| Background:Cardiac remodeling is the key link of HF and generally accepted as a critical pathological process in the progression of HF heart failure,and also is the main cause of diastolic heart failure.Cardiac remodeling is a process secondary to the long-term cardiac overload induced by diseases,It is a compensatory change at the initial stage,followed by massive myocardial fibrosis and cardiomyocyte apoptosis and necrosis,which further lead to cardiac dysfunction.Persistent cardiac hypertrophy can eventually lead to dilated cardiomyopathy,heart failure and sudden death.Exploration of the mechanism for cardiac regeneration to inhibit the adverse cardiac remodeling is an important strategy for the treatment of HF due to the regeneration ability of the heart and the proliferation ability of myocardial cells,which will help to develop new strategies to replace apoptotic and necrotic myocardium cells.It has been reported that various signaling pathways are involved in the process of cardiac remodeling.Many drugs are currently available for the clinical treatment of cardiac remodeling in HF and these drugs target all process of cardiac remodeling.Therefore,the discovery of novel therapeutic targets against ventricular remodeling has become a research focus in the treatment of HF from the cellular molecular level.Caffeic acid phenethyl ester(CAPE)is one of the main components in propolis and a member of citrus flavonoids.It is a phenolic acid among other organic acids,with the structure of hydroxyphenylacrylic acid.CAPE exerts great anti-inflammation,antioxidation,antiapoptotic,anti-tumor,and immune regulation effects and is used in the prevention and treatment of various diseases related to inflammatory response,oxidative stress,ad viral infection,including cardiovascular disease,brain damage,human immunodeficiency virus(HIV)infection,leukopenia and thrombocytopenia,due to its antioxidant and anti-apoptotic functions.However,the role of CAPE in cardiac remodeling has not been reported yet.Therefore,this study will further investigate the effects of CAPE on cardiac hypertrophy and fibrosis during cardiac remodeling.Objective:The present study was designed to determine the changes of expression of CAPE under normal physiological state and pathological state corresponding to cardiac remodeling in an animal model established by AB to verify the function of CAPE in the process of cardiac remodeling and fibrosis;to clarify the definite mechanism of CAPE in the regulation of cardiac remodeling via in vitro experiments in H9C2 cells;and finally to discuss whether CAPE can serve as a novel therapeutic targets for cardiac remodeling based on the experimental data obtained.Protocols and Results:Section I:Myocardial hypertrophyAnimal experiment:8-10-week old healthy wild-type C57 male mice weighing 23.5-25.5g were randomly divided into four groups,i.e.,the sham operation control group(CON),the CAPE sham operation group(CAPE),the control AB group(AB),and the CAPE AB group(AB+CAPE).Mouse model was established by using AB method and then treated after the successful operation according to the groups assigned.CAPE was orally administered 3 days after the operation and samples were collected 6 weeks after the intervention.Expression of CAPE protein in the heart tissue was detected by Western blot.The following measurements were used to detect the process of cardiac remodeling and the related molecular mechanism.1.LVSd,LVDd,LVPWD,LVPWS,LVEF,and FS of mice in each group were measured by echocardiography.2.EF,FS,ESP,and dPdtmax and dPdtmin of mice in each group were detected by hemodynamic monitoring method.3.Heart weight(HW),lung weight(LW),and ratios of heart weight/body weight(HW/BW)and heart weight/tibial length(HW/TL)in each group were measured by anatomical methods.4.Heart volume and cross section area(CSA)of myocyte in mice in each group were detected by the HE staining of the tissue section and PSR staining.5.Levels of mRNA expression of proliferation markers,including ANP,BNP,a-myosin heavy chain(a-MHC)and ?-myosin heavy chain(?-MHC),were determined by real-time quantitative PCR.6.Expression of molecules in various signaling pathways in heart tissue was studied in each group.ResultsSection I:Myocardial hypertrophy1.Ultrasonic testing and hemodynamic monitoring method results:controlg roup LVDd(3.31 ±0.12)?LVDs(2.46± 0.13).LVPWd(90.68±0.07)?LVPWs(1.01 ±0.02)?ESP(100.82±3.80),EDP(12.04±0.23)?EF(81.25± 2.16),FS(46.00±1.99),dPdtmax(9653±262.97),dPdtmin(9138.5± 304.88)(P<0.01 vs CON),AB g roup LVDd(5.00±0.13).LVDs(3.80±0.07)?LVPWd(1.17±0.05)?LVPWs(1.33±0.03)?ESP(127.27±3.8),EF(58.87±2.62),FS(23.75±0.99)dPdtmax(6520.16±529.95),dPdtmin(6142.16±413.53)the difference is significant.(P<0.01 vs AB),After CAPE treatmen groupvLVDd(4.37± 0.14)?LVDs(3.27± 0.14)?L VPWd(0.816±0.02)?LVPWs(1.20±0.37)?ESP(139.11±5.2)?EDP(20.90±1.46)?EF(66.37± 1.32),FS(30.00±0.72)dPdtmax(8057.83±356.54),dPdtmin(7707.66±563.05)the difference is significant.(P<0.01 vs AB)2.Tissue morphology Results:control group BW(22.43±0.58),HW(114.87±8.45),LW(136.75±3.73),HW/BW(4.21 ±0.08),HW/TL(6.57±0.39);AB group(27.57 ±0.46),HW(215.87.87 ± 12.1),LW(168.23 ±3.94),HW/BW(7.62 ± 0.67),HW/TL(11.68±0.62)the difference is significant.(P<0.01,vs CON);After CAPE treatment group,weight gain to BW decreased to(25.38±0.50),H W(182.875±6.65),LW(155.87 ±4.24),HW/BW(6.43± 0.21),HW/TL(9.82± +0.32)the difference is significant.(P<0.01 vs.AB).3.HE staining of pathological tissue sections showed that the heart volume and the CSA of myocardial cells increased after AB,which were both significantly redu-ced by CAPE treatment.4.Real-time quantitative PCR indicated that the expressions of proliferation markers,including ANP,BNP,a-MHC and ?-MHC,were significantly increased in mice after AB compared with those of the C57 mice.the difference is significant.(P<0.01 VS CON),and CAPE significantly reduced the expression of these markers(P<0.01 VS AB).5.Western blot showed that the MEK/ERK signaling pathway was activated in the myocardium of mice by the pressure overload after AB and CAPE inhibited the activation of this signaling pathway.The difference is significant.(P<0.01 VS AB).Section ?:Animal experiment:8-10-week old healthy wild-type C57 male mice weighing 23.5-25.5g were selected to establish the model of pressure overload-induced myocardial remodeling using the AB method.CAPE was orally administered and samples were collected 6 weeks after the intervention.Animals were sacrificed by cervical dislocation and hearts were immediately removed and weighted.Lung weight and body weight of the mouse were recorded at the same time.The hearts collected were divided into two groups,and left ventricles in one group were collected and stored at-80? for Western blot ad RT-PCR assays and the hearts in the other group were dehydrated,embedded,and sectioned for pathological staining after they were arrested at the diastolic phase in 10%KCl.The HE staining was used for the CSA of myocyte in mice,PSR staining for the fibrosis area,and RT-PCR for the expression of fibrosis related biomarkers,such as TGF-?,collagen I,and collagen ?,in myocardial tissue of these mice.The following measurements were used to detect the process of myocardial fibrosis and the related molecular mechanism.1.Myocardial fibrosis in mice was detected by PSR staining in each group.2.Expression of mediators of myocardial fibrosis in mice was determined by PSR staining in a quantitative and real-time manner.3.Expression of the Smad cascade-related proteins was measured by Western blot.Results:1.PSR staining showed that fibrosis area in the left ventricle as well as was the volume and content of collagen were increased by the AB-induced pressure overload,which were significantly reduced by CAPE.2.Real-time quantitative PCR demonstrated that the expression of collagen I and collagen III increased significantly in myocardial tissue by pressure overload and CAP significantly decreased the expression of these fibrotic mediators,The difference is significant(P<0.01VS AB).3.Western blot results showed that the expressions of phosphorylated TGF-?,Smad 1/5,and Smad3 were increased significantly by pressure overload.Compared with the AB group,thelevels of phosphorylated TGF-?,Smad1/5,and Smad3 were significantly reduced in the CAPE +AB group,The difference is significant(P?0.01 VS AB).Section III:Cell experiment in vitroCell experiment:H9C2 rat cardiomyocytes were used to establish the isolated hypertrophy cell model through phenylephrine(PE)induction.H9C2 cells were divided into four groups,namely the PE stimulation group groups?the control group?CAPE groups and AB+CAPEgroups,and cultured in complete medium with or without PE(50?M),respectively,for 24 hours.Changes of CAPE protein in H9C2 cells over time were determined and the expression of CAPE was detected in the cells on slides by immunofluorescence.The expression of CAPE protein in myocardial tissue was detected by Western blot and the expression and localization of CAPE protein in myocardial tissue were determined by immunofluorescence assay.Results:1.The results of immunofluorescence assay showed that the surface area of H9C2 cells was enlarged after 24 hours of culture,and the surface area of H9C2 cells treated with CAPE in each group was decreased compared with that of the PE stimulation model group.2.The results of Western blot indicated that PE significantly activated the MEK/ERK signaling pathway and CAPE treatment inhibited the activity of the activated MEK/ERK signaling pathway,The difference is significant(P<0.01 VS AB).Conclusion:1.Chronic stress caused by aortic constriction can cause myocardial hypertrophy and myocardial fibrosis in mice.2.CAPE can be used to suppress the pressure load induced cardiac hypertrophic,which is achieved by blocking the MEK/ERK signaling pathway;3.CAPE can reduces the myocardial fibrosis caused by chronic stress load through the cascade protein signaling pathway TGF-beta,smad1/5 and Smad3.4.CAPE can inhibit the hypertrophy of myocardial cells induced by PE;The effect is achieved by blocking the MEK/ERK signaling pathway and further verifying the body research. |
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