| Background and ObjectivesCurrent studies have demonstrated that in the progression of cardiac insufficiency caused by various cardiac disorders, such as chronic ischemic heart disease, myocardial infarction, valvular heart disease,and myocarditis,there are a series of changes including myocardium hypertrophy and vascular hyperplasia. These changes, called ventricular remodeling, are one of the major causes that eventually induce heart failure. In particular, left ventricular hypertrophy in the course of ventricular remodeling has been proved as an independent risk factor for heart failure . In addition, hypertrophic ventricular cardiomyocytes have been found to undergo transition from compensatory hypertrophy to apoptosis. It has been shown that during the progression of compensatory left ventricular hypertrophy to failure, loss of cardiomyocyte plays an important role and the development of cardiac disorders is usually accompanied with cardiomyocyte apoptosis . Meanwhile,myocardial fibrosis act an importment role in ventricular collagen remodeling .It has become a new target of heart failure therapy.Peroxisome proliferator activated receptors (PPARs) is one kind of nuclear transcription factors, the activation of which is mediated by their corresponding ligands. There are three subtypes of PPARs: PPARα,PPARβ(also known as PPARδ) and PPARγ. Their ligands are both receptor- and tissue-specific, and show various biological effects. PPARβ/δis found in almost all tissues, but its physiologic functions remain unclear. PPARαand PPARγcan promote adipocyte differentiation and fat production, enhance the sensitivity of body to insulin, modulate sugar balance, inhibit inflammatory factor production and inflammation occurrence, etc. In recent years, PPARαand PPARγhave also been found to have a capacity of protecting the cardiovascular system, which, however, is paradoxically.The aim of this study is to research the effects of Fenofibrate and Pioglitazone, two activators of PPARαand PPARγ, on the cardiac hypertrophy in vitro and in vivo. To explore the role of the PPARs signal pathway in hypertrophy, apoptosis and fibrosis of cardiomyocytes during ventricular remodeling.Methods1. Cell culture:1).The neonatal rat cardiomyocytes were cultured by the method of digestion step by step and adherence with different speeds,and with Angiotensin II(final concentration10-7mmol/L ) stimulation ,a model of hypertrophy and apoptosis of neonatal rat cardiac myocytes was established. PPARαligands, Fenofibrate and PPARγligands,pioglitazone pretreatment ,10μmol/L,24h prior to AngII. With the aid of Leica Qwin Image software, the surface area of cardiac myocytes was analysis. The mRNA expression of PPARα,PPARγ,α-MHC andβ-MHC was measured by reverse transcription-polymerase chain reaction(RT-PCR) and the cultured myocyte viability was estimated by MTT assay .Annexin V-FITC and PI staining and then Flow cytometry was used to monitor the apoptosis cells. Using Western blot , the proto-oncogene BCL-2/Bax and Fas/Fas-L expression was observed; 2). Cardiac fibroblasts(CFs) were isolated by trypsin digestion method. CFs in third passage in this study. Then the cultured rat cardiac fibroblasts were divided randomly into five groups :control group ,AngII stimulation group(10-7mol/L), Fenofibrate pretreatment group(10μmol/L), Pioglitazone pretreatment group(10μmol/L) and concomitant fenofibrate and pioglitazone group . Cell cycle kinetics of CFs was analyzed by flow cytometry. Collagen synthesis rate was determined by measuring MTT.2. Animal experiment: To study associated pathologic and pathophysiologicchanges of myocardial hypertrophy, the pressure overloading model was established by the constriction of abdominal aorta in Wistar rats. Forty-eight hours after the procedure , the surviving rats were randomly divided into three time stages to breed, that is, 4, 8 weeks.Then the rats in every time stage were also randomly divided into five groups: (1) coarctation of abdominal aorta(CAA)controls group, (2) fenofibrate(F group,30 mg·kg- 1·d - 1 ), (3) pioglitazone(P group, 3 mg·kg- 1·d - 1) , (4) concomitant fenofibrate and pioglitazone (F+P group, 30mg·kg- 1·d - 1and 3mg·kg- 1·d - 1)groups ,and (5) sham-operated(SH) group was selected to serve as non-pressure overload controls.There are ten groups in all.Fenofibrate and pioglitazone were delivered by direct gastric gavage.Heart rate (HR), left ventricular end diastolic pressure (LVEDP), left ventricular systolic pressure (LVSP), mean arterial pressure (MAP) and left ventricular pressure maximal rising and declining velocities(±dp/dtmax), and the ratio of left ventricular weight to body weight (LVW/BW) and the ratio of right ventricular weight to body weight(RVW/BW). And the rennin activity,level of angiotensin II and aldosteron in Plasma and myocardial were detected by radioimmunity. The morphologic features of myocardiac cells were observed by transmission electron microscopy. DNA fragmentations were determined semiquantitatively by in situ TDT-mediated dUTP nick end labeling ( TUNEL). After Sirius red trinitrophenol staining, myocardial collagen volume fraction (CVF) were determined by Leica analysis software. PPARα,PPARγ,α-MHC/β-MHC , AT1, types I and III collagen mRNA expressions were assessed with reverse transcription polymerase chain reaction (RT-PCR). Using Western blot, the proto-oncogene Bcl-2/Bax and Fas/FasL expression were observed.Results1.Fenofibrate and pioglitazone pretreatment 24h prior to AngII, Significantly (p<0.01-0.05) reduced AngII-induced cardiac hypertrophy,increased expression ofα/β-MHC mRNA,and inhibited the effect of Ang II on the cardiac myocyte viability .There were no significant differences in the above mentioned indices between fenofibrate and pioglitazone group(P >0. 05) .2.Fenofibrate and pioglitazone pretreatment significantly(P<0.05) reduced angiontensinII-induced cardiocyte apoptosis by Flow cytometry analysis.Significantly inhibited the expression of Fas/FasL and Bax, while enhanced the expression of Bcl-2 and Bcl-2/Bax ratio.3. AngII significantly increased the proliferation index [(S+G2/M )/(S+G2M +G0/G1 )] of CFs with low concentration (final concentration10-7mmol/L ). Fenofibrate and pioglitazone pretreatment ,10μmol/L,24h prior to AngII, inhibited the effects of the proliferation induced by AngII (P<0. 01). Fenofibrate and pioglitazone pretreatment decreased the OD numerus of MTT assay induced by AngII for 24 h (P < 0. 05).4.In CAA8W group characteristic features of apoptotic myocardiac cells was recognized by transmission electron microscopy. We discovered the two drugs treatment significantly(P<0.05) reduced cardiocyte apoptosis in pressure-overload induced when they treatment 8 weeks. Compared with sham-operated rats ,the Bax/Bcl-2 and Fas/ FasL expressions increased at protein levels in all CAA groups (all P < 0. 05) . Compared with the CAA group , Bax/Bcl-2 and Fas/ FasL expressions were significantly decreased in 8 weeks treatment groups ( P < 0. 05 - 0. 001 ). There were no significant differences in the above mentioned indices among the four treatment groups in 4 weeks(all P > 0. 05) .5. At 4, 8 weeks after the operation, LVW/BW and CVF were increased significantly in CAA group,fenofibrate group and pioglitazone group compared with those in sham operated group, but those in fenofibrate and pioglitazone 8 weeks group were significantly lower than that of CAA group.Meanwhile,fenofibrate and pioglitazone significantly decreased the types I and III collagen mRNA expressions of the left ventricular myocardium induced by CAA. There were no significant differences in the above mentioned indices among the four treatment groups in 4 weeks(all P > 0. 05) .6. Compared with sham-operated rats , the left ventricular end diastolic pressure (LVEDP) , left ventricular systolic pressure(LVSP),mean arterial pressure(MAP)and the maximum left ventricular pressure rising rates (+dp/ dt),and the LVW/BW , mRNA of AT1 as well as plasma and myocardial angiotensin II and aldosteron were significantly increased in all CAA groups (all P < 0. 05) ,accompanied with heart rate(HR) and the maximum left ventricular pressure declining rates (-dp/ dt) were decreased.RVW/BW is no change in all groups, indicating that left ventricular remodeling(LVRM) occurred after CAA.Compared with the CAA group ,LVW/BW ,MAP ,LVSP ,LVEDP,HR were all significantly decreased when drug treatment 8 weeks ( P < 0. 05 - 0. 001 ), while corrected -dp/dt was significantly enhanced ( P < 0. 05 - 0. 001). The three drug treatment groups had no effect on plasma and myocardial angiotensin II and aldosteron activity(p>0.05). Pioglitazone treatment 8 weeks can downregulation the mRNA expression of AT1 while fenofibrate group had no effect on it. There were no significant differences in the above mentioned indices among the three drug treatment groups except AT1 mRNA (all P > 0. 05).Conclusions1. It is suggested that PPARα,PPARγactivators inhibit hypertrophy of cardiac myocytes and PPARs-dependent pathway be involved in the inhibitory course.2. Chronic treatment with the PPARαand PPARγagonist may be useful in preventing cardiac hypertrophy and apoptosis in vitro(24h) and vivo(8weeks), which is related the regulation of the changes of proto-oncogene Bax/Bcl-L , Fas and FasL expression.3. AngII can enhance proliferation in CFs and increase basal collagen syntheses. PPARαand PPARγactivators may attenuate myocardial fibrosis induced by AngII by inhibiting the above effects.4. PPARαligands(fenofibrate) and PPARγligands(pioglitazone) chronic treatment(8 weeks) can ameliorate pressure overload rat's left ventricular hypertrophy and hemodynamic parameters. 5. PPARαand PPARγagonist can attenuate remodeling of myocardial collagen network in pressure overloaded rats.6. Peroxisome proliferator-activated receptors ligands had no effect on plasma and myocardial angiotensin II and aldosteron activity. But mRNA expression of AT1 was upregulated by activing PPARγsignaing pathway . PPARα,γligands can improve left ventricular remodeling (LVR).7. There is no additive effect when the two drugs were used in combination.
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