Effects And Mechanism Of Wogonin On Cardiac Hypertrophy

BackgroundMyocardial hypertrophy is characterized by thickening of the ventricle wall in the heart,an adaptive response to,for example,mechanical and neurohumoral stimulation.At the early stage of myocardial hypertrophy,cardiomyocytes grow in length and/or width,the main reason for ventricular thickenin.However,if the stimuli continually exist,apoptosis of cardiomyocytes would occur and leads to heart failure,arrhythmia and sudden death.Thus myocardial hypertrophy is considered as the pathological foundation for multiple cardiac events.?-adrenoceptors,located in the membranes of the three major cardiac cell types,i.e.cardiomyocytes,fibroblasts,and endothelial cells,are members of the G protein-coupled receptors superfamily.Their stimulation activate downstream signaling pathways regulating different intracellular,sarcolemmal and myofibrillar substrates.Under neurohumoral stimulation or binding of catecholamine to ?-adrenoceptors,the related heterotrimeric G proteins dissociate into G?s/G?? and G?i/G?? subunits.The G?s is free to activate the adenylyl cyclase to generate the second messenger c AMP,leading to an increase in heart rate and myocardiac contractility.While G?i subunit activates PI3K/Akt and MAPK pathways signaling.Both pathways promote myocardial hypertrophy.Therefore,there lie potential targets for myocardial hypertrophy therapy in ?-adrenoceptors signaling pathway.Wogonin,5,7-dihydroxy-8-methoxyflavone,a natural dihydroxyl flavonoid compound isolated from the roots of Scutellaria baicalensi Georg,Scutellaria amoena C.H.Wright or Scutellaria rivularis Wall,processes of a variety biological activities,including anti-oxidation,anti-inflammation,neuroprotection andanti-carcinoma.A recent report shows that wogonin attenuates diabetic cardiomyopathy,however,whether wogonin attenuates ?-adrenoceptors mediated myocardial hypertrophy and the detailed molecular mechanism for its antihypertrophic effect remains unknown.Research content and Objective 1.Establishment of myocardial hypertrophy in vivo and examine theanti-hypertrophy effects of wogonin.2.Establishment of myocardial hypertrophy model in vitro and study theanti-hypertrophy molecular mechanism of wogonin.3.Investigate the anti-hypertrophy target of wogoninExperimental methods and Results The First Part Establishment of myocardial hypertrophy in vivo and examine the anti-hypertrophy effects of wogonin.Materials and Methods 1.Samples collection and preparation:(1)8-week-old mice(n =29)were under thedelivery of isoprenaline(5 mg /kg/day)was achieved by implanting an osmoticminipump.Immediately after implantation,wogonin was administered byintraperitoneal injection for two weeks with the dose of 1 mg/kg or 10 mg/kgonce daily.(2)The mice were divided into 5 groups,control,wogonin,ISO,lowdose of wogonin treatment group(Wog 1mg/kg+ISO),high dose of wogonintreatment group(Wog 10mg/kg+ISO)2.Echocardiographic assay to to evaluate the cardiac function and chamber size.The heart and body weight were measured after mice were sacrificed.3.Histology of heart was examined by hematoxylin and eosin(H&E).4.Real-time quantitative PCR was used to detect the expression of ANP and BNPm RNA levels in heart.Results 1.Wogonin reverse the increase in heart weight,although wogonin failed to preventthe isoprenaline-mediated reduction in body weight.Wogonin alone had noeffects on body weight or heart weight.Isoprenaline treatment increased HW,HW/BW,IVSd,LVPWd,LV Mass,which were all reversed by wogonin.But EF,the index of left ventricular systolic function,was neither worsen by isoprenalinenor affected by wogonin.2.Histologic analysis also displayed that the wogonin mediated attenuation on theenlargement in heart size and cross sectional area of myocardial fiber afterisoprenaline treatment.3.Wogonin alone did not affect the cardiac m RNA levels of ANP and BNP,but itreduced those elevated by isoprenaline treatment in dose dependent manner inmice.Summary Wogonin can improve isoproterenol induced cardiac hypertrophy in mice models.The Second Part Establishment of myocardial hypertrophy model in vitro and study the anti-hypertrophy molecular mechanism of wogonin.Materials and Methods 1.The rat cardiomyocyte cell line(H9C2)was stimulated with ISO(10 ?M)toestablish a cardiomyocyte hypertrophy model.The treatment group wasco-incubated with wogonin(1 ?M,10 ?M)for 24 h.2.Real-time quantitative PCR was used to detect the expression of ANP and BNPm RNA levels in H9C2 cell.3.?-SMA straining was used to observe the morphology of H9c2.4.Western blot was used to detect the expression of p-Akt,Akt,p-CREB,CREB,p-JNK,JNK,p-p38 MAPK,p38 MAPK,p-ERK1/2 and ERK1/2.5.H9c2 cells were treated with wogonin,isoprenaline and wortmannin for 24 h,p-Akt,Akt,p-Fox O1,Fox O1,p-Fox O3 a,Fox O3 a,p-CREB and CREB werefollowed by immunoblots.Results 1.Compared to controls,ISO(10 ?M)can significantly increase the m RNAexpression levels of ANP and BNP.Compared with the low-dose group Wug(1?M),the high-dose group wogonin(10 ?M)significantly improved ISO-inducedincreases in ANP and BNP expression.2.The detection of ?-SMA by immunofluorescence showed that ISO stimulationcould increase the size of H9C2 cells significantly,and that it could be reversedby wogonin.3.The phosphorylation levels of Akt,CREB,JNK,p38,and ERK weresignificantly increased with ISO stimulation,whereas the phosphorylation of Aktand CREB was reversed only by wogonin.To verify the PKC signaling pathway,H9c2 Cells were treated with wogonin and PDBU as indicated for 24 h.wedetected m RNA levels of ANP and BNP.However,wogonin has no effect thatPKC-induced transcription of ANP and BNP.4.The phosphorylation of CREB under the simulation of ISO is regulated by bothc AMP/PKA and PI3K/Akt pathways.Wogonin failed to reduce thephosphorylation of CREB induced by DB-CAMP.It implies that wogoninreduces CREB phosphorylation via PI3K/Akt instead of c AMP/PKA pathway.5.Wogonin inhibited the phosphorylation of Akt and its downstream Fox O1,Fox O3 a and CREB.Combined with wogonin and wortmannin.However,thecombination of wogonin and wortmannin did not further enhance their inhibitoryeffect.Thus we reasoned that wogonin specifically worked on the PI3K/Aktsignaling pathway.Summary Wogonin attenuated the hypertrophy of H9C2 induced by isoproterenol byinhibiting the PI3K/Akt signaling pathway..The Third Part Investigate the anti-hypertrophy target of wogoninMaterials and Methods 1.H9c2 cells were transfected by p USEamp(+)-Akt(CA)or p USEamp(+)-Pik3caand grew for 48 h.Then wogonin was added for 24 h before harvesting.Westernblot was used to detect the expression of Pik3 ca,p-Akt,Akt,p-Fox O1,Fox O1,p-Fox O3 a,Fox O3 a,p-CREB and CREB.2.H9c2 cells were transfected by p USEamp(+)-Pik3 ca and grew for 48 hours.Thenwogonin was added for 24 hours before harvesting.?-SMA straining was used toobserve the morphology of H9c2.3.H9c2 cells were treated with the wogonin(10?M)and isoprenaline(10?M)for 24hours.Then cells were treated by MG132(5 ?M)for 8 hours before harvesting.Cell extracts were immunoblotted by Pik3 ca antibody.The m RNA level of Pik3cawas determined by q RT-PCR.H9c2 cells over-expressing HA-tagged PI3 KCA,were treated with wogonin(10 ?M,24 h)and MG132(5 ?M,8 h)subsequently.Cell extracts were immunoprecipitated by HA-beads and immunoblotted withantibodies against ubiquitin and HA.4.H9c2 Cells were treated with wogonin and isoprenaline as indicated for 24 hours.The protein expression and m RNA level of Nedd4 l was determined byimmunoblots and q RT-PCR.H9c2 cells were transfected withp Adeno-MCMV-MCS-3Flag-Nedd4 l expression vectors and grew for 48 h.Cellswere treated with wogonin(10 ?M)for 24 h before harvesting.Cell extracts were immunoblotted with Flag and ?-actin antibody.H9c2 cells were transfected with p GL3--2000/+1LUC Nedd4 l constructs with wogonin(10 ?M)treatment for 24 h.Nedd4 l promoter driven luciferase activity were detected.Results 1.Akt(CA)significantly enhanced the phosphorylation of Fox O1,Fox O3 a andCREB,which were not affected by wogonin.Wogonin reversed the PI3K/Aktactivation via reducing Pik3 ca protein level.Consistently,?-SMA stainingshowed that Pik3 ca overexpression amplified the size of H9c2 cells and wogoninalso reversed that pathological change.2.Pik3 ca got significant increase in protein and m RNA levels under isoprenalinetreatment in H9c2 cells.While,wogonin reduced the protein level of Pik3 ca,butfailed to decrease its m RNA level.3.We utilized MG132 found that wogonin induced the extensive enhancement inubiquitination of proteins,while MG132 reversed wogonin-induceddownregulation of Pik3 ca.HA-tagged Pik3 ca was transfected into H9c2 cells andtreated by wogonin and MG132 subsequently,immunoprecipitation suggestedthat the precipitated Pik3 ca got higher ubiquitination level after wogonintreatment.4.Isoprenaline treatment significantly reduced the m RNA and protein levels ofNedd4l in H9c2 cells.Wogonin reversed the inhibitory effect of isoprenaline onNedd4l expression at transcriptional and post-transcriptional levels.We expressedFlag-tagged Nedd4 l in H9c2 cells by transient transfection,and found thatwogonin treatment did not affect the protein levels of exogenous Nedd4 l.It isconformed in the luciferase assay,since wogonin significantly enhanced theNedd4l promoter-driven luciferase activity.SummaryWogonin?Ehanced the expression of Nedd4l-kisspeptin expression?Improvedthe ubiquitination of Pik3ca?Promoted protein degradation of Pik3ca?Inhibited PI3K/Akt signaling pathway.ConclusionThe present investigation reveals that wogonin targets Nedd4 l and elevates its expression of Nedd4 l in cardiomyocytes,by which promotes the ubiquitination and degradation of its substrate,Pik3 ca.Thus wogonin suppresses the signaling transduction in ?-adrenoceptor/PI3K/Akt pathway medicating the expression of hypertrophy-related genes,and thus ameliorates myocardial hypertrophy induced by isoprenaline treatment in mice.