPWTs,LVEF,LVFS in Model group were significantly decreased(P<0.01).Compared with Model group,LVDd,LVDs in YQHXLSF-M and YQHXLSF-H groups were improved(P<0.05,P<0.01),and LVEF,LVFS were significantly increased(P<0.05).(3)Cardiac morphology and the pathomorphological changes in rats:In the Model group,the shape of heart in the Model group was irregular with thinner ventricular,light red color and decreased elasticity compared with Sham group.In YQHXLSF-M and YQHXLSF-H groups,the shape,color and elasticity of heart were improved.HE staining results showed that:Compared with Sham group,In Model group,at the edge of infarction the number of myocardial cells area in Model groupwas reduced and the nucleus was missing.Myocardial cell was observed with hypertrophy,edema,loose cytoplasm and a large number of vacuoles.Myocardial fiber fracture and inflammatory cell infiltration were observed in the interstitium;In YQHXLSF-M and YQHXLSF-H groups,the morphological changes of myocardial cell hyperplasia and edema at the edge of infarction area were improved,and the myocardial fiber fracture,disorder of ordering and inflammatory cell infiltration were alleviated.(4)The extent of myocardial fibrosis in rats:Masson staining results showed that:In Model group,normal myocardial cells were reduced,myocardial cells were distributed,myocardial cell structure and morphology were changed,myocardial cells were disordered,and the interstitium of cells was filled with blue stained collagen fibers compared with Sham group.In YQHXLSF-M and YQHXLSF-H groups,the changes of myocardial cell morphology and structure were improved,the collagen fiber deposition was improved.(5)Apoptosis of cardiomyocytes in rats:TUNEL staining results showed that:Compared with Sham group,the apoptotic Index increased significantly in Model group(P<0.01).Compared with Model group,the apoptotic Index decreased significantly in TMZ group,YQHXLSF-M group and YQHXLSF-H group(P<0.05).(6)ROS in rats:Compared with Sham group,the peak of Model group moved to the right,and the ROS content in cardiomyocytes increased significantly(P<0.01);Compared with Model group,the peak of YQHXLSF-M and YQHXLSF-H groups moved to the left,and the ROS content in cardiomyocytes decreased significantly(P<0.01).(7)The ultrastructure of the myocardial mitochondria:Compared with Sham group,the number of myocardial fibers was reduced,the arrangement was disordered,the intercalation disc was blurred or disappeared,the number of adhesion between mitochondria was reduced,the mitochondria was swollen and deformed,the internal crists were deformed and the internal were vacuolated in Model group.The myocardial cells of YQHXLSF-M and YQHXLSF-H groups were arranged orderly,the number of mitochondria was increased,and the disorder of crest morphology was improved.(8)Activity of respiratory chain complex in mitochondria:Compared with Sham group,the enzymatic activity of mitochondrial respiratory chain complexes(Ⅰ-Ⅳ)was decreased in model group(P<0.05);The enzymatic activity of mitochondrial respiratory chain complexes(Ⅰ-Ⅳ)was increased in YQHXLSF-M and YQHXLSF-H groups(P<0.01).(9)Mitochondrial membrane potential:Compared with Sham group,the mitochondrial membrane potential in Model group was decreased(P<0.01).In YQHXLSF-H group,the mitochondrial membrane potential was increased compared with Model group(P<0.05).(10)Mitochondrial ATP production:Compared with Sham group,mitochondrial ATP production in Model group was decreased(P<0.01).Compared with Model group,mitochondrial ATP production in YQHXLSF-M and YQHXLSF-H groups was increased(P<0.01).(11)Regulation of YQHXLSF on AMPK/PGC-1α signaling pathway in rats:Western Blot results showed that:In Model group,AMPK protein expression was not changed compared with Sham group(P>0.05),p-AMPK,PGC-1α,NRF-1 and TFAM protein expression were decreased(P<0.05,P<0.01).After treatment with YQHXLSF(P<0.05,P<0.01).Real-time PCR results showed that:Compared with Sham group,mtDNA copy number and mRNA expression of AMPK,PGC-1α,NRF-1 and TFAM in Model group were significantly decreased(P<0.05,P<0.01).In YQHXLSF-M and YQHXLSF-H groups,mtDNA copy number and the mRNA expression of AMPK,PGC-1α,NRF-1 and TFAM were significantly increased compared with Model group(P<0.05,P<0.01).Conclusion:1 YQHXLSF can reduce NT-proBNP and GLS,increase LVEF and improve the clinical symptoms of CHF patients with qi deficiency,blood stasis and water stasis syndrome.GLS has a certain correlation with the cardiac function and condition of CHF patients,it can be considered as an auxiliary indicator to evaluate the condition and efficacy of CHF patients in clinic.2 YQHXLSF contains 116 active ingredients and involving 205 targets.The mechanism of action involves many aspects,it involved in energy regulation include PI3K/Akt signaling pathway,AMPK signaling pathway and cAMP signaling pathway.3 YQHXLSF has a diuretic effect,and its effect on maintaining the stability of mitochondrial structure and function,promoting mitochondrial biogenesis,regulating mitochondrial energy metabolism,increasing ATP production,and improving the structure and function of failing heart may be related to the activation of AMPK/PGC-1α signaling pathway. |
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