Pomegranate Flower Extract Diminishes Cardiac Fibrosis in... : Journal of Cardiovascular Pharmacology (original) (raw)
Myocardial extracellular matrix (ECM) is composed of many different proteins, including collagen and the components of the pericellular matrix (fibronectin and proteoglycans). Dysregulation of the myocardial ECM contributes to abnormal cardiac muscle function. Fibrosis reflects an increase in collagen concentration. In diabetes the heart shows a persistent increase in development of ECM deposition,1 associated with interstitial fibrosis of ventricle in diabetic patients.2 Enhanced myocardial stiffness results from cardiac fibrosis as collagen I is a very rigid protein, which also generates arrhythmias because fibrosis creates myocardial electrical heterogeneity and hampers systolic ejection by increasing the myocardium heterogeneous.3 Myocardial fibrosis is one of the vital determinants involved in cardiac remodeling, congestive heart failure, severe arrhythmias, and sudden death.3 Therefore, reversal of fibrosis may improve cardiac function and survival. There are two different types of fibrosis, namely, reparative and reactive fibrosis. Reparative fibrosis occurs as a reaction to a loss of myocardial material (due to necrosis or apoptosis, after myocardial ischemia or senescence), and it is mainly interstitial. In contrast, reactive fibrosis is observed in the absence of cell loss as a reaction to inflammation and is primarily perivascular.3
The causes of cardiac fibrosis are multifactorial. Endothelin (ET)-1, a vasoconstrictor peptide, plays an important role in the pathophysiology of cardiovascular diseases. ET-1 acts through two specific receptors: type A (ETA) and type B (ETB). Besides increasing tissue fibronectin mRNA expression in the diabetic heart,4 ET-1 also increases both collagen (I and III) synthesis and cardiac fibroblast proliferation and reduces collagenolytic activity through ETA in adult rat cardiac fibroblasts.5 Activator protein (AP)-1 has been implicated in a large variety of biologic processes including cell differentiation, proliferation, apoptosis, and oncogenic transformation. AP-1 activation is most pronounced in the retina, kidney, and heart of streptozotocin-induced diabetic rats, which is involved in fibronectin synthesis.4 Nuclear factor (NF)-κB has been implicated in diverse processes such as apoptosis and inflammation.6 NF-κB has been suggested to mediate a high level of glucose-induced fibronectin synthesis in human macrovascular and microvascular endothelial cell lines.7 Diabetes-induced increased tissue fibronectin mRNA expression requires activation of NF-κB, which appears to be the predominant mechanism in the heart.4
Punica granatum (commonly known as pomegranate) flower (PGF) has been using in Unani and Ayurvedic medicines for diabetes.8 It has been reported that pomegranate juice and wine have cardiovascular protective effects.9 Recently, we have reported that PGF extract activates peroxisome proliferator-activated receptor (PPAR)-α and -γ to improve glucose tolerance,10 hyperlipidemia, and excess cardiac triglyceride accumulation11 in Zucker diabetic fatty (ZDF) rat, a genetic model for obesity and type 2 diabetes. As a potent α-glucosidase inhibitor, PGF extract also improves postprandial hyperglycemia in ZDF rats.12
In the current study we have continued studies to investigate the effects of PGF extract on cardiac fibrosis and its underlying molecular mechanisms in ZDF rats, as well as in an in vitro cell line system.
MATERIALS AND METHODS
Materials
Gallic acid (3,4,5-trihydroxybenzoic acid, GA), oleanolic acid (3β-hydroxy-olea-12-en-28-oic acid, OA), ursolic acid (3β-hydroxy-urs-12-en-28-oic acid, UA), and lipopolysaccharide (LPS) were obtained from Sigma (Australia). PGF was collected in June 2002 in Maharashtra state, India, and identified. A voucher specimen was deposited at the herbarium of the Herbal Medicines Research and Education Center, Faculty of Pharmacy, the University of Sydney, Australia. PGF extract was prepared as previously described.10,11 Briefly, air-dried and powdered PGF (1 kg) were extracted with 5 volume of methanol (W/V) at room temperature three times. The solvent was evaporated under reduced pressure below 50°C to give an extract (yield: 40%). The extract has been demonstrated to contain OA, UA, and GA by using the methods of column chromatography, high performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance, by comparison to standard OA, UA, and GA.10,11
Animals and Treatment
All animal experimental procedures have been approved by the Animal Ethics Committee of the University of Sydney, Australia. Male Zucker lean rats (ZL, Fa/?) and ZDF (fa/fa) aged 13 to 15 weeks (Monash University Animal Services, Victoria, Australia) were housed in an air-conditioned room at 23 ± 1°C with a 12-hour light/dark cycle and were provided with standard food and water ad libitum. Animals were allowed free access to standard food and water for 1 week before starting the experiments.
Plasma glucose levels were determined under non-fasted conditions before treatment. The results demonstrated that ZDF rats had hyperglycemia and hyperlipidemia (data not shown). Animals were divided into (ZL) control (receiving vehicle only), ZL PGF (receiving PGF extract), ZDF control (receiving vehicle only), and ZDF PGF (receiving PGF extract) groups (5 animals for each group); 500 mg/kg was chosen as the dosage applied in the present study, according to our previous experience.12 Test sample (suspended in 5% acacia) or vehicle (5% acacia) was given orally by a gavage method once daily for 6 weeks. (The animals were fed standard food and water ad libitum during the experiment).
Measurement of Heart Weight and Determination of Left Ventricular Fibrosis
After rats were killed under halothane anesthesia (non-fasted conditions) at week 6, and the hearts were rapidly excised and washed with saline on ice. The hearts were accurately weighed after the excess water on the surface was removed with filter paper. The ratio of the heart weight to body weight (HW/BW) was calculated. One part of the left ventricle was frozen in liquid nitrogen and stored at −80°C for the analysis of mRNAs. Another part of the left ventricle was fixed in 10% formalin and prepared for pathologic examination. Cardiac fibrosis was measured essentially as described previously.13-15 To determine the degree of collagen fiber accumulation, 40 fields in three individual sections were randomly selected and the areas of van Gieson-stained interstitial collagen deposit and the total left ventricular area were measured, respectively, by using an image analyzing system (KS 400 Imaging System; Carl Zeiss Vision, Eching, Germany). The ratio of the areas of interstitial collagen deposit to the total left ventricular area was calculated. Perivascular fibrosis was assessed by calculating the ratio of the area of van Gieson-stained collagen deposit immediately surrounding the coronary artery to the wall area (media). An average of >10 coronary artery regions was used for each animal.
Tissue Culture
RAW264.7, a mouse macrophage cell line, was obtained from the American Type Culture Collection. Cells were cultured in DMEM supplemented with antibiotics (100 U/mL of penicillin A and 100 U/mL of streptomycin) and 10% heat-inactivated fetal bovine serum (Invitrogen, Australia) and maintained at 37°C in a humidified incubator containing 5% CO2.
Gene Expression Analysis
Total mRNA was prepared from the left ventricle using TRIzol (Invitrogen, Australia). The relative levels of specific mRNAs were assessed by reverse transcription polymerase chain reaction (RT-PCR), as described previously.16 Single-stranded cDNA was synthesized from 1 μg of total RNA using SuperScript II RNase H-Reverse Transcriptase, as per the instructions of the manufacturer (Invitrogen, Australia). PCR was performed on a Thermocycler, PTC-200 DNA engine (MJ Research Inc, MA). The required cDNA was synthesized with the Platinum® Pfx DNA Polymerase method (Invitrogen, Australia). The genes examined from the left ventricle were collagen type I, collagen type III, fibronectin, inhibitor (I)-κBα, I-κBβ, c-fos, c-jun, ET-1, ETA, and ETB. The sequences of the sense and antisense primers used for amplification are shown in Table 1. The PCR samples were electrophoresed on 3% agarose gels and stained with ethidium bromide. The gel images were digitally captured with a CCD camera and analyzed with the Image J 1.29x (NIH, USA). RT-PCR values are presented as a ratio of the specified gene signal in the selected linear amplification cycle divided by the β-actin signal.
Primers Used in this Study
Transfection and Nuclear Factor-κB Luciferase Assay
Lipopolysaccharide is a well-known pro-inflammatory agent used to activate the NF-κB pathway in cell lines.17,18 The transfection and luciferase procedures were performed using the RAW264.7 cell line, as described previously.19 The plasmids used for transfection were p-NF-κB-Luc (a kind gift from Dr Sheridan Henness, University of Sydney, Australia) and pSV-β-Galactosidase Control Vector (Promega) to normalize transfection efficiencies. Cells were transfected with FuGENE 6 transfection reagent (Roche, Australia) in accordance with the manufacturer's instructions. The cells were pretreated for 2 hours with either vehicle (DMSO), PDTC (100 mM) (a known inhibitor of NF-κβ), or increasing concentrations of PGF extract (10 μg/mL to 100 μg/mL) and GA, OA, and UA (10 μM to 300 μM) prior to 23 hours treatment with LPS (100 ng/mL). Unstimulated RAW264.7 cells acted as a negative control. After 24 hours, the cells were lysed and assayed for luciferase and β-galactosidase activities using the Bright-Glo Luciferase Assay System and Beta-Glo Assay System (Promega, Australia), respectively. The results were expressed as relative luciferase activity (fold difference compared with negative control).
Statistics
All results are expressed as means ± SEM. Data was analyzed by 1-factor ANOVA. If a statistically significant effect was found, the Newman-Keuls test was performed to isolate the difference between the groups. Values of P < 0.05 were considered statistically significant.
RESULTS
Effects of PGF Extract on Heart Weight/Body Weight, and Interstitial and Perivascular Fibrosis in Left Ventricle of Zucker Lean and Zucker Diabetic Fatty Rats
Punica granatum flower has been traditionally used in diabetes. We have recently demonstrated that PGF extract improves hyperglycemia, hyperlipidemia, and excess cardiac triglyceride accumulation, whereas it did not affect body weight, in ZDF rats.10-12 As ZDF rats also show excess cardiac fibrosis,15 we now observed the effects of PGF extract on heart mass and cardiac fibrosis in ZDF rats. The results showed that the weights of the heart in ZDF rats were significantly increased, but the ratios of HW/BW were significantly decreased, compared with those in ZL rats (Table 2). Treatment with PGF extract showed little effect on HW and HW/BW (Table 2). Consistent with a previous report,15 control ZDF rats exhibited wider area of van Gieson-stained collagen deposit (red color) in the interstitia of the left ventricles (Fig. 1D) compared with control ZL rats (Fig. 1B). The ratio of the area of interstitial collagen deposit to the total left ventricular area was much higher in ZDF rats than in ZL rats (Fig. 1A). The collagen deposit immediately surrounding the coronary artery was also increased in ZDF rats (Fig. 1I) compared with ZL rats (Fig. 1G). The ratio of perivascular fibrosis area to media area in ZDF rats was increased (Fig. 1F). Treatment with PGF extract (500 mg/kg, p.o.) for 6 weeks decreased both interstitial (Figs. 1A and 1E) and perivascular (Figs. 1F and 1J) collagen accumulation in ZDF rats, whereas it had no effect in ZL rats (Figs. 1A, 1C, 1F, and 1J, respectively).
Effects of Punica Granatum Flower Extract on Heart Mmass in Zucker Lean and Zucker Diabetic Fatty Rats
Effect of Punica granatum flower (PGF) extract on cardiac interstitial and perivascular fibrosis in male Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats. ZL PGF group and ZDF PGF group received PGF extract (500 mg/kg, p.o.) treatment, whereas ZL control group and ZDF control group were given vehicle only for 6 weeks. The cardiac van Gieson-stained interstitial collagen deposit area, the total left ventricular area, perivascular collagen deposit area, and the media area of coronary artery were analyzed by using an image analyzing system (KS 400 Imaging System; Carl Zeiss Vision, Eching, Germany). A, Interstitial fibrosis (%) (the ratio of the area of interstitial collagen accumulation to the total left ventricular area); B-E, Representative examples of interstitial fibrosis (red) in ZL control, ZL PGF, ZDF control and ZDF PGF groups, respectively (magnification 200×); F, Perivascular fibrosis (%) (the ratio of the area of perivascular collagen accumulation to the media area); G-J, Representative examples of perivascular fibrosis (red) in ZL control, ZL PGF, ZDF control, and ZDF PGF groups, respectively (magnification, 100×). All values are means ± SEM (n = 5). *P < 0.05, **P < 0.01. Con: control.
Changes in Gene Expression Profiles in Animals
Fibronectin, collagen I and III are major components of the ECM. They play an important role in abnormal cardiac muscle function. Therefore, we determined the expression of their genes in the heart. The results showed that expression of fibronectin (Fig. 2A), and collagen I and III (Figs. 2B and 2C) mRNAs were markedly enhanced in ZDF rats. Treatment with PGF extract reduced the expression to normal (ZL rat) levels (Figs. 2A-2C).
Cardiac expression of mRNAs encoding for fibronectin (FBN) (A), collagen (Col)s I (B), and III (C) in Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats after 6-week treatment with vehicle or PGF extract (500 mg/kg, p.o.). Total mRNA was prepared from the left ventricle using the TRIzol method. The relative levels of specific mRNAs were assessed by reverse transcriptase polymerase chain reaction (RT-PCR). Results were normalized to β-actin. Levels in ZL control were arbitrarily assigned a value of 1.0. All values are means ± SEM (n = 5). *P < 0.05, **P < 0.01. Con: control.
ET-1 is involved in fibronectin and collagen (I and III) synthesis and cardiac fibroblast proliferation (see Introduction). To investigate the molecular mechanism underlying the anti-fibrogenic effect of PGF extract in ZDF rats, we investigated the cardiac expression of ET-1 and its receptors. Compared with ZL rats, the hearts of ZDF rats showed higher mRNA expression of ET-1 (Fig. 3A), ETA (Fig. 3B), and ETB (Fig. 3C). Chronic oral administration of PGF extract suppressed the cardiac overexpression of ET-1 (Fig. 3A), ETA (Fig. 3B) mRNAs, whereas it did not affect ETB mRNA (Fig. 3C) in ZDF rats. In contrast, the treatment showed little effect in ZL rats (Figs. 3A-3C).
Cardiac expression of mRNAs encoding for endothelin (ET)-1 (A), ETA (B), and ETB (C) in Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats after 6-week treatment with vehicle or PGF extract (500 mg/kg, p.o.). Total mRNA was prepared from the left ventricle using the TRIzol method. The relative levels of specific mRNAs were assessed by RT-PCR. Results were normalized to β-actin. Levels in ZL control were arbitrarily assigned a value of 1.0. All values are means ± SEM (n = 5). *P < 0.05, **P < 0.01. Con: control.
As NF-κB activation is involved in inflammatory and apoptotic processes, we investigated cardiac gene expression of I-κBs, the intrinsic inhibitors of NF-κB. The results showed that cardiac I-κBα (Fig. 4A) mRNA expression was decreased, whereas I-κBβ (Fig. 4B) was increased in ZDF rats, compared with those in ZL rats. Treatment with PGF extract normalized these abnormalities (Figs. 4A and 4B).
Cardiac expression of mRNAs encoding for inhibitor (I)-κBα (A) and I-κBβ (B) in Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats after 6-week treatment with vehicle or PGF extract (500 mg/kg, p.o.). Total mRNA was prepared from the left ventricle using the TRIzol method. The relative levels of specific mRNAs were assessed by RT-PCR. Results were normalized to β-actin. Levels in ZL control were arbitrarily assigned a value of 1.0. All values are means ± SEM (n = 5). *P < 0.05, **P < 0.01. Con: control.
One of the mechanisms required for AP-1 activation is transcriptional induction of certain AP-1 encoding genes, including c-jun and c_-fos_.20 We investigated potential changes of cardiac c-jun and c_-fos_ mRNA expression in the tested animals. The RT-PCR results showed no significant difference in cardiac c-fos mRNA expression between ZL and ZDF rats (Fig. 5A), but cardiac c-jun expression was markedly increased in ZDF rats (Figs. 5B). PGF extract suppressed the overexpressed c-jun (Fig. 5B) in ZDF rats, whereas it did not affect c-jun in either type of animal (Fig. 5A).
Cardiac expression of mRNAs encoding for c-fos (A) and c-jun (B) in Zucker lean (ZL) and Zucker diabetic fatty (ZDF) rats after 6-week treatment with vehicle or PGF extract (500 mg/kg, p.o.). Total mRNA was prepared from the left ventricle using the TRIzol method. The relative levels of specific mRNAs were assessed by RT-PCR. Results were normalized to β-actin. Levels in ZL control were arbitrarily assigned a value of 1.0. All values are means ± SEM (n = 5). *P < 0.05. Con: control.
Effects of Punica granatum Flower Extract and its Components on Lipopolysaccharide-Induced NF-κB Activation in Cell Lines
To further understand the underlying regulatory roles on gene expression of NF-κB activity, we investigated the effects of PGF extract and its components OA, UA, and GA on LPS-induced NF-κB luciferase reporter expression. As expected, LPS evoked NF-κB promoter activity, whereas PTDC (100 mM) completely abolished this activation of NF-κB promoter in macrophages (Fig. 6). PGF extract (100 μg/mL) and its components OA, UA, and GA (300 μM) all inhibited NF-κB promoter activity (Fig. 6).
Effects of PGF extract and its prominent components gallic acid (GA), oleanolic acid (OA), and ursolic acid (UA) on lipopolysaccharide (LPS, 1 μg/mL)-induced nuclear factor (NF)-κB activation in RAW264.7 macrophages transfected with NF-κB luciferase reporter gene. The results were expressed as relative luciferase activity (fold difference compared with negative control). Pyrrolidine dithiocarbamate (PDTC) was used as a positive control. All values are means ± SEM (duplicate, n = 3). *P < 0.05, **P < 0.01. Con: control.
DISCUSSION
ET-1 can be synthesized in endothelial cells and fibroblasts.21 Cardiac levels of ET-1 were increased in Otsuka Long-Evans Tokushima Fatty rats, a spontaneous model of human type 2 diabetes.22 It has been demonstrated that high glucose concentration induces ET-dependent fibronectin synthesis in human macrovascular and microvascular endothelial cell lines, in which the AP-1 pathway is involved.7 ET-1 levels in the left ventricle have also been shown to increase in hypercholesterolemic and hypertriglyceridemic animals.23 These previous results suggest an important role of hyperglycemia and hyperlipidemia in the development of cardiac fibrosis via the ET-1 pathway. PPAR-α and -γ are well known to play an important role in maintaining homeostasis of glucose and lipid metabolism, and their agonists have been clinically used for the treatment of hyperglycemia and hyperlipidemia, respectively. It has been reported that PPAR-α activator fenofibrate and PPAR-γ activator rosiglitazone can prevent cardiac fibrosis and abrogate the increase in prepro-ET-1 mRNA content in the left ventricle of DOCA-salt rats (a model of overexpressing ET-1), suggesting a modulatory role of PPAR activators on cardiac remodeling, in part associated with decreased ET-1 production.24 Fenofibrate also attenuates collagen type I and type III mRNA expression, and interstitial and perivascular fibrosis through suppression of AP-1-mediated ET-1 gene augmentation in the rat hearts with aortic banding-induced pressure-overload.25 We have recently reported that PGF extract possesses dual activator properties of PPAR-α and -γ to improve hyperglycemia and hyperlipidemia in ZDF rats.10-12 The present study demonstrated that mRNA levels of fibronectin, collagen I and III, ET-1, ETA, and ETB were increased in cardiac samples of ZDF rats. Although cardiac c-fos mRNA levels were unchanged, c-jun mRNA levels were up-regulated in ZDF rats. These results suggest the involvement of the ET-1 pathway, at least in part, in increasing cardiac fibronectin and collagen I and III synthesis, thus exacerbating cardiac fibrosis. Interestingly, treatment of ZDF rats with PGF extract suppressed the overexpressed cardiac fibronectin, collagen I and III, ET-1, ETA, but not ETB mRNA expression. Furthermore, PGF extract restored c-jun mRNA expression, suggesting the modulation of AP-1 activation. These results suggest that PGF extract may modulate cardiac ET-1 (ETA) pathway, probably via AP-1, to reduce cardiac fibrosis in ZDF rats. However, a definite direct effect of PGF extract on the heart remains to be established with further studies on cultured cardiomyocytes or on animal models with largely cardiac limited pathologic development.
Nuclear factor-κB is sequestered in the cytoplasm through its association with its inhibitors, p105 or I-κB-like proteins, such as I-κBα, I-κBβ, I-κBγ, Bcl-3. It has been suggested that an increase in I-κBα expression is a common mode of action for the therapeutic interventions of some non-steroidal anti-inflammatory drugs and agonists of PPAR-γ.26-28 Non-phosphorylated I-κBβ can bind to NF-κB but does not mask its nuclear localization site nor transcription activation domain. NF-κB·I-kBβ complexes can enter the nucleus and exhibit transcriptional activity, and it has been proposed that this mechanism is responsible for persistent NF-κB activation.29-31 It was found that pomegranate wine inhibits tissue necrosis factor (TNF)-α-induced NF-κB activation in vascular endothelial cells.32 Quercetin, catechin, and GA, three components isolated from pomegranate wine, have been reported to inhibit TNF-α-induced NF-κB (p65) translocation in vascular endothelial cells, GA being the most potent inhibitor.32 OA and UA have anti-diabetogenic and anti-inflammatory activities.33 UA has been reported to inhibit NF-κB activation induced by carcinogenic agents through suppression of Iκ-Bα kinase and p65 phosphorylation.34 In the present study, ZDF rats showed decreased Iκ-Bα mRNA and enhanced Iκ-Bβ mRNA in the heart. Treatment of ZDF rats with PGF extract increased I-κBα mRNA and suppressed I-κBβ mRNA in the heart. Furthermore, PGF extract dose-dependently inhibited LPS-induced NF-κB promoter activity in RAW264.7 macrophages in luciferase reporter assay. OA, UA, and GA, the three prominent components contained in PGF extract,10,11 also suppressed NF-κB activation. Taken together, the in vivo and in vitro results suggest that the cardiac NF-κB regulating pathway is involved in reducing fibrosis in diabetic hearts by PGF extract, in which OA, UA, and GA, at least in part, are responsible.
CONCLUSION
Our findings demonstrate that PGF extract diminishes cardiac fibrosis in ZDF rats mainly by modulating cardiac ET-1 and NF-κB pathways. These data suggest that PGF may be promising as an antifibrogenic agent in diabetic and obese heart. The plant may help prevent or delay the onset of diabetes-related cardiac complications caused by fibrosis.
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Keywords:
diabetes; endothelin; fibrosis; heart; NF-kappaB; pomegranate
© 2005 Lippincott Williams & Wilkins, Inc.