The Role And Underlying Mechanisms Of Icariside Ⅱ In Myocardial Remodeling

BackgroundMyocardial remodeling is characterized by cardiac myocyte hypertrophy and myocardial fibrosis.At the early stage of myocardial hypertrophy,the nuclei and mitochondria of hypertrophic cardiomyocytes increased and needed energy.With the development of myocardial remodeling,insufficient energy supply and energy utilization of myocardial cells lead to apoptosis and necrosis of myocardial cells.The necrosis of myocardial cells leads to the decrease of the number of myocardial cells and the decrease of myocardial contractility,which triggers the inflammatory cascade reaction,clears the damaged cells and necrotic cells and activates the repair process.The excessive inflammatory reaction increases the cardiac injury,exacerbates the process of fibrosis,and causes the development of fibrosis.Contraction dysfunction.The degree of extracellular interstitial fibrosis was aggravated by accumulation of extracellular matrix(Extracellular matrix,ECM),increased myocardial stiffness,decreased ventricular compliance and caused diastolic dysfunction.Myocardial structural remodeling results in changes of electrophysiological characteristics of cardiac myocytes such as automatism,excitability and conduction,induces various arrhythmias,affects cardiac pump function,exacerbates hemodynamic disturbance,and easily complicates cardiogenic shock and even sudden death.Myocardial ischemia due to myocardial structural remodeling and electrical remodeling(Electrical remodeling)Injury and reperfusion injury,malignant remodeling aggravation,eventually irreversible heart failure.Therefore,it is important to explore the mechanism of myocardial remodeling and find potential targets to prevent and delay myocardial hypertrophy in prevention and treatment of heart failure.With the development of molecular biology,myocardial remodeling focuses on the regulation of signaling pathways such as gene transcription,translation,protein synthesis and modification.For example,adenosine monophosphate activated protein kinase/rapamycin target protein(AMPK/mTOR),)associated with interstitial fibrosis growth factor(TGF β),mitogen-associated mitogen-activated protein kinase(MAPK),)signal transduction Signaling pathways such as transcriptional activator(JAK/STAT).Multiple signaling pathways interact with each other through upstream and downstream molecules to form complex and variable cellular signal networks System,precise regulation of myocardial remodeling.Abnormal regulation of energy metabolism,inflammation,autophagy,apoptosis,endoplasmic reticulum stress and oxidative stress can lead to poor myocardial remodeling.Therefore,early detection of myocardial remodeling related signal pathway proteins targeted intervention may prevent the occurrence and development of heart failure.Icariin Ⅱ(ICA Ⅱ),also known as icariin,is one of the flavonoids extracted from Epimedium.It exerts cell cycle regulation and apoptosis through many signal pathways such as JAK2-STAT3,MAPK-ERK,and PI3k-Akt-mTOR.Biological efifects such as angiogenesis and oxidative stress.ICA Ⅱ exerts antioxidant activity through ROS/GSK-3b mitochondrial signaling pathway and alleviates oxidative stress induced autophagy in neural PC 12 cells.ICAⅡ inhibits PI3K/Akt in vitro and in vivo.By inhibiting cardiomyocyte apoptosis and oxidative stress,ICAⅡ has protective effect on myocardial cell apoptosis and ischemia-reperfusion injury induced by hypertension.Based on the cardiovascular protection of ICAⅡ,ICAⅡ has attracted much attention in the field of cardiovascular research.In our laboratory,we have found that icariin protects cardiomyocytes from inflammatory damage and apoptosis induced by LPS by inhibiting reactive oxygen species dependent on JNK/NF-κ B pathway.However,whether ICA Ⅱ can relieve cardiac remodeling caused by pressure overload,It is not clear before.In this study,we will investigate the protective effect and possible mechanism of ICA Ⅱ on myocardial hypertrophy induced by pressure overload in mice and cardiomyocyte hypertrophy induced by PE in neonatal rats.In this study,Real time-PCR was used to detect the expression level of mRNA associated with hypertrophy gene.;Western Blot was used to detect p-AMPK α,T-AMPK α,p-Akt,T-Akt,pmTORC1,T-mTORC1,p-P70S6K,T-P70S6K,P-4EBP1,T-4EBP1.Related signal pathway protein level,further clarify the possible molecular mechanism of ICAⅡ to improve cardiac remodeling.Research ObjectiveTo explore the effect and mechanism of ICAⅡ on myocardial remodeling,the effects and mechanisms of ICA Ⅱ on myocardial hypertrophy induced by pressure overload in vivo and ICA Ⅱ on phenylephrine(PE)induced myocardial hypertrophy in neonatal rats in vitro were studied by animal and cell experiments.Determine whether ICA ii is a potential drug for the treatment or adjunctive treatment of malignant myocardial remodeling.MethodsPart one(Animal experiments):Select 8-9 weeks old,weight 23.5-24.5g healthy male C57BL/6J mice.The tress-induced myocardial hypertrophy model was established by aortic constriction and was intervened with ICAⅡ.Myocardial mast cells,myocardial fibrosis and cardiac function were detected in 4 groups of Sham+vehicle(Veh)(n=16),Sham+ICAⅡ(ICAⅡ)(n=16),AB+vehicle(AB)(n=16)and AB+ICAⅡ(n=16).The experimental animals were randomly assigned to the sham Operation Group and the experimental group,each group had 16 animals.For Sham+ICAⅡ(ICAⅡ)(n=16),AB+vehicle(AB)(n=16)and AB+ICAⅡ(n=16),ICAⅡ is dissolved with 60%ethanol,then diluted with 0.9%normal saline,and ICAⅡconcentration is 1mg/ml for intragastric administration.Meanwhile,as for Sham+ICAⅡ and AB+vehicle gave the same amount of saline for 6 weeks.The changes of cardiac function in mice were evaluated by M-type two-dimensional echocardiography with index-measurement of HR,IVSd,IVSs,LVPWd,LVPWs,LVDd,LVDs,FS and EF.The mouse 1VESP,LVEDP,dp/dt max and-dp/dt min indexes were detected by hemodynamics to evaluate the left ventricular pressure-volume parameters.Immediately after the death of a broken neck,the heart of each mouse was quickly removed and weighed,while the weight of the mouse(body weight,BW),heart weight(heart weight,HW),lung weight(lung weight,LW)and tibial length(Tibial length,TL)were recorded.To calculate the LW left ventricular mass,HW/BW total Heart mass index and LW/BW ventricular mass index,the collected heart was randomly divided into two groups,one group took left ventricle in-80 ℃ for western-blot and RT-PCR detection,and the other group was stopped in diastolic period by 10%KCl,after being dehydration,embedding and slicing,it was used for pathological staining.Hematoxylin(HE)staining was used to observe the changes of the cross-sectional area ’of cardiac myocytes in mice,heart-tissue section was stained using picricsiriusred staining(PSRstaiining)to evaluate the myocardial interstitial and vascular fibrosis in mice and.to detect the fibrosis area,and a-SMA staining was applied to detect the activation and transformation of muscle fibroblasts.RT-PCR was used to detect the cardiomyocyte hypertrophy related biomarkers,such as atrial natriuretic peptide(ANP),B-type natriuretic peptide(BNP),alpha myosin heavy chain/β-myosin heavy Chain(a-MHC/β-MHC)and detect the fibrosis-related markers(TGF-β),collagen protein Ⅰ(Coll Ⅰ),collagen Ⅲ(Coll Ⅲ),and connective tissue growth factor(CTGF).Myocardial tissue related protein expression was detected by Western blot,such as p-Akt,T-Akt,p-AMPKa,T-AMPKa,p-mTORCl,T-mTORC1,p-P70s6k,T-P70s6k,p-4EBP1,T-4EBP1and GAPDH.For real-time quantitative polymerase chain reaction(RT-PCR),RNA extraction kit was used to extract total RNA from mouse heart tissue.Part Two(Cell experiment):Neonatal rat cardiac myocytes(NRCM)culture:Prepare NRCM from 1-3-day-old Sprague-dawley rats in the laboratory.PE induces NRCM hypertrophy.RT-PCR was used to detect the cardiomyocyte hypertrophy related biomarkers,such as atrial natriuretic peptide(ANP),B-type natriuretic peptide(BNP),alpha myosin heavy chain/β-myosin heavy Chain(α-MHC/β-MHC),and detect fibrosis-related biomarkers,such as transforming growth factor-p(TGF-β),collagen protein I(Coll Ⅰ),collagen Ⅲ(Call Ⅲ)and connective tissue growth factor(CTGF).Myocardial tissue related protein expression was detected by Western blot,such as p-Akt,T-Akt,p-AMPKα,T-AMPKα,p-m TORC1,T-mTORC1,p-P70s6k,T-P70s6k,p-4EBP1,T-4EBP1 and GAPDH.In real-time quantitative polymerase chain reaction(RT-PCR),RNA extraction assay was used to extract total RNA from NRCM.To explore the concentration-dependent and time-dependent for the effection of ICAII,the gene expression of p-AMPKα,T-AMPKα,p-Akt and T-Akt was evaluated by RT-PCR,at the same time,the effects of solvents,RCV,ICAⅡ and AICAR on p-AMPKα,T-AMPKα,p-Akt and T-Akt were also evaluated.ResultsPart one:1.ICAⅡ reduces cardiac hypertrophy induced by pressure overload:compared with VEH or ICAⅡ group,pressure overload significantly induces cardiac hypertrophy in mice in AB group.AB+ICAⅡ group significantly reduces the ratio of HW,HW/BW,and LW/BW(P<0.05).Heart-tissue HE staining showed that AB group significantly induce the left ventricular hypertroph and myocardia cell surface increasing.Compared to VEH group and ICAⅡ group,hypertrophy related genes(ANP and BNP)are up regulated(P<0.05)in AB group.Compared to AB group,the AB+ICAⅡ group has a down-regulation of ANP and BNP.AB+ICAⅡ group is able to prevent the currence of β-MHC.2.ICAⅡ reduces myocardial fibrosis induced by pressure overload:compared with VEH or ICAⅡ group,fibrosis emerges both in myocardial interstitium and perivascular(P<0.05)in AB group,and AB+ICAⅡ group has significantly suppression of such kind of fibrosis.In AB group,the TGF-β,Coll Ⅰ,Coll Ⅲ and CTGF are all up-regulated(P<0.05),however,compared to AB group,all of these genes are down-regulated in AB+ICAⅡ group(P<0.05).3.ICAⅡ improves cardiac insufficiency induced by pressure overload:The changes of cardiac function in mice were evaluated by M-type two-dimensional echocardiography.Increasing of LVEDD and LVESD results in systolic dysfunction,LVEF decrease and FS decrease.Left ventricular hypertrophy takes form of increase of LVPWs and LVPWd,and all of parameters are improved by the treatment of ICAⅡ(P<0.05).HR is not significantly different between different groups(P>0.05).Besides,cardiac hemodynamic changes of different groups are obsearved by PV-loop,results show that LVESP and LVEDP are up-regulated in AB group,as well as dp/dt-max and dp/dt-min are down-regulated.All of these parameters are improved by the treatment of ICAⅡ(P<0.05).4.ICAII treatment activates AMPK-a and suppresses Akt.ICAⅡ treatment suppresses the dysfunction of AMPK-a and over-activated Akt induced by pressure overload(P<0.05).ICAⅡ treatment suppresses the downstream of AMPK-a and Akt,mTORC 1 and 4EBP1(P<0.05).After ICAⅡ treatment,AMPK-κ is activated in baseline level,while the phosphorylation state of Akt is not affected in baseline level.Part two:1.ICAⅡ treatment activates AMPK-α and suppresses mTORC 1.ICAⅡ treatment activates AMPK-α in a concentration-dependent style,and different concentrations of ICAⅡ have no impact on phosphorylation of Akt.ICAⅡ treatment activates AMPK-αin 0.5h,and p-AMPKα/T-AMPKα is increasing as time goes by,and after one-hour ICAⅡ treatment,the increasing phosphorylation of AMPKκ does not exist and the p-AMPKα/T-AMPKα seems to be stable.Meanwhile,ICAⅡ treatment does not regulate the phoshporylation of Akt.The activity of ICAⅡ is better than RSV in activating AMPKα,and there is no difference between ICAⅡ and AICAR(P<0.05).In final,in NRCM stimulated by PE,ICAⅡ treatment reverses the activation of AMPKαand suppresses activation of mTORC 1/p70S6K/4EBP1 pathways.2.ICAⅡ suppresses hypertrophy of NRCM induced by PE via ativation of AMPKa.PE stimulation results in hypertrophy of NRCM,which is prevented by ICAⅡtreatment.When the activity of AMPKα is suppresses by CPC,ICAⅡ doesn’t prevent the hypertrophy of NRCM.ICAⅡ treatment suppresses the NRCM-hypertrophy related genes expression,such as ANP,BNP and β-MHC,and the effect of CPC could totally eliminate the therapeutic effect of ICAII.ConclusionICAⅡ can inhibit myocardial remodeling of cardiac and neonatal rat cardiac myocyte(NRCM)hypertrophy in vivo and in vitro by regulating AMPK/mTORC pathway.ICAⅡ may be a potential drug for treating or assisting in the treatment of malignant myocardial remodeling.