Total Flavones From Psidium Guajava Leaves Depress Angiotensin Ⅱ-induced Cardiomyocyte Hypertrophy Through AT₁-PKC Pathway

BackgroundCardiomyocyte hypertrophy refers to the increase of cell’s volume and myocomma; and changes of types of contracting proteins at the same time. In its early stage, it is a compensatory response, but if prolonged, the heart may undergo a transition to heart failure. Therefore, it is important to prevent the process of cardiomyocyte hypertrophy induced by extracellular signals for any proposed therapy to regulate the myocardial hypertrophic responses.As an important neuroendocrine factor, AngⅡcan not only regulate the physiological functions of cardiovascular system, but also make a vital role in physiopathologic processes such as myocardial hypertrophy or heart failure.It is well known the role of AngⅡto cardiomyocyte is mainly mediated by the angiotensinⅡ1 receptor.AT1 receptor is a G protein-couple receptors, which activated can activate phospholipid membrane enzyme system to generate inositol triphosphate (Inositol trisphosphate, IP3) and diacylglycerol (Diacylglycerol, DG).IP3 and DG can heighten intracellular Ca2+ concentration and activate protein kinase-C,which translocating into the nucleus to regulate the nuclear gene expression to promote cardiomyocyte hypertrophy. Previous studies have shown that, PKC signaling is a rate-limiting molecular switch and a common signaling pathway response to hypertrophic aignal transduction which plays an important role in the process.Chinese traditional medicine Psidium guajava L is the Myrtaceae Psidium plant widely planted in Guangxi,Fujian,Taiwan,Hainan,Guangdong and so on.Its leaves neutral in nature,sweet flavor,have an effect on astringing to stop diarrhea and eliminating inflammation and hemostasis,collected on summer and autumn. Guava leaf,Myrtaceae plant dry guava leaves and tender stems with leaves, is found in Guangdong, Guangxi, Sichuan, containingβ-sitosterol, quercetin, guava glycosides, colorless cyanidin, guava acid composition,of which flavonoids as the main component.More studies on flavonoids have showed that it can anti-cardiovascular diseases,anti-cerebrovascular disease, antioxidant, analgesic, anti-virus, anti-tumor, immunomodulatory and so on in recent years.Its effect on anti-hypertrophy of cardiomyocyte presents no report, yet to be elucidate.ObjectiveTo investigate the effect and the possible mechanism of total flavones from Psidium guajava leaves on cultured neonatal rat cardiomyocyte hypertrophy induced by Ang II.Methods1. Neonatal cardiomyocyte culturesPrimary cultures of 1-3 day old neonatal Wistar rat myocytes were prepared and cut into 1mm×1mm×1mm size. Minced ventricular myocardium was placed into D-Hanks’salt solution, pH 7.4. The cells were dissociated by a trypsin (0.01%) digestion in D-Hanks’salt solution. After each of five to six successive 6 min incubations, the dissociated cells were mixed with DMEM containing 10% FBS and were centrifuged and pooled. The dissociated cells were enriched for cardiomyocytes by the technique of differential adhesion for 90 min and plated at a concentration of 106 cells/well. Cultures were incubated in a humidified 5% CO2-95% O2 at 37℃Bromodeoxyuridine (0.1 mM) was added into the medium to inhibit proliferation of nonmyocytes. This procedure yielded cultures with 90-95% myocytes, as assessed by microscopic observations of cellular contractions. After a 2 overnight incubation in DMEM containing 10% FBS the attached cells were rinsed and maintained in DMEM containing 0.1% FBS.After 48 h of serum starvation, cardiomyocytes weretreated with various agents.2、Morphological assessment of myocyte apoptosisMyocardial cells were seeded in 6 well plate, at a concentration of 2x105cells/well, different concentrations of total flavones from Psidium guajava leaves (50ug/ml,100ug/ml,150ug/ml,200ug/ml,250ug/ml) were added into different wells. cultured for 24h, Cardiomyocytes were fixed in 4% paraformaldehyde for 30min at room temperature, Cells were then washed twice in D-Hanks and were incubated with Hochest 33258 (5 mg/L) for 30min. Then cells were stained with mounting liquor (20 mmol/L citric acid,50 mmol/L disodium hydrogen phosphate,50% glycerol, pH 5.5) after washed by D-Hanks.Immunostained cardiomyocytes were viewed by fluorescence microscopy.3. Experimental protocolsCells made quiescent in serum-free medium for hours were treated with study drugs. Six groups were studied:(1) Control. Cells were treated without any drug intervention; (2) AngⅡ(0.1μmol/L); (3) AngⅡ+total flavones from Psidium guajava leaves (50ug/ml). Pretreatment with total flavones from Psidium guajava leaves 50ug/ml for 24h, Ang II was then added; (4) AngⅡ+total flavones from Psidium guajava leaves (100ug/ml). Pretreatment with total flavones from Psidium guajava leaves 100ug/ml for 24h, AngⅡwas then added; (5) AngⅡ+total flavones from Psidium guajava leaves (150ug/ml). Pretreatment with total flavones from Psidium guajava leaves 150ug/ml for 24h, Ang II was then added; (6) AngⅡ+Losartan (10umol/L)Pretreatment with Losartan for 30 minutes, AngⅡwas then added.4. ImmunofluorescenceAfter treated with 0.1μmol AngⅡalone or with total flavones from Psidium guajava leaves for 24 h, supernatants were removed. Cardiomyocytes were fixed in 4% paraformaldehyde for 15min at room temperature, Cells were then washed twice in ice-cold PBS and were incubated with confining liquid for 60min. Then cells were incubated with Actinin antibody dissolved in 1% BSA-PBS(1:500) for 2h at room temperature slow-moving on shaking table and Goat anti-mouse IgG-FITC was added (1:300) for 1h. Immunostained cardiomyocytes were viewed by fluorescence microscopy. Quantitation of cell surface area was performed on actin-stained cardiomyocytes. The cell size of cardiomyocytes was measured by Image-Pro Plus 5.0. The experiment was performed four times.5、Protein content assayCardiomyocytes were treated with different drugs for 24 hours, harvested in centrifuge tube, and centrifuged in 1000 r/min for 6 minutes, supernatants were removed. Cells were then washed twice in ice-cold D-Hanks and suspended with 2 ml D-Hanks, counting the number of myocardial cells per well,150μl NP-40 lysis buffer was added to dissolve the cardiac cell membrane and centrifuged in 12000r for 5min. The total protein content of myocardial cells of each well was detected with BCA method by measuring bovine serum albumin (BSA 0.5mg/ml) as the standard curve. Operation was conducted by agents instructions. The experiment was performed four times. 6. Western-Blot analysisTo explore the molecular mechanisms of the antihypertrophic effect of total flavones from Psidium guajava leaves, we determined whether total flavones from Psidium guajava leaves inhibits signaling through AT1-PKC pathway. After various treatments, myocytes were harvested and lysed in 150μl NP-40 lysis buffer. The protein concentration was determined by the BCA method. Equal protein was loaded in each lane, resolved by SDS-PAGE, blotted on nitrocellulose membrane. Membranes were blocked in 5% nonfat milk powder in Tris-buffered saline (TBS)/0.1% Tween-20 for 90min at room temperature, and then incubated with specific antibodies in 5% BSA in TBS for overnight. Membranes were incubated with peroxidase conjugated second antibody in blocking buffer for 2 h. The labeled proteins were detected with enhanced chemiluminescence. The results were analyzed by KODAK Image Station 4000MM exposure imaging system. The experiment was performed four times.7. Statistical MethodsSPSS 13.0 statistical soft was used. Statistical analysis Results were expressed as mean±S.D.Statistical significance was determined using one-way ANOVA, multiple comparisons using Fisher’s LSD post hoc tests. When the variance of arrhythmia, the use of robust estimation Welch, then the use of T3 compared methods. The differences were considered statistically significant at a value of P<0.05.Result1. Neonatal cardiomyocyte culturesJust isolated cardiac cells were spherical, then begin to adherent to wells after 4h cultured, showing round, then into a spindle. Cells continue to expand in the wells, extending pseudopods, and become irregular; A small number of individual adherent cells were spontaneously beating in a different frequency and rhythm after 12h cultured;Two days later cells are into a single layer, after 3 to 4 days cells extend pseudopodia contacting with each other into a network, and gradually forme cell clusters or single cells layer, into a radial array, beating rhythmically;sat a rate of 30 to 120 times/min (Figure 1).2、Different concentrations of total flavones from Psidium guajava leaves on myocardial apoptosis.Myocardial cells were seeded in 6 well plate, at a concentration of 2×105cells/well, different concentrations of total flavones from Psidium guajava leaves (50ug/ml,100ug/ml,150ug/ml,200ug/ml,250ug/ml) were added into different wells. cultured for 24h. The nucleolus of the control group and different concentrations of total flavones from Psidium guajava leaves (50ug/ml, 100ug/ml,150ug/ml) showed a diffuse homogeneous nuclear fluorescence. Total flavones from Psidium guajava leaves of 200ug/mL,250ug/mL group showed a morphological features of apoptotic death (cell shrinkage, chromatin condensation, and fragmentation). It shows that the 200ug/ml and 250ug/ml groups begin to show a trend to inhibit the activity of myocardial cells.in a dose-dependent way (Figure 2).3. Different concentrations of total flavones from Psidium guajava leaves on cell size of myocardial cells.Cardiomyocyte hypertrophy refers to the increase of cell’s volume and myocomma; changes of types of contracting proteins and embryonic gene expression (such as ANF) at the same time. After treated with 0.1μmol AngⅡalone or with total flavones from Psidium guajava leaves for 24h, AngⅡgroup were higher than control group, AngⅡmarkedly increased cell size, which was prevented by Losartan (both P<0.01). Different concentrations of total flavones from Psidium guajava leaves have a significant difference on inhibition the cell size (P<0.01)(Figure 3、4,Table 1). 4. Different concentrations of total flavones from Psidium guajava leaves on protein content of myocardial cells.After treated with 0.1μmol AngⅡalone or with total flavones from Psidium guajava leaves for 24h, Ang II group markedly stimulated protein synthesis compared with control group (P<0.05).Different concentrations of total flavones from Psidium guajava leaves significantly depresss the protein synthesis compared with AngⅡgroup ((P<0.05) (.Table 2, Figure 5)5. Different concentrations of total flavones from Psidium guajava leaves on AT1 activity of myocardial cells.To explore the molecular mechanisms of the antihypertrophic effect of total flavones from Psidium guajava leaves, we determined whether total flavones from Psidium guajava leaves inhibits signaling through AT1 pathway induced by Ang II. After treated with O.1μmol AngⅡalone or with total flavones from Psidium guajava leaves for 24h, AT1 activation was detected by Western-Blot.The result shows that Ang II group markedly stimulated AT1 activation compared with control group (P <0.001).Different concentrations of total flavones from Psidium guajava leaves significantly depresss the protein activation compared with Ang II group in a dose-dependent way (P<0.001) (Table 3, Figure 6、7).6. Different concentrations of total flavones from Psidium guajava leaves on PKC activity of myocardial cells.To further investigate the inhibition mechanisms of total flavones from Psidium guajava leaves on AT1 activation,this study has verified the effect of guava leaves flavonoids on PKC activation in myocardial cells. The result shows, AngⅡwas significantly increases protein levels of PKC (P＜0.001). Different concentrations of total flavones from Psidium guajava leaves significantly inhibited the levels, compared with the Losartan group (P＜0.001) (Table 4, Figure 8、9). Conclusions1. The experiment uses cultured neonatal rat cardiomyocyte hypertrophy induced by AngⅡto investigate the effect and the possible mechanism of total flavones from Psidium guajava leaves, the results showed that total flavones from Psidium guajava leaves significantly decreases the cell size and protein synthesis of myocardial cells.2. To further investigate the possible mechanism of total flavones from Psidium guajava leaves on cultured neonatal rat cardiomyocyte hypertrophy induced by AngⅡ, the results showed that total flavones from Psidium guajava leaves markly depressed the protein levels of AT1 and PKC in a dose-dependent way.3. Total flavones from Psidium guajava leaves may depress angiotensinⅡ-induced cardiomyocyte hypertrophy through AT1-PKC pathway.