| As one of the chronic and refractory diseases which seriously threat human health,chronic heart failure is a complex pathological process in which many factors interact with each other.The value of clinical guidance of traditional Chinese medicine has become more obvious.The vessels-collateral theory of traditional Chinese medicine guides the research on prevention and control of chronic heart failure,and points out that lost balance of ying wei and disorder of activities of qi was the basic pathogenesis.Lack of warmness of yang qi,weak blood transportation,disordered qi of tertiary collateral at the end of collaterals,fluid and blood exchange barrier causes phlegm and blood stasis accumulation in collateral.Body fluid cannot return and gather outside the collaterals,leading to “qi separation”,“blood separation” and “water separation” lesion.On this basis,the principle of “common treatment and separated elimination of qi,blood and water” and the treatment method of “reinforcing qi and warming yang,promoting blood circulation to remove meridian obstruction,and inducing diuresis to alleviate edema” were established,and Qiliqiangxin Capsule composition was developed.The research chose representative Compound Qiliqiangxin Capsule theory as intervention drug,and revealed its intervention effect and related mechanism on Cardiac function,RAAS system,myocardial microvascular,and renal water metabolism and others in rats with chronic heart failure.At the same time,it used the metabonomics research methods and bioinformatics analysis technology to study the correlation between endogenous metabolites in serum and therapeutic effects,and provide methodology for the modern study of traditional Chinese medicine,and provide the support of research data to the scientific value of the doctrine of traditional Chinese medicine.Part I Study on the Effect of Qiliqiangxin Capsule on Cardiac Function and Mechanism with Heart Failure after Myocardial InfarctionObjective: To explore Qiliqiangxin Capsule's influence on cardiac function,myocardial microvascular and renal aquaporin in rats with chronic cardiac failure.Methods: The method of ligation of left anterior descending coronary artery was used to establish the model of rats with heart failure after acute myocardial infarction(AMI).The decline of ST segment of electrocardiogram(ECG)before and after ligation was used to evaluate whether AMI model was successful.After 4 weeks,the left ventricular ejection fraction less than 45% in ultrasonic cardiogram of small animals was used to judge that the heart failure model was successful.After 4 weeks,6 weeks and 8 weeks,the change of cardiac function,NT-proBNP and RAAS system level was detected.After the intervention of 8 weeks,the ultrastructure of myocardial tissue,myocardial fibrosis and other changes of tissue morphology were observed;with ELISA method,the level of NRG1 and AngPTL4 at different time points was tested.After 8 weeks of intervention,myocardial microvascular endothelial ultrastructure,and protein/gene expression of NRG1 and Ang PTL4 in myocardial tissue revealed that Compound Qiliqiangxin Capsule could protect cardiac microvessels;through the testing of calcitonin generelated peptide(CGRP)level in plasma and protein/gene expression of eNOS/ guanylate cyclase(cGMP)/ protein kinase G(PKG)at different time points,it was further verified whether vasodilatation of Qiliqiangxin Capsule was related to NO-mediated cGMP-protein kinase system approach;and through the testing of the level of arginine vasopressin in plasma(AVP),and protein/gene expression of V2 R and AQP2 of kidney receptor,the influence of Qiliqiangxin Capsule on water retention of kidney was observed.Results: 1 It was confirmed that Qiliqiangxin Capsule could inhibit ventricular remodeling and activation of RAAS system,reduce myocardial cell injury,improve heart function,delay the course of heart failure and improve prognosis in rats with chronic heart failure.Results of ultrasonic cardiogram showed that Qiliqiangxin Capsule significantly reduced ventricular cavity,improved the ventricular wall motion coordination,increased the ventricular systolic and diastolic amplitude,increased blood flow,effectively reduced the left ventricular end-systolic and end-diastolic diameter and volume,and increased left ventricular ejection fraction in rats with heart failure.It showed that Qiliqiangxin Capsule could improve cardiac function in rats with heart failure,and with the passage of time the effect was remarkable.Results of projection electron microscopy showed that Qiliqiangxin Capsule significantly reduced pathological injury of myocardial tissue,inhibited cardiomyocyte hypertrophy and inflammatory cell infiltration,protected myocardial tissue ultrastructure,and inhibited collagen proliferation in rats with chronic heart failure.It showed that Qiliqiangxin Capsule could protect myocardial cell structure,and inhibit ventricular remodeling of chronic heart failure.Results of detection of NT-proBNP level in plasma showed that compared with the Sham Group the level of NT-proBNP in plasma of rats of Model Group significantly increased at different time points(P<0.01);compared with the Model Group,the level of NT-proBNP in plasma of QLQX Group reduced with time-dependent(P<0.05,P<0.01).Results of detection of RAAS system level in plasma showed that compared with the Sham Group the level of RI,Ang? and ALD in plasma of rats of Model Group significantly increased at different time points(P<0.01),and with the passage of time RI,Ang? and ALD gradually increased;compared with the Model Group,the level of RI,Ang? and ALD in plasma of QLQX Group reduced in different degrees(P<0.05,P<0.01).2 It was revealed that Qiliqiangxin Capsule could protect myocardial microvascular structure,and the vasodilation mechanism might be related to eNOS/sGC/c GMP/PKG pathway.Results of the transmission electron microscopy(TEM)showed that rats of Model Group had serious damage of cardiac microvascular endothelial cells,damage of basilar membrane and linking protein,and capillary lumen stenosis or deformity.Qiliqiangxin Capsule could significantly protect linking protein,and improve the ultrastructure of microvascular endothelial cells.Results of detection of NRG1 and AngPTL4 level in plasma and myocardial tissue showed that compared with the Sham Group the level of NRG1 and Ang PTL4 significantly reduced,protein and gene expression of NRG1,and Ang PTL4 in myocardial tissue decreased significantly(P<0.05,P<0.01),and with the passage of time the level of NRG1 and Ang PTL4 in plasma continuously reduced in rats of the Model Group;compared with the Model Group,the level of NRG1 and AngPTL4 in plasma of QLQX Group increased in different degrees,protein and gene level of NRG1 and AngPTL4 in myocardial tissue increased(P<0.05,P<0.01),and with the passage of time the level of NRG1 and AngPTL4 in plasma significantly increased.Results of detection of the calcitonin generelated peptide(CGRP)level in plasma and aorta level showed that compared with the Sham Group the CGRP level in plasma and aorta in rats of the Model Group significantly reduced(P<0.01);compared with the Model Group,the CGRP level in plasma and aorta of QLQX elevated Group increased(P<0.05,P<0.01).Results of detection of eNOS/ sGC/ cGMP/ PKG pathway showed that compared with the Sham Group CGRP and eNOS protein expression in aorta,and s GC and PKG mRNA expression significantly reduced in rats of Model Group(P<0.01);compared with the Model Group,CGRP and eNOS protein level and sGC and PKG mRNA level significantly increased in QLQX Group(P<0.05,P<0.01).3 It was revealed that collateral-dredging compound Qiliqiangxin Capsule improved water metabolism of kidney by reducing arginine vasopressin(AVP)and V2 R and AQP2 level of kidney.Results of detection of AVP level in plasma and analysis on its correlation with RAAS system showed that compared with the Sham Group,AVP level in plasma in rats of the Model Group significantly increased at different time points,and with the passage of time,AVP in plasma always maintained a high level(P<0.01);compared with the Model Group,AVP level in plasma of QLQX Group reduced in different degrees(P<0.05,P<0.01).The correlation analysis showed that AVP level and AngII level in plasma had significantly positive correlation(P<0.001).Results of detection of V2 R and AQP2 expression of kidney showed that compared with the Sham Group V2 R and AQP2 protein and mRNA expression significantly increased in rats of the Model Group(P<0.01);compared with the Model Group,V2 R and AQP2 protein and mRNA level of QLQX Group reduced in different degrees(P<0.05,P<0.01).Summary:In the process of onset and development of chronic heart failure,microvascular endothelium and myocardial cells are damaged,RAAS system is activated,vascular resistance increases,blood flow is insufficient,ventricular cavity expands,ventricles are remodeled,ventricular wall has poor coordination,cardiac functions decrease,and kidney has water retention.At the same time,NT-proBNP level increases and the prognosis is poor.The representative collateral-dredging drug Qiliqiangxin Capsule significantly protects microvascular endothelial cells and myocardial cells,improves the pathological damage,inhibits RAAS system activation and ventricular remodeling,expands blood vessels,improves water retention of kidney and heart functions,effectively delays the course of chronic heart failure,improves prognosis,has good therapeutic effects,and further proves the guidance value of vein-collateral doctrine for prevention and treatment of chronic heart failure.Part II Research on Plasma Metabonomics and Qiliqiangxin capsule Intervention Effect in Rats with Heart Failure after Myocardial InfarctionObjective: The changes of endogenous metabolites in plasma after different active ingredients of Qiliqiangxin Capsule were given to rats with chronic heart failure were revealed.At the same time,combined with the bioinformatics method the differences between therapeutic effects and metabolites were analyzed,which provided data support to reveal the prevention and treatment effect of different bioactive substances in the development of chronic heart failure.Methods: The chromatography separation method of large pore resin absorption column was used.Qiliqiangxin was eluted with ethyl alcohol with different concentrations(0%,30%,50%,70% and 90%),respectively.The eluant was collected.QLQX-H,QLQX-30%,QLQX-50%,QLQX-70% and QLQX-90% powder were obtained and administered to rats with heart failure after AMI.Three metabolome platforms,including GC-MS,Oxylipins and Oxidative and Nitrosative Stress,were used to analyze features of endogenous metabolites in plasma of rats;based on the differences between extracts of Qiliqiangxin Capsule obtained with different concentration of ethanol and plasma metabolites of rats,the main component analysis,partial least squares and other bioinformatics methods were used to analyze its correlation with protection of cardiac microvessels,vasodilation,diuresis,inhibition of RAAS system activation and other therapeutic effects.Results: 1 Potential metabolic markers of chronic heart failure disease were revealedThree metabolome platforms,including GC-MS,Oxylipins and Oxidative and Nitrosative Stress,were used to analyze and compare features of plasma metabolites in rats of Sham Group and Model Group.It was found that compared with the Sham Group the Model Group had a high level of disease markers,and the difference was significant(P<0.05).It showed that in rats with chronic heart failure increased plasma metabolites included lysophosphatidic acid(LPA C14:0,C18:1,C18:2,C18:3,C20:1,C20:3,C20:5,C22:5,C22:6),annular lysophosphatide acid(cLPA C16:0,C18:0),lipids(5,6-DiHETrE,12,13-DiHODE,12,13-DiHOME,9-HOTrE,13-KODE,9-KODE),and organic acids(glyceric acid and pyruvic acid);decreased metabolites included lipids(11,12-EpETrE,14,15-EpETrE,8,9-Ep ETrE,8-iso-PGA1,12S-HEPE,20-HETE,13,14-dihydro-PGF2a,PGE2),and organic acids(2-hydroxy butyric acid,3-hydroxy butyric acid,3-hydroxy isobutyric acid,3-hydroxy isovaleric acid,glycolic acid,methyl malonic acid,focal glutamic acid),which could be used as potential disease markers of chronic heart failure.2 Potential biomarkers of Qiliqiangxin Capsule in the intervention of chronic heart failure were revealed.Results showed that compared with the Model Group at T3 time point acid metabolites in plasma had significant difference,and after 8 weeks of intervention,the level difference of 2-hydroxybutyric acid,methylmalonic acid and 3-hydroxybutyric acid was significant in rats of QLQX Treatment Group.It showed that the organic acid metabolites could be used as potential biomarkers of Qiliqiangxin Capsule in the intervention of rats with chronic heart failure.3 The correlation between therapeutic effects and potential biological metabolic markers of Qiliqiangxin Capsule was revealedThree metabolome platforms totally detected 67 kinds of metabolic markers,which were negatively related to arginine vasopressin(AVP)and the activation of RAAS system,and positively related to calcitonin generelated peptide(CGRP),AngPTL4 and NRG1.It indicated that these metabolites were related to inhibition of the activation of RAAS system,diuresis,vasodilation and protection of cardiac microvessels.It revealed that the above potential biological active compounds were related to disorder of lipid and lysophosphatide acid.Adjustment of lipid metabolism disorder may be an important way for Qiliqiangxin Capsule to delay the development of chronic heart failure.Summary:In the onset and development process of chronic heart failure,there were lipid energy,amino acids and other metabolic disorders.Among them,the lipid metabolic disorder was the most severe;in the intervention of Qiliqiangxin Capsule 2-hydroxybutyric acid,methylmalonic acid,3-hydroxybutyric acid and other organic acids could be used as potential metabolic markers for the treatment of chronic heart failure;Qiliqiangxin Capsule was significantly related to protection of cardiac microvessels,inhibition of the activation of RAAS system,diuresis,vasodilation and metabolism of lipids and lysophosphatide acid.It revealed that inhibition of lipid disorders might be an important way for Qiliqiangxin Capsule to delay the onset and development of chronic heart failure.Part III Effect of Qiliqiangxin Capsules on the Lipid Metabolism in Rats with Heart Failure after Myocardial Infarction and the Study of Relative MechanismObjective: To investigate the interference of Qiliqiangxin Capsules on peroxidation/ nitrative stress of lipid in chronic heart failure,energy metabolism of fatty acid,metabolism of cholesterol and important pathway of lipid transformation.Methods: ELISA and molecular biological methods were adopted to determine the level of 8-isoprostanes F2?(8-iso-PGF2?),3- nitrotyrosine(3-NT)in the serum and the 3-NT level in the cardiac muscle tissue and reveal the lipid peroxidation/ nitrative stress and the interference of dredging collaterals in the cardiac muscle tissue in rats with chronic heart failure;gas chromatographic method was adopted to determine the composition of fatty acid in the cardiac muscle tissue to understand the correlation between the n-3 and n-6 series PUFAs level in the cardiac muscle tissue and the free fatty acid(FFA)in the serum;by detecting the protein/gene expression level in the cardiac muscular tissue centering on the key transcription factors of lipid metabolism like cholesterol and fatty acid and key activation/ inhibitory enzyme of energy metabolism of fatty acid and LOX and COX routine of lipid transformation,the metabolic process and mechanism of dredging collaterals of rats with chronic heart failure was revealed.Results: 1 Reveal Qiliqiangxin capsule could inhibit the lipid peroxidation/ nitrative stress reactionCompared with Sham group,the level of 8-iso-PGF2? and 3-NT in serum and 3-NT protein expression in cardiac muscular tissue in rats from Model group increased significantly(P<0.05,P<0.01);compared with Model group,the level of 8-iso-PGF2? and 3-NT in serum and 3-NT protein expression in cardiac muscular tissue in rats from QLQX group decreased significantly(P<0.05,P<0.01).2 Reveal Qiliqiangxin capsule could improve the energy metabolism of fatty acidGas chromatography showed that the cardiac muscular tissue was mainly composed of unsaturated fatty acid.Serum FFA level detection and correlation analysis showed that compared with Sham group,the PUFAs in n-6 series and serum FFA in the cardiac muscular tissue of rats from Model group increased significantly and PUFAs in n-3 series decreased obviously(P<0.05,P<0.01),and the FFA and PUFAs in n-6 series was positively related(P<0.001)and negatively related with PUFAs in n-3 series(C18:3,C22:5)(P<0.01),suggesting that the PUFAs and FFA level was closely related in the development of chronic heart failure.The PUFAs in n-6 series increased and the FFA released into blood was increased and the cytotoxicity was strengthened,PUFAs in n-3 series was decreased and the composition of unsaturated fatty acid in cardiac muscular tissue was disordered.Qiliqiangxin capsule could significantly decrease the serum FFA composition and energy metabolism,reduce the toxic lipid accumulation and play the role of postponing chronic heart failure.Inspection result of FAT,CPT-1,FAS,PARP-1 protein and mRNA of cardiac muscular tissue showed that,compared with Sham group,the FAT,CPT-1 protein and mRNA expression was decreased and the FAS,PARP-1 protein and mRNA expression was up-regulated in Model group(P<0.005);compared with Model group,the FAT,CPT-1 protein and mRNA expression was increased to some extent and the FAS,PARP-1 protein and mRNA expression was decreased(P<0.05,P<0.01),indicating that Qili Qiangxin capsule could inhibit the synthesis of fatty acid by promoting the transfer and utilization of fatty acid so as to reduce the accumulation of fatty acid and the energy consumption of cardiac muscle and improve the energy metabolism of the cardiac muscle.3 Reveal Qili Qiangxin capsule could regulate the cholesterol metabolism by SCAP/ SREBP-1c/ ACC1 routineThe inspection result of SCAP/ SREBP-1c/ ACC1 protein and mRNA of cardiac muscular tissue showed that,compared with Sham group,the expression of SCAP,SREBP-1c/ p-SREBP-1c,ACC1/ p-ACC1 protein and/ or mRNA was up-regulated to some extent in the Model group(P<0.01,P<0.05);compared with Model group,the expression level of SCAP,SREBP-1c/ p-SREBP-1c,ACC1/ p-ACC1 protein and/ or mRNA was decreased(P<0.05,P<0.01),indicating that under chronic heart failure condition,the synthesis of cholesterol was over activated and the synthesis of lipid was increased,which would cause the lipid accumulation of cardiac muscular tissue and led to lipid toxicity.Qiliqiangxin capsule could inhibit the over activation of cholesterol synthesis,improve the lipid metabolism.Regulation of cholesterol by SCAP/ SREBP-1c/ ACC1 way might be one of the important mechanisms in improving lipid metabolism.4 Confirm that Qiliqiangxin capsule could improve lipid metabolism by LOX and COX routines and play the role of protecting chronic heart failure.The inspection result of 15-LOX,cPLA2,p38 MAPK and COX-2 protein and mRNA of cardiac muscle tissue showed that compared with Sham group,the cPLA2,p38 MAPK and its phosphorylation,COX-2 protein and/ or mRNA expression was up-regulated in Model group(P<0.01,P<0.05);compared with Model group,the cPLA2,p38 MAPK and its phosphorylation,COX-2 protein and/ or mRNA expression was decreased to some extent in the QLQX group(P<0.01,P<0.05),suggesting that under the pathological condition of chronic heart failure,the activation of LOX routine by p38 MAPK and its phosphorylation pathway could promote the high expression of cPLA2 and the lipid peroxidation was worsened.The COX-2 routine was activated,which pushed that inflammation reaction induced by AA synthesized prostaglandin.Qili Qiangxin capsule could down-regulate the expression of 15-LOX,cPLA2 and COX-2 protein and/or gene,inhibit the p38 MAPK and its phosphorylation pathway,indicating that Qili Qiangxin capsule could improve lipid metabolism by LOX and COX routines.Summary:The lipid peroxidation/ nitrative stress reaction of rats with chronic heart failure strengthened and the level of PUFAs of n-6 series was increased and the fatty acid was accumulated.The FFA level that was released into blood and the cytotoxicity strengthened.The PUFAs level of n-3 series decreased and the construction of fatty acid was disordered.The synthesis of two lipid metabolisms of cholesterol,LOX and COX was over-activated and the synthesis of lipid increased,causing accumulation of fatty.Qiliqiangxin capsule could down-regulate the expression of 8-iso-PGF2? and 3-NT,inhibit the lipid peroxidation/ nitrative stress reaction,improve the construction of PUFAs and energy metabolism,promote the transfer,uptake and utilization of fatty acid,decrease the synthesis and accumulation of fatty acid,improve energy metabolism and regulate cholesterol metabolism by SCAP/ SREBP-1c/ ACC1 routine and inhibit LOX routine by p38 MAPK and phosphorylated routine,reduce the COX routine activation and improve the lipid metabolism.Conclusions:1 Investigate the pathogenic characteristics of chronic heart failure from the perspective of traditional Chinese medicine vessels-collateral theory as the instruction.It was pointed out that qi and yang defect caused by lost balance of ying wei and disorder of activities of qi was the basic pathogenesis.The yang and qi lost pushing and warming with inability of blood delivery,static blood blocking collaterals and obstruction of exchange of liquid and blood,which may cause the obstruction of collaterals by phlegm stagnation and the liquid could not reflux and accumulated outside the collaterals.Therefore,the pathogenesis was divided into “gas related,blood related and water related”.The three pathogenesis interacted with each other and formed reality and led to the masses obstruction of heart collaterals.Based on the above,the treatment principle of “common treatment and separated elimination of qi,blood and water” and Qiliqiangxin Capsule composition was developed.2 Reveal the cardiomyocytes and microvascular endothelial cells damage during the development course of chronic heart failure,activation of RAAS system,reconstruction of ventricle,dysfunction of heart;cardiac vessel damage and dysfunction of secretion;pathological changes like renal water metabolism.Qiliqiangxin capsule can protect the ultrastructure of cardiac microvessel,inhibit the activation of RAAS system and ventricle reconstruction,dilute the vessels by eNOS/ sGC/ cGMP/ PKG routine and improve renal water retention by inhibiting the expression of AVP and receptor V2 R,and AQP2 so as to improve the heart function and play the role of “reinforcing qi and warming yang,promoting blood circulation to remove meridian obstruction and inducing diuresis to alleviate edema”,which further confirmed the inner value of preventing chronic heart failure with venation theory.3 By taking advantage of the metabonomics research method,and analysis of biological information,the metabolic disorder of lipid,energy and amino acid during the development course of chronic heart failure is revealed.The protection of cardiac microvessel,inhibition of RAAS system activation,dieresis and dilation of blood vessel is significantly correlated with the lipid and lysophosphatidic acid metabolism,revealing that Qiliqiangxin capsule can inhibit the metabolic disorder of lipid materials and postpone chronic heart failure,which provide methodological reference for the modern study of compound traditional Chinese medicine.4 Reveal that Qiliqiangxin capsule inhibits the lipid peroxidation/ nitrative stress by down-regulating the expression of 8-iso-PGF2? and 3-NT;improve the composition of unsaturated fatty acid of cardiac muscle,decrease FFA level,reduce the lipid accumulation,protect cardiac muscle cells and decrease the synthesis and accumulation of cardiac muscle fatty acid and improve energy metabolism by promoting the transfer,uptake and utilization of fatty acid;regulate the cholesterol metabolism by SCAP/ SREBP-1c/ ACC1 routine;reveal that Qili Qiangxin capsule can improve lipid metabolism by LOX and COX routines. |
PDF Full Text Request