Effects Of Benefiting Qi,Activating Blood And Promoting Diuresis On Energy Metabolism Remodeling For Treatment Of Heart Failure

Chronic heart failure(HF)is a global public health problem.With the development of modern medical technology,the mortality of cardiovascular disease has been controlled because of several cardiovascular drugs widely used in clinic.However,once the disease progresses into HF,the mortality rate is still high.The aging process of the population in China is speeding up,and the number of people suffering from hypertension,coronary heart disease is huge,which has become a potential population of HF and aggravates the medical burden.Therefore,in-depth understanding of the pathogenesis of heart failure,and the development of effective prevention and treatment of HF drugs,is the most important task of prevention and treatment of HF.Currently,energy metabolism regulation therapy has raised increasing interest for prevention and treatment of HF.By improving mitochondrial function and myocardial energy metabolism,ventricular remodeling and cardiac function can be widely improved.However,there are still some limitations of the modern drugs for improving cardiac energy metabolism,and it is difficult to handle such complex pathologies of HF.Multi-target drugs have raised increasing interest in the last decade owing to their advantages in additive and synergistic effects.Therefore,effective combination therapies for HF,which achieve comprehensive efficacy by interacting with multiple targets and pathways,are needed.The theory of traditional Chinese medicine highlights the comprehensively regulation according to syndrome differentiation.The development of multiple-component treatments derived from Chinese herbal formulae represents a novel approach for the concept of combination therapy.Recently,in very broad studies,Chinese herbal formulae have significant clinical efficacy in HF treatment.The understanding of the pathogenesis of HF in traditional Chinese medicine tends to be consistent.It is believed that the basic pathogenesis of heart failure is the deficiency of cardiac qi and yang,therefore interrupts the normal blood stasis,and induces phlegm drink and water dampness.The key of HF pathogenesis is the deficiency and decline of cardiac qi and yang.Unable to maintain the stasis of blood.Benefiting qi,activating blood and promoting diuresis has become the basic method for the treatment of HF.Nuanxinkang(NXK),is a representative Chinese herbal formula which formed by our research group under the guidance of the method of benefiting qi,activating blood and promoting diuresis,and through streamlining and optimizing according to numerous clinical experience and basic research data.Several additional studies have also indicated that NXK has comprehensive efficacy for HF treatment.This paper mainly studies the effect and mechanism of benefiting qi,activating blood and promoting diuresis derived representative herbal formula NXK on energy metabolism remodeling for the treatment of HF.Objective:To investigate the effect of NXK on cardicac function and ventricular remodeling of HF.In addition,to explore the potential mechanisms and chemical basis of NXK in regulating metabolic remodeling of HF by transcriptome,metabonomics analysis and network pharmacology.Then to validate the effect on NXK in metabolic regulation of HF,and to examine role of ADRA1A-AMPK axis on NXK effect in HF.Methods:1.The mouse model of HF was established by the surgery of coarctation of aorta.The mice of Sham and TAC group were treated with normal saline,the mice of NXK and TMZ group were treated with NXK and trimetazidine respectively.After 6 weeks of administration,the cardiac function of mice was measured by echocardiography.Cardiac hypertrophy was evaluated by heart weight ratio.Myocardial fibrosis was measured by Masson staining.Cardiomyocytes apoptosis was measured by TUNEL staining,and the content of cardiac neuroendocrine hormones was measured by fluorescence quantitative PCR(q-PCR).Above to evaluate the effects of NXK on cardiac function and ventricular remodeling of HF.2.Extracted the total RNA from the heart tissue of Sham,TAC,NXK group,to conduct the RNA-sequence assay.Extracted the serum of TAC and NXK group for UHPLC-MS metabonomic-sequence assay.Comprehens the results of the differential genes of transcriptome analysis and the metabonomics by Omicsnet,to construct upstream differential genes and downstream metabolite networks related to energy metabolism of HF.3.By comparing UHPLC-MS method with database and literature,the effective active substances in NXK were identified,and then the network of "effective component-target-pathway" of NXK was constructed by using network pharmacology method.The important active substances,key targets and signaling pathways of NXK were analyzed.Fluorescence quantitative PCR(FQ-PCR)was used to verify the coincident targets of network pharmacology and transcriptology,and the target alpha la(Adrenergic receptor alpha la(ADRA1A)was selected to construct ADRA1A protein model.Molecular docking simulation was carried out with 21 active compounds identified by NXK.4.The total ATP production of mice hearts of each group were measured by kit,the energy production efficiency of mitochondrial electron chain was evaluated by measuring the ratio of NAD+/ADH,and the structure of mitochondria in each group was observed by transmission electron microscope(TEM).The expression of PGC-1α in the hearts of each group was determined by q-PCR.The primary cardiomyocytes of each group were extracted and stained with Mitotraker and MitoSox to observe the expression of mitochondria and mitochondrial ROS in cardiomyocytes.H9c2 cardiomyocytes were treated with NXK-containing serum or control serum.Seahorse energy metabolism method was used to detect mitochondrial respiratory function in cardiomyocytes,including basic respiration,maximum respiratory consumption,proton leakage,energy production coupling and so on.Finally,the expression of glycolipid related transporter CPT-1,GLUT4 and MPC1 in each group was determined by qPCR to observe whether NXK optimized the utilization of glycolipid energy substrates.5.The hypoxic H9c2 cardiomyocyte model was established.The protein expression of related markers were detected by Western-Blotting,and the mRNA level of related makers were detected by qPCR.ADRA1A overexpression,ADRA1A knockout and ADRA1A overexpression were given and AMPK signal wasinhibited,respectively.The effects of ADRA1A on AMPK signaling pathway,total ATP production,mitochondrial production and the effect of the utilization of glycolipid energy substrate in hypoxic cardiomyocytes were observed.Then the hypoxic cardiomyocyte model was treated with NXK-containing serum or control serum,respectively,when ADRA1A was blocked or AMPK signal was inhibited,Whether NXK can still play a protective role on cardiomyocytes,whether the total ATP production,mitochondrial production ability and glycolipid energy substrate utilization switching of cardiomyocytes have corresponding changes under this condition.Results:Part Ⅰ.The effects of NXK in cardiac function and ventricular remodeling of HF1.The low dose of NXK significantly increased LVFS and LVESD(P<0.01,P<0.05),but showed no significant changes in LVEF and SV(P>0.05,P>0.05).Middle dose and high dose of NXK could significantly increase LVEF(P<0.001,P<0.001),LVFS(P<0.001,P<0.001),significantly down-regulate LVESD(P<0.001,P<0.001),middle dose of NXK could increase SV(P<0.001),while high dose of NXK showed more significant increase of SV(P<0 01).2.High dose of NXK could significantly reduced the heart to weight ratio of failed heart(P<0.05).3.Both NXK and trimetazidine significantly improved myocardial fibrosis of HF(P<0.05).NXK showed better effects on myocardial fibrosis than that of trimetazidine.4.NXK significantly decreased cardiomyocytes apoptosis in mice heart of HF(P<0.001).NXK showed better effects on apoptosis than that of trimetazidine.5.NXK could improve the mRNA expression level of ANP and BNP in the failed heart(P<0.001).NXK showed better effects on neuroendocrine hormone environment than that of trimetazidine.Part Ⅱ.The systems biological study of NXK effects on energy metabolism of HF1.The biological process of NXK effects was very similar to that of HF,including the regulation of apoptosis,the formation of extracellular matrix,the response to hypoxia,mitochondrial formation,ATP decomposition and so on.2.The effects of NXK on energy metabolism of HF might focus on major biological processes including ATP catabolism,mitochondrial tissue,mitochondrial respiratory electron chain,glycolipid metabolism,mitochondrial membrane potential,mitochondrial calcium homeostasis and mitochondrial apoptosis;Major molecular function including ATP binding,protein binding,zinc ion binding;Major cell components including mitochondrial membrane,mitochondrial respiratory chain complex,mitochondrial synthase,mitochondrial pyruvate dehydrogenase.3.Lots of signaling pathways,such as PI3K-AKT,Notch,FoxO,cAMP,MAPK,epinephrine signal and AMPK in cardiomyocytes,are involved:in the regulation of NXK on energy metabolism of HF.4.NXK significantly changed the metabolic microenvironment of serum of mouse model of HF.Combining upstream transcriptome differential genes and downstream metabonomics metabolites,it was found that NXK could significantly change the metabolic microenvironment of serum in mouse model of chronic heart failure.The most important metabolite that regulated by differential genes of NXK related to energy metabolism is adenosine diphosphate(AMP).Part Ⅲ.Network pharmacological study of NXK effects of energy metabolism of HF1.21 active ingredients of NXK were identified.2.After analyzed the key nodes of the ingredients-target-pathway network of NXK effects,the key effective ingredients of NXK were found,including 20(R)-ginsenoside Rg3,ginsenoside Rs3,Notoginsenoside H,ilexsaponin A1,Chikusetsusaponin V,ginsenoside Rd and ginsenoside Rs6.Key direct targets included MAPT,STAT family genes(STAT1～STAT4),CHRM2 and CHRM3,and BCL2 family genes(BCL2,BCL2L1,BCL2L2).The key signaling pathways included PI3K-AKT signaling pathway,RAS signaling pathway,MAPK signaling pathway,cAMP signaling pathway,calcium signaling pathway and adrenergic signaling pathway.The results suggested that these active components,targets and signaling pathways may be highly involved in the effects of NXK.3.Overlapped the differentially expressed genes of transcriptome analysis and key targets predicted by network pharmacologg,14 overlapped genes were found,among which 13 genes were further identified in the mouse heart of HF by qPCR.Those targets included Bcl2,Bcl211,Chrm2,Adoral,Statl,Mme,Ctss,Dct,Hpse,Hsdllbl,and Adrala.4.The energy metabolism related target ADRA1A was selected as the key target,and the structural protein of ADRA1A protein was obtained by Swiss Model simulation.Six potential active ingredients interacting with ADRA1A were obtained by molecular docking simulation with 21 active components of NXK,including Ginsenoside Rd,Ginsenoside Re,Ginsenoside Rs3 and Notoginsenoside H,Indole-3-acetic acid,and Raffinose.Part IV.NXK effects on cardiac energy metabolism of HF1.NXK significantly increased the production of ATP in heart tissu of mouse model of HF(P<0.001).2.NXK increased the NAD+/NADH ratio in heart tissue of mouse model of HF(P<0 05),as well as increasing the mRNA expression of PGC-1 a in heart tissue of mouse model of HF(P<0.001).NXK significantly improved the ultrastructure of mitochondria in heart tissue of mouse model of HF.NXK also increased the expression of mitochondria and decreased the expression of mitochondrial ROS in primary cardiomyocytes of failed heart.3.NXK-containing serum significantly decreased BR(P<0.001),MR(P<0.001),PL(P<0.001)and NMR(P<0.01),and significantly increased ATP(P<0.001)production and CE(P<0.01)in H9c2 cadiomyocytes.There was no significant change in SR.4.NXK dynamically changed the mRNA expression level of CPT-1,GLUT4,MPC1 at 1 day,7 days,14 days,28 days and 42 days after the surgery of coarctation of aorta.Part V.The mechanisms of NXK effects on energy metabolism of HF1.ADRA1A shRNA could knock down the expression of ADRA1A protein in cardiomyocytes.Compound C can inhibit the phosphorylation of AMPK αprotein in cardiomyocytes.2.ADRA1A up-regulated the phosphorylation of AMPK a signal in H9c2 cardiomyocytes during hypoxia,and ADRA1A increased the ATP production in H9c2 cardiomyocytes during hypoxia(P<0.05),AMPK inhibitor could block the up-regulated effect of ADRA1A on ATP production.The total ATP production of cardiomyocytes was significantly decreased by 47%compared with the control group.ADRA1A significantly increased the mRNA expression of PGC-1α GLUT4 and MPC1 in H9c2 cardiomyocytes(P<0.05,P<0.05,P<0.05),and significantly decreased the mRNA expression of CPT-1(P<0.05).AMPK inhibitor could partially or completely block the regulation effects of ADRA1A activation on PGC-1α,CPT-1,GLUT4 and MPC1(P<0.05,P<0.05,P<0.05,P<0.05).3.NXK up-regulated the protein expression of ADRA1A and the phosphorylation of AMPK α protein in mice’ failed hearts.4.NXK-containing serum significantly increased the protein expression level of the phosphorylation of AMPK a in H9c2 cardiomyocytes,and increased the ATP production of H9c2 cardiomyocytes(P<0.001),as well as increasing the mRNA levels of PGC-1α,GLUT4 and MPC1(P<0.05,P<0.05,P<0.05)and decreased the expression of CPT-1 in H9c2 cardiomyocytes(P<0.05).After adding the AMPK inhibitor or ADRAA shRNA,there was no significant difference in ATP production between NXK group and control group(P>0.05).After adding AMPK inhibitor,there was no significant difference in the mRNA expression of PGC-1α,CPT-1,GLUT4 and MPC1 between NXK group and control group(P>0.05,P>0.05,P>0.05,P>0.05).After adding ADRA1A shRNA,there was no significant difference in the expression of PGC-1α,CPT-1 and MPC1 in H9c2 cardiomyocytes between NXK group and control group(P>0.05,P>0.05,P>0.05).However,ADRA1A knockout even further decreased the expression of GLUT4 in H9c2 cardiomyocytes in Wenxinkang group(P<0.05).Conclusion:1.Middle and high doses of NXK can improve the cardiac function in mouse model of HF,and the effect of high dose of NXK was better.NXK significantly improved myocardial fibrosis,reduced myocardial apoptosis,improve the neuroendocrine hormone environment.And NXK showed better improvement in the treatment of HF than trimetazidine group.2.NXK may play a role in the pathologies and the pathogenesis of HF to treat HF.NXK can widely affect the composition,function and energy metabolism environment of mitochondria,and simultaneously regulate glycolipid substrates metabolism and ATP biosynthesis,which is likely to regulate the remodeling of energy metabolism through these pathways.NXK might affected lots of signaling pathways in the failed heart.AMPK signaling pathway may play an important role in the regulation of NXK on the remodeling of energy metabolism in failed heart.3.Twenty-one active components of NXK were identified.Network pharmacological analysis further confirmed that NXK regulated multi-target and multi-pathway.NXK may activate ADRA1A through six important active ingredients.4.NXK significantly improved the myocardial energy supply in mouse model of HF,improved the efficiency of mitochondrial electron chain energy supply in failed heart,enhanced the biogenesis of mitochodria in failed heart and cadiomyocytes,and improved the ultrastructure of mitochondria in mouse model of HF.NXK attenuated mitochondrial oxidative stress in cardiaomyocytes in mouse model of HF.NXK can improve the total energy supply and productivity of mitochondria of cardiomyocyte during hypoxia,and reduce the oxygen consumption of mitochondria of cardiomyocyte,which will not be accompanied by the changes of mitochondrial spare respiratory potential and non mitochondrial respiratory such as glycolysis.NXK can dynamically regulate the utilization of glycolipid energy metabolism substrates in the progress of HF.The substrates utilization mode changed from aerobic oxidation of fatty acids or glycolysis of glucose to aerobic oxidation of glucose during HF.These results suggested that NXK may improve the quantity,quality and respiratory function of mitochondria and optimize the utilization of energy metabolism substrates in the heart of mouse model of HF.In conclusion,NXK improved the remodeling of energy metabolism in failed heart.5.ADRAIA increased the production of ATP and switched the utilization of the energy metabolism substrates in cardiomyocytes during hypoxia by regulating AMPK signaling pathway.By activating the ADRA1A-AMPK signal axis,NXK improved the ATP production and switch the utilization of the energy metabolism substrates in cardiomyocytes during hypoxia,so as to improve the process of energy metabolism remodeling of cardiomyocytes.