Experimental Study On Qindan Capsule And The Effective Components Quercetin Reversing Ventricular Hypertrophy In Spontaneous Hypertensive Rats

BackgroundHypertension is one of the familiar risk factors for the human health. Ventricular hypertrophy is a common complication of hypertension, and it is an independent risk factor for sudden cardiac death and the other cardiovascular events. Ventricular hypertrophy is consist of cardiomyocyte hypertrophy, myocardial interstitial cell proliferation, extracellular matrix accumulation pathologically. Therefore, to explore ventricular hypertrophy mechanisms of hypertension is important for prevention and treatment of hypertensive target organ damage and reduce mortality.Peroxisome proliferator-activated receptor y (peroxisome proliferater-activated receptor-y, PPAR-y) is a ligand-activated nuclear transcription factor superfamily members. Recent studies have confirmed that PPARy plays a critical role in inhibition of cardiac hypertrophy both in vitro and in vivo. NF-κB activation was essential to the development of cardiac hypertrophy in recent years and inhibition of NF-κB activation may be important targets for myocardial hypertrophy. Furthermore, we also have shown previously that PPAR-y regulates gene expression in a DNA-independent fashion by interfering with other signaling pathways, such as the NF-κB pathwayQindan Capsule(QC), a prepared compound of traditional Chinese medicine, has been used as an anti-hypertensive agent for years in clinic. It has shown that QC plays an important role in attenuating vascular remodeling in our previous study.In the present study,the changes of the pathology of myocardial hypertrophy were observed in SHR, and the variation of ANP and BNP mRNA expression was detected by RT-PCR, and the variation of PPARy and NF-κB was detected by immunohistochemistry. In order to discover novel agents on inhibiting myocardial hypertrophy and improving cardiac function, the possible mechanism of QC on improveing ventricular hypertrophy in the spontaneously hypertensive rats was investigated. It could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.Aim1. To investigate the anti-hypertrophy effects of QC through observing the changes of the morphology and the ultrastructure in myocardial tissues.2. To investigate the possible mechanism of QC in reversing myocardial hypertrophy in hypertension through observing the changes of the PPARy and NF-κB and the variation of ANP and BNP mRNA expression.Methods40male (8weeks old) SHRs and8male (8weeks old) Wistar Kyoto (WKY) rats were randomly divided into5groups(n=8):the WKY control group, the SHR control group, the Telmisartan (SHR+Tel)group, the high dosage QC group (SHR+QCL), and the low dosage QC group (SHR+QCL).The SHR+Tel group was treated with Telmisartan for10mg/kg per day, the SHR+QCH group was treated with high dosage (750mg/kg per day) of QC, and the SHR+QCL group was treated with low dosage(150mg/kg per day)of QC. The WKY group and the SHR group were treated with equal volume of distilled water instead. All the treatments were administered by gastrogavage once a day for12successive weeks.After the last administration, all rats were fasted with free access to water for24h, and anesthetized with intraperitoneal injecfion of3%pentobarbital (30mg/kg)which was followed by other experiments.The detection contents listed below:(1) Measurement of the systolic blood pressure (SBP);(2) The left ventricular weight (LVW) and body weight (BW) ratio was determined;(3) Observation of cardiomyocyte morphological by means of HE;(4) The ultrastrueture of myocardial cell was observed with transmission electron microscope;(5) Determination of the levels of PPARy,NF-κB in myocardial by immunohistochemistry;(6)Analysis of ANP and BNP mRNA expression by real-time PCR. Statistical analysisThe data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared. Differences were considered to be statistically significant when the P values were less than0.05.Results1. Comparison of SBPThe SBP of rats in the SHR group with8weeks old was much higher than those in the WKY group(P<0.05). The rats in the SHR group continuously developed further hypertension to a high pressure level. After12weeks, the SBP of rats in the SHR group with20weeks old was significantly higher than those in the WKY group (P＜0.05). From the second week of treatment, the SBP was lower in the SHR+Tel group, the SHR+QCH group, and the SHR+QCL group than that in the SHR group(all P＜0.05). After six weeks of treatment,the SBP in the SHR+QCH group was decreased significantly compared with that in the SHR+QCL group(P＜0.05). After twelve weeks of treatment, the SBP in each group was decreased in different degree compared with the SHR group(all P＜0.05). The SBP in the SHR+QCH group and the SHR+Tel group was lower than that in the SHR+QCL group(P＜0.05), but no significant difference was shown in comparison among these two groups (P＜0.05).2. Left ventricular relative mass(LVM/BW)At the end of the experiment, the left ventricular weight index (LVW/BW) in SHRs was significantly greater than in WKY rats. This parameter was dose-dependently and significantly decreased in QC or Tel-treated SHRs compared with distilled water-treated SHRs (P＜0.05).3. Observation of seetions of myocytes stained with HEPathological changes were significantly attenuated by QC or Tel. Hematoxylin and eosin staining was performed for all groups. Compared with the WKY group, cardiac muscle fibers were enlarged and disorganized in the SHR group. Hematoxylin and eosin-stained myocardial tissue in the QC or Tel-treated SHR groups showed reductions in the size of the cardiomyocytes and reduced fibrosis. In addition, cardiac muscle fibers were also better-arranged.4. Ultrastructural changes of myocytes observed by transmission electron microscopy.In the WKY group, arrays of myofibrils were closely arranged in an orderly manner within the sarcomere, and mitochondria were of normal size and present in normal numbers. However, in the SHR group, the mitochondrial structure was damaged severely and cellular or tissue swelling was apparent in myocardium. Most myofibrils had either disappeared or were poorly arranged. Mitochondria were noticeably swollen and loosely arranged. In addition, mitochondrial membranes were vague or partly ruptured and cristae were clearly loose or dissolved with many vacuoles. The significant change in ultrastructural organization of mitochondria and myofibrils was attenuated in the SHR+QCH group, the SHR+QCL group and SHR+Tel group with more obvious improvements noted in the high-dose group.5. Immunohistochemistry detection(1) Comparison of expression of PPAR-y protein:The expression of PPAR-y protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the three treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).(2) Comparison of expression of NF-κB protein:The expression of NF-κB protein was higher in the SHR group than that in the WKY group (P＜0.05).The expression of NF-κB protein was decreased in the three treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).6. Comparison of expression of ANP and BNP mRNA(1) Comparison of expression of ANP mRNA:The expression of ANP mRNA was higher in the SHR group than that in the WKY group (P＜0.05).The expression of ANP mRNA was decreased in the QC or Tel treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).(2) Comparison of expression of BNP mRNA:The expression of BNP mRNA was higher in the SHR group than that in the WKY group (P＜0.05).The expression of BNP mRNA was decreased in the QC or Tel treatment groups than that in the SHR group and QC revealed a dose-dependent manner (P＜0.05).Conclusions(1) A reversing effect of Qindan capsule on the hypertensive ventricular hypertrophy in SHR was observed in our present study. Qindan capsule could improve the morphological index of myocardial tissue and reduce myocardial tissue collagen content. Furthermore, it improves cardiac function and myocardial ultrastructure.(2) Qindan capsule may inhibit cardiac hypertrophy by enhancing PPAR-y expression partly and by suppressing the NF-κB signaling pathway, resulting inhibition the transeription of its downstream gene expression of ANP and BNP. BackgroundHypertension is one of the familiar risk factors for the human health. Cardiac hypertrophy results in congestive heart failure and is a major world-wide cause of sudden death. The hypertrophic response in cardiomyocytes is characterized by an enlargement of myocytes, an increase in the content of contractile proteins, and expression of embryonic genes such as atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP). The hypertrophic response is compensatory at an early stage of various cardiac diseases, but sustained extracellular stimuli may lead to excessive cardiac hypertrophy and finally to heart failure.Peroxisome proliferator-activated receptors (PPARs) are transcription factors belonging to the nuclear receptor gene family. In recent study also demonstrated the PPAR-y-dependent pathway is critical in the inhibition of cardiac hypertrophy both in vivo and vitro. One major transcription factor is the activator protein1(AP-1) complex, which is composed of the Jun and Fos family of DNA binding protooncoproteins. The c-Jun and c-Fos proteins form dimmers that bind DNA through specific response elements to transactivate the transcription of genes downstream from these enhancer elements.The activation of AP-1has been shown to be involved in the regulation of a variety of cellular processes such as proliferation, differentiation, and transformation. Moreover, recent studies have demonstrated that myocardial AP-1DNA binding activities are significantly increased in experimental cardiac hypertrophy in cardiovascular disease. It can regulate mang hypertrophy gene including such as ANP and BNP. Recently, it has been reported that a dominant negative mutant lacking the transactivating domain of c-Jun (DNJun) inhibits endothelin1(ET)-1and phenylephrine (PE)-induced cardiomyocyte hypertrophy. Furthermore, we also have shown that PPAR-y regulates gene expression in a DNA-independent fashion by interfering with other signaling pathways previously, such as the activator protein-1(AP-1) pathway.Quercetin (3,3’,4’,5,7-pentahydroxyflavone) is a member of a group of naturally occurring compounds and is one of the most widely distributed bioflavonoids. The very extensive biological effects of flavonoids, including their anti-inflammatory, anti-oxidant, anti-atherosclerotic, and anti-hypertensive properties facilitate an important role for quercetin in the prevention of cardiovascular diseases. Furthermore, it has also been revealed to show agonistic effects on peroxisome proliferator activated receptors and attenuates Monocyte Chemoattractant Protein-1gene expression in glucose-primed aortic endothelial cells via AP-1.In the present study, the changes of the pathologies of myocardial hypertrophy were observed in SHR rats, and the variation of PPAR-y and AP-1(C-Jun, C-Fos) was detected by immunohistochemistry or western blot, and the variation of its downstream target genes including ANP and BNP mRNA expression was detected by real-time RT-PCR. In order to discover novel agents on inhibiting myocardial hypertrophy and improving cardiac function, the possible mechanism of quercetin on improveing ventricular hypertrophy in the spontaneously hypertensive rats was investigated, which could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.Aim1. To investigate the anti-hypertrophy effects of quercetin through observing the changes of the morphology and the ultrastructure in myocardial tissues.2. To investigate the possible mechanism of quercetin in reversing myocardial hypertrophy in hypertension through observing the changes of the PPAR-y and AP-1(c-fos, c-jun) and the variation of ANP and BNP mRNA expression. MethodsTwenty four8-week-old male SHRs and eight8-week-old male Wistar-Kyoto (WKY) rats obtained from Slaccas (Shanghai, China) were used in this study. Animals were housed in a temperature-controlled (22±0.5℃) room, and rats had free access to standard rodent chow and water. A12-h/12-h light/dark cycle was maintained. Spontaneously hypertensive rats were randomly divided into three groups:one group (SHR) was treated with vehicle (1ml of1%methylcellulose) and the other two groups were treated with either a low dose of quercetin (SHR+QL;5mg.kg-1, mixed in1ml of1%methylcellulose) or a high dose of quercetin (SHR+QH;10mg.kg-1, mixed in1ml of1%methylcellulose). Age-matched WKY rats were treated with vehicle (1ml of1%methylcellulosa) and used as a control group. Quercetin or vehicle was delivered by gavage at the same time once daily for12weeks.After the last administration, all rats were fasted with free access to water for24h, and anesthetized with intraperitoneal injection of3%pentobarbital(30mg/kg)which was followed by other experiments. The detection contents listed below:(1)Measurement of the systolic blood pressure (SBP);(2)The common echocardiographic indexes were detected before at the first and at the end of the experiment;(3) the left ventricular weight (LVW) and body weight (BW) ratio was determined;(4)Measurement of the morphological index of cardiomyocyte diameter of myocytes and volume fraction of collagen;(5) The ultrastrueture of myocardial cell was observed with transmission electron microscope.(6) Analysis of PPAR-γ、 AP-1(c-fos, c-jun) protein expression and location by immunohistochemical detection.(7)Analysis of PPAR-γ AP-1(C-Jun, C-Fos) protein expression by western blot.(8)Analysis of PPAR-γ,AP-1(C-Jun, C-Fos),ANP and BNP mRNA expression by real-time PCR;Statistical analysisThe data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared.Differences were considered to be statistically significant when the P values were less than0.05.Results1. Comparison of SBPIn the week prior to treatment with quercetin, systolic blood pressure in the SHRs was significantly increased compared with the WKY (P＜0.05). Twenty weeks of quercetin administration induced a significant reduction in SBP in the SHRs, and this effect reached statistical significance after the first week of treatment (P＜0.05). From the second week of treatment, SBP was significantly decreased in the quercetin-treated groups compared with the SHR group (P＜0.05), and the decline in the SHR+QH group was distinct from that of the SHR+QL group (P＜0.05) and was significantly distinct from the7th to the12th week (P＜0.05).2. Left ventricular weight index (LVW/BW)Neither SHRs nor WKY rats showed a change in body weight after treatment with quercetin or vehicle. The left ventricular weight index (LVW/BW) in SHRs was significantly greater than in WKY rats. This parameter was dose-dependently and significantly decreased in quercetin-treated SHRs compared with vehicle-treated SHRs (P＜0.05).3. Echocardiographic detectionCompared with the WKY group, LVPWd and IVSd were both significantly increased at week1or12of the experiment but LVIDd decreased by the12th week in the SHR group (P＜0.05). After12weeks of quercetin treatment, LVIDd was significantly increased but IVSd and LVPWd were apparently decreased compared with the SHRs (P＜0.05). Furthermore, the mid-wall fractional shortening and E/A decreased significantly in SHRs and were significantly improved at week12in SHRs treated with quercetin (P＜0.05).4. Observation of seetions of myocytes stained with HEPathological changes were significantly attenuated by quercetin. Hematoxylin and eosin staining was performed for all groups. Compared with the WKY group, cardiac muscle fibers were enlarged and disorganized in the SHR group. Hematoxylin and eosin-stained myocardial tissue in the quercetin-treated SHR groups showed reductions in the size of the cardiomyocytes and reduced fibrosis. In addition, cardiac muscle fibers were also better-arranged.5.Comparison of Mean cardiomyocyte diameter of myocytesMean cardiomyocyte diameter was increased significantly in the SHR group compared with the WKY group(P＜0.05). Mean cardiomyocyte diamete was decreased significantly in the SHR+QH and the SHR+QL groups compared with that in the SHR group(P＜0.05).Quercetin administration dramatically and dose-dependently attenuated the enlargement in myocyte size compared with SHRs (P＜0.05).6.Ultrastructural changes of myocytes observed by transmission electron microscopy.In the WKY group, arrays of myofibrils were closely arranged in an orderly manner within the sarcomere, and mitochondria were of normal size and present in normal numbers. However, in the SHR group, the mitochondrial structure was damaged severely and cellular or tissue swelling was apparent. Most myofibrils had either disappeared or were poorly arranged. Mitochondria were noticeably swollen and loosely arranged. In addition, mitochondrial membranes were vague or partly ruptured and cristae were clearly loose or dissolved with many vacuoles. The significant change in ultrastructural organization of mitochondria and myofibrils was attenuated in the quercetin-treated SHR groups with more obvious improvements noted in the high-dose group.7. Observation of sections of myocytes stained with MassonMasson’s trichrome staining was performed to assess the effect of quercetin on cardiac fibrosis. The WKY myocardium showed a normal array of myocardial fibers and very little interstitial collagen. The SHR group showed many newly formed collagen deposits between myocardial interstices and myocardial disarrangement and cellular swelling as compared with the WKY group. Myocardial collagen deposition was greater in the SHR+QH group and the SHR+QL group than that in the WKY group, but was less than that in the SHR group.8. Comparison of the volume fraction of collagenThe VFC of myocytes in the SHR group was higher than that in the WKY group(P＜0.05). The VFC in the SHR+QH group and the SHR+QL group were lower than that in the SHR group (P＜0.05).Quercetin administration dramatically and dose-dependently attenuated the increase in collagen volume fraction compared with SHRs (P＜0.05).9.Immunohistochemical detection(1) Comparison of expression of PPAR-y protein:The nucleus expression of PPAR-y protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The nucleus expression of AP-1(C-Jun, C-Fos) protein was higher in the SHR group than that in the WKY group(P＜0.05).The expression of AP-1(C-Jun, C-Fos) protein was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner.(P＜0.05).10. The mRNA expression of related to hypertrophy gene were detected by RT-PCR(1) Comparison of expression of PPAR-y mRNA:The expression of PPAR-γ mRNA was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y mRNA was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).(2) Comparison of expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA:The expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA was higher in the SHR group than that in the WKY group(P＜0.05).The expression of c-fos mRNA, c-jun mRNA, ANP mRNA and BNP mRNA was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05). 11. The Protein levels of PPAR-y and AP-1(C-Jun, C-Fos) were detected by western blot(1) Comparison of expression of PPAR-y protein:The expression of PPAR-γ protein was lower in the SHR group than that in the WKY group(P＜0.05).The expression of PPAR-y protein was increased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).(2) Comparison of expression of AP-1(C-Jun, C-Fos) protein:The expression of AP-1(C-Jun, C-Fos) protein was higher in the SHR group than that in the WKY group (P＜0.05).The expression of AP-1(C-Jun, C-Fos) protein was decreased in the two treatment groups than that in the SHR group in a dose-dependent manner (P＜0.05).Conclusions:(1) A reversing effect of quercetin on the hypertensive ventricular hypertrophy in SHR was observed in our present study. Quercetin could improve the morphological index of myocardial tissue and reduce myocardial tissue collagen content. Furthermore improves cardiac function and myocardial ultrastructure.(2) Quercetin may inhibit cardiac hypertrophy by enhancing PPAR-γ expression partly and by suppressing the AP-1signaling pathway, resulting inhibition the transcription of its downstream gene expression of ANP and BNP. BackgroundThe changes of cardiac hypertrophy are the adaptive reaction to the disorders of hemodynamics, neurohumor and local endocrinic factors in hypertension.The hypertrophic response in cardiomyocytes is characterized by an enlargement of myocytes, an increase in the content of contractile proteins, and expression of embryonic genes such as atrial natriuretic peptide(ANP) and brain natriuretic peptide (BNP). The hypertrophic response is compensatory at an early stage of various cardiac diseases, but sustained extracellular stimuli may lead to excessive cardiac hypertrophy and finally to heart failure. Therefore, it is important to reverse the cardiac hypertrophy for prevention and treatment of hypertension and target organ damage.The renin-angiotensin system(RAS) plays a major role in the regulation of blood pressure. AngiotensinⅡ is one of the most important factors in RAS. It is shown one of the important factors to myocyte hypertrophy both in vivo and in vitro experiments. Ang Ⅱ has direct growth-promoting effects on the cardiac and vascular cells, in part via induction of the expression of various growth factors, extracellular matrix proteins and cytokines. In vitro, Ang II could activates a number of inducible transcription factors such as activator protein-1(AP-1), which in turn make impact on the expression of genes that regulate cell growth and extracellular matrix protein biosynthesis. Activator protein-1could be made up of c-fos and c-jun, which together make a heterodimer complex that plays a significant role in cardiomyocyte hypertrophy, and direct inhibition of AP-1activity significantly declined cardiac hypertrophy in myocardial tissue. Therefore, initiation of myocyte hypertrophic growth is accompanied by a rapid and transient expression of immediate-early genes such as c-fos, c-jun at the genetic level, followed by an activation of the fetal gene regulatory program with reexpression of genes for atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP),increase contractile proteins and transformation to embryonal Protein Phenotype.These observations have contributed to speculation that inhibition of these hypertrophic signals may be effective, patent strategies in the treatment of pathological hypertrophy and heart failure.Quercetin (3,3’,4’,5,7-pentahydroxyflavone) is a member of a group of naturally occurring compounds and is one of the most widely distributed bioflavonoids. The very extensive biological effects of flavonoids, including their anti-inflammatory, anti-oxidant, anti-atherosclerotic, and anti-hypertensive properties facilitate an important role for quercetin in the prevention of cardiovascular diseases. Furthermore, it has also been revealed to show agonistic effects on peroxisome proliferator activated receptors and attenuates Monocyte Chemoattractant Protein-1gene expression in glucose-primed aortic endothelial cells via AP-1.The above results suggest the quercetin may inhibits cardiac hypertrophy by blocking AP-1(c-fos, c-jun) and activating PPAR-y signaling pathways in vivo, but the possible mechanism of quercetin direct inhibition of cardiac hypertrophy are still unknown in vitro. In the present study, the changes of the protein synthesis and surface area of cells were observed in H9C2cells, and the variation of PPAR-γ and AP-1(c-fos,c-jun) was detected by immunofluorescence. The variation of PPAR-y and AP-1(c-fos,c-jun) was detected by western blotting and real time-PCR, and the variation of ANP and BNP mRNA expression was detected by real-time RT-PCR after transfection with PPAR-γ siRNA in H9C2cells. In order to discover novel agents on inhibiting H9C2hypertrophy, the possible mechanism of quercetin on improveing cardiac hypertrophy in the H9C2cells cell hypertrophy was investigated, which could provide scientific evidence for the treatment of myocardial hypertrophy heart disease in clinic.Aims(1) This study used the H9C2cells induced by angiotensin Ⅱ as cardiac hypertrophy model to observe the influence of H9C2cell surface area, the [3H] leucine incorporation at different concentration of quercetin stimulus.(2) To observed the quercetin impact on transcription factor C-Fos, C-Jun protein expression, detection of specific indicators of cardiac hypertrophy such as ANP and BNP mRNA expression by mean of siRNA blocking PPAR-y pathway.Methods1. Culture H9C2cell line routinely. The hypertrophy of H9C2cell was induced with Angiotensin Ⅱ, and intervened with quercetin at different concentration.2. As the index of cardiomyocyte hypertrophy, the cell size was determined by phase contrast microscope and protein synthesis rate was measured by [3H]-Leucine incorporation.3. Immunofluorescence was used to detect the protein expression and location of PPAR-y and AP-1(c-fos, c-jun).4. Based on the Genebank cDNA accession number, design and synthesize PPAR-y siRNA oligonueleotides lines. Design and synthesize one negative siRNA oligonueleotides, which are not specific for any known gene.5. Culture H9C2cells line routinely.Transfect the siRNA to H9C2cells, three groups was designed, negtive, test and control group.6. After transfection of siRNA24h, western blot was used to detect the expression of PPAR-y transfection efficiency.7. After H9C2cells were transfected with each siRNA for48h, cells were incubated with quercetin and Angll alone or in combination for24h. Cells were incubated for another24h prior to RNA isolation for quantitative real-time PCR analysis and48h prior to isolating protein samples for western blotting analysis.8. Statistical analysisThe data are expressed as the mean±Sem and were analyzed using SPSS17.0. For multiple comparisons between groups, one or two-way ANOVAs were used followed by Bonferroni corrected post-hoc tests. An independent-samples t-test was applied, when only two groups were compared.Differences were considered to be statistically significant when the P values were less than0.05.Results1. Comparison of [3H]leucine incorporation[3H]leucine incorporation was increased significantly in the Ang Ⅱ-induced group compared with the control group(P＜0.01). However, in quercetin pretreated cells, Ang Ⅱ-mediated incorporation was markedly reduced in a concentration-dependent manner (P＜0.01). At100μg/ml, compared with Ang Ⅱ-induced group protein synthesis was reduced to almost basal levels (64%reduction of Ang Ⅱ stimulated [3H]leucine incorporation)(P＜0.01).2. Comparison of the surface area of H9C2cellsThe surface area of H9C2cells was increased significantly in the Ang Ⅱ-induced group compared with the control group(P＜0.01). However, Quercetin (100μg/mL) pretreatment significantly attenuated this increase in H9C2cells compared with the Ang Ⅱ group (P＜0.01).3. Immunofluorescence detection(1) Comparison of expression of PPAR-γ protein:The expression of PPAR-γ protein was declined in the Ang Ⅱ group than that in the control group(P＜0.01) However, the fluorescence of PPAR-γ protein was significantly increased in the quercetin and AngⅡ combination treatment group compared with the Ang Ⅱ group (P＜0.01;).(2) Comparison of expression of AP-1(c-fos, c-jun) protein:The expression of AP-1(c-fos, c-jun) protein was increased in the Ang Ⅱ group than that in the control group(P＜0.01) However, the fluorescence of AP-1(c-fos, c-jun) was significantly decreased in the quercetin and AngⅡ combination treatment group compared with the Ang Ⅱ group (P＜0.01).4. Western blotting detection(1) Comparison of expression of PPAR-γ protein:The expression of PPAR-γ protein was lower in the PPAR-γ NCsiRNA group than that in the control group(P＜0.01) However, the fluorescence of PPAR-γ protein was significantly decreased in the PPAR-γ siRNA group compared with the Ang Ⅱ group (P＜0.01). (2) Comparison of expression of AP-1(c-fos, c-jun) protein:The expression of AP-1(c-fos, c-jun) protein was higher in the AngⅡ group than that in the control group(P＜0.01). However, the fluorescence of AP-1(c-fos, c-jun) was significantly decreased in the PPAR-y siRNA reatment group compared with the Ang Ⅱ group (P＜0.01).5. Comparison of expression of ANP and BNP mRNA in H9C2cells(1) Comparison of expression of ANP mRNA:Transfection siRNA after24hours, the expression of ANP mRNA of PPAR-γ siRNA group was obviously increased compared with the Ang Ⅱ group (P＜0.01). No significant difference was shown in comparison between the Ang Ⅱ group and the PPAR-y NCsiRNA group (P＞0.01).(2) Comparison of expression of BNP mRNA:Transfection siRNA after24hours, the expression of BNP mRNA of PPAR-y siRNA group was obviously increased compared with the Ang Ⅱ group (P＜0.01). No significant difference was shown in comparison between the Ang Ⅱ group and the PPAR-y NCsiRNA group (P＞0.01).Conclusions:(1) A reversing effect of quercetin on Ang Ⅱ induced H9C2cells hypertrophy was observed in our present study.(2) Quercetin may inhibit Ang Ⅱ induced H9C2cells hypertrophy by enhancing PPAR-y expression partly and by suppressing the AP-1signaling pathway, resulting inhibiting the transcription of its downstream gene expression of ANP and BNP.