Erectile dysfunction (ED) is associated with diabetes mellitus (DM). Pomegranate juice (PJ) is a potent antioxidant in diabetes induced oxidative stress. The aim of this study was to evaluate whether the administration of PJ ameliorates ED in streptozotocin (STZ)-diabetic rat model. Adult male Sprague-Dawley rats were divided into three groups (n=10–12, each): (1) Control, (2) STZ (25–35 mg kg−1, intravenously, 10 weeks) induced DM, and (3) PJ (100 mg kg−1 day−1, 10 weeks) treated DM rats. The in vivo erectile [a ratio of intracavernosal pressure (ICP)/mean arterial pressure (MAP)] and ex vivo corpus cavernosum (CC) responses were evaluated. Immunohistochemistry and Masson’s trichrome staining were performed. Malondialdehyde (MDA) levels were measured. The ICP/MAP value in diabetic rats was lower than controls, which was partially improved by PJ treatment. Electrical field stimulation (EFS)-induced relaxant responses in CC from the diabetic group were significantly decreased that were ameliorated by treatment. Phenylephrine- and EFS-induced contractions were not altered in diabetic rats. PJ treatment normalized raised MDA levels of diabetic CC samples. Although the intensities of endothelial nitric oxide synthase (NOS) and neuronal NOS enzymes were decreased, inducible NOS protein levels were stronger in diabetic slides than controls. This is the first study to show that PJ treatment ameliorates partially ED and completely oxidative stress and fibrosis in a diabetic rat model. Our results highlight the success of antioxidant mechanism of PJ in ED with diabetes and open the way for future understanding in alternative treatment combinations with PDE5 inhibitors.
Diabetes mellitus (DM) is a major risk factor for ED.1 Clinical studies indicate that ED affects 49% of diabetic men aged >60 years.2, 3 Diabetic men are considered to progress to ED between 10 and 15 years earlier than men who do not suffer from the disease.4 Also, ED develops three times more in diabetic men than in non-diabetics.5
Multiple mechanisms involving vasculogenic and neurogenic factors are involved in ED associated with diabetes.6 The generation of reactive oxygen species leads to increased vascular dysfunction via nitric oxide (NO) scavenging and other direct or indirect mechanisms that have been observed in diabetic vascular complications.7
Although PDE5 inhibitor is a first-line therapy for patients with ED,8 >30% of diabetic patients with ED are non-responsive to PDE5 inhibitor therapy.7 Thus alternative treatment option may be phytotherapy for ED in diabetic patients. Antidiabetic activity of flower and juice of the pomegranate seeds9 has been observed in earlier studies.
The pomegranate is a distinctive fruit with a medicinal history, a symbol of life, longevity and health.10 Pomegranate contains polyphenols that are potent antioxidants.11 Recently, pomegranate juice (PJ) has been shown to exhibit potent inhibitory effect on the formation of advanced glycation end products compared with several other commonly consumed fruit juices.12 In addition, PJ modified hyperglycemia by inhibiting oxidative stress-induced pancreatic damage13 and had marked free radical scavenging activity.14 In earlier studies, atherogenic and ischemic ED in the rabbit by giving PJ restored cavernous response by increasing intracavernosal blood flow, smooth muscle relaxation and decreasing oxidative stress.14, 15 In addition, erectile tissue fibrosis was delayed by the treatment.14, 15 In a recent study, PJ had significant erectile activity and induced the relaxation of isolated rat corpus cavernosum (CC).16 The goal of this study was to investigate the possible preventive effect of PJ treatment on ED in streptozotocin (STZ)-induced diabetic rat.
Materials and methods
Adult male Sprague-Dawley rats (350-400 g) received a low dose of STZ (25–35 mg kg−1, intravenously), which was dissolved in citrate buffer (pH=5.5) on the day of use. Blood glucose levels in the diabetic groups were measured after 3 days of STZ injection using an one-touch glucometer (ACCU-CHEK, Roche, Mannheim, Germany). Animals with blood glucose level ⩾250 mg dl−1 were included in the study. Rats were divided into three groups (n=10–12, each): (1) Control, (2) diabetic, and (3) PJ-treated diabetic. Animals were housed in separete cages on a 12-h light–dark cycle and were fed standard chow and water ad libitum. In a previous study, we demonstrated that PJ contains 7.98 mg ml−1 polyphenol antioxidants after high pressure liquid chromatography analysis.16 PJ (100% pure, Atatürk Forest Farm, Ankara, Turkey) was given in their drinking water that was equivalent to 100 mg kg−1 day−1 of total polyphenols for 10 weeks after induction of diabetes.15 The desired concentrations were calculated based on body weight and daily water intake of the animals.15 This study was approved by the Institutional Animal Care and Use Committee of Ankara University (2013-18-134).
In vivo studies
To measure intracavernous pressure (ICP), the rats were anesthetized with sodium pentobarbital (50 mg kg−1, intraperitoneally). The trachea was cannulated using polyethylene-240 tubing to maintain constant airway. The carotid artery was also cannulated (polyethylene-50 tubing) to measure mean arterial pressure (MAP) using a transducer (Statham, Oxnard, CA, USA) attached to a data acquisition system (Biopac MP 100 System, Santa Barbara, CA, USA). A 25-G needle filled with 250 U ml−1 of heparin and connected to the polyethylene tubing was inserted into the right crura of the penis and connected to the pressure transducer to continuously measure ICP. The right major pelvic ganglion and cavernous nerve (CN) were identified. A stainless steel bipolar hook electrode was placed around the CN posterolateral to the prostate for stimulation. The CN was stimulated (2.5, 5 and 7.5 V, 15 Hz, 30 ms pulse width) with a square-pulse stimulator (Grass Instruments, Quincy, MA, USA), and the MAP and ICP were continuously measured.17
Ex vivo studies
The cavernosal tissue was removed and placed in a Petri dish containing Krebs-bicarbonate solution (containing, mM: NaCl: 118.1, KCl: 4.7, KH2PO4: 1.0, MgSO4: 1.0, NaHCO3: 25.0, CaCl2: 2.5 and glucose: 11.1). The strips (1 × 1 × 9 mm3) were dissected and mounted under 1 g of resting tension in a 20-ml organ bath with one end attached to an electrode holder and the other to a wire connected to a force transducer. The organ chamber temperature was maintained at 37 °C by bubbling with a mixture of 95% O2 and 5% CO2. The tissues were allowed to equilibrate for 60 min. Electrical field stimulation (EFS) of the autonomic nerves (duration: 15 s; amplitude: 50–90 V; frequency: 1–20 Hz; pulse width: 0.5 ms) was accomplished by the use of platinum electrodes, positioned on either side of the tissue strip (Grass Instruments). Preparations were preincubated for 30 min with guanethidine (5 μM) to eliminate noradrenergic effects and with atropine (1 μM) to prevent cholinergic responses. After phenylephrine (PE) precontraction under these conditions, EFS tissue relaxation was mediated by nitrergic fibers.
In the first series of experiments, EFS (1–20 Hz), acetylcholine (ACh; 10−8–10−3 M) and sodium nitroprusside (SNP, 10−8–10−3 M) induced relaxation responses were evoked after precontraction of CC strips with phenylephrine (PE, 10−5 M).
In the second series of experiments, cumulative dose–response curves for PE (10−8–10−3 M) induced contractile responses were evaluated. The direct sympathetic stimulation-related contraction was induced with EFS (1–40 Hz) in the tissue strips.
Bivalved penises were fixed in 10% formalin and processed for paraffin embedding. Tissue cross sections (8–10 μm) were deparaffinized in xylene and hydrated with graded alcohol. Endogenous peroxidases were quenched with 3% hydrogen peroxide, and sections were washed with phosphate-buffered saline. Non-specific binding was blocked using normal horse serum (1:50 dilution) in 0.1% bovine serum albumin in phosphate-buffered saline. Slides were treated with 0.1% Triton X-100 for 20 min, washed in phosphate-buffered saline for 5 min and then incubated with rabbit primary polyclonal antibody (BD Transduction Laboratories, San Diego, CA, USA) at a dilution of 1:100 for 1 h at room temperature. Samples were then washed and incubated for an additional 30 min with biotinylated secondary antibody (DAKO, Carpinteria, CA, USA). Following a further 30 min incubation with an avidin–biotin-conjugated horseradish peroxidase (DAKO), the 3,3′diaminobenzidine substrate was added for 5 min. The endothelial NO synthase (eNOS), neuronal NOS (nNOS) and inducible NOS (iNOS) protein-positive cells appeared brown against Harris hematoxylin counterstain. Negative control slides, stained with only secondary antibody, were carried out for each tissue specimen (data not shown). Images were visualized under light microscopy (DM4000B and DFC280 color digital camera system, Leica Microsystems, Wetzlar, Germany) and semiquantitative histomorphological assessment was performed on all of stained specimens in a blinded manner using light microscopy. Three observers scored staining of brown area (0–1, no positive staining; 1–3, increasing degrees of intermediate staining; and 4, extensive staining).17
Masson’s trichrome staining
The proportions of collagen and smooth muscle in the tissue samples (n=4–6) from the rat groups were determined by staining tissue sections with Masson’s trichrome, staining the extracellular matrix (collagen and connective tissue elements) blue and smooth muscle red as in previous studies.18, 19 As collagen represents the majority of the extracellular matrix, blue-staining areas were referred to as stained collagen. Bouin’s Solution (Sigma Chemical (St Louis, MO, USA)) preheated at 56 °C for 15 min was used as a mordant in the tissue sections. Slides cooled with tap water were stained in working Weigert’s Iron Hematoxylin Solution for 5 min. After rinsing in deionized water, slides were stained with Biebrich Scarlet-Acid Fucshin (Sigma) for 5 min, then in Working Phosphotungstic/Phosphomolybdic Acid Solution (Sigma) for 5 min, followed by Aniline Blue Solution (Sigma) for 5 min and then 1% acetic acid for 2 min. Finally, the tissues were rinsed and dehydrated with alcohol, cleaned with xylene and mounted. Image analyses of the Masson’s trichrome stains were performed using the Image-J software (NIH, Bethesda, MD, USA) to identify the mean proportion of collagen and smooth muscle fibers.20
Measurement of malondialdehyde (MDA) levels
Lipid peroxidation was quantified spectrophotometrically by measuring the formation of thiobarbituric acid reactive substances (TBARS) expressed as MDA.21 Penile tissue homogenate (w/v, 20%) was prepared in buffer containing 0.02 M Tris and 0.15 M KCl using the ultrasonic homogenizer (BioLogics, VA, USA). Thereafter, the homogenate was centrifuged (3000 r.p.m., 15 min) and H3PO4 (1%) and TBA (0.6% in 0.5 M Na2SO4) solution were added. The mixture was placed in boiling water for 45 min and cooled with cold water. TBARS adducts were extracted into n-butanol by vortex and centrifuged (5000 r.p.m., 5 min). The absorbance of the supernatant was measured at 535 nm.
All data values were expressed as mean±s.e.m. Statistical differences were determined by analysis of variance followed by the complementary analysis of Bonferroni using the GraphPad Prism 4 (GraphPad Software, San Diego, CA, USA). A P-value <0.05 was considered to be significant. At the end of the experiment, each CC strip was weighed. All contractile responses were expressed as mg of tension developed per mg of corporal tissue and relaxant responses were calculated as a percentage of PE contraction.
All drugs were purchased from Sigma Chemical and PJ (100% pure) was provided from Atatürk Forest Farm (Ankara, Turkey).
Characteristics of animals
The blood glucose levels in diabetic rats were higher than in untreated rats. Treatment with PJ partially returned blood glucose levels in the diabetic group (data not shown).
Effects of PJ treatment on erectile function
ICP/MAP values by stimulation of CN at 5 and 7.5 voltage levels were decreased in diabetic rats (P<0.001) when compared with control rats, which was partially reversed at 7.5 voltage by PJ treatment (P<0.01, Figure 1a).
Ex vivo relaxant and contractile responses
As shown in Figure 1b, EFS-induced relaxation in strips contracted with PE was significantly reduced in diabetic rats when compared with control rats. In treating rats, EFS-induced relaxation response was completely reversed as seen in Figure 1b. At 5 voltage level, CC from the PJ treated group displayed potentiated response (P<0.05) when compared with control rats.
ACh-induced endothelium-dependent relaxation responses was significantly decreased in CC strips from diabetic rats compared with untreated rats (Figure 1c). CC strips from treated diabetic rats did not show any improvement in the relaxant responses of ACh (Figure 1c).
Endothelium-independent relaxation induced by SNP (NO donor) served normally in the groups (Figure 1d).
There was no difference in EFS-induced maximum contractile responses in the groups (Figure 2a). However, contractile response by direct EFS in PJ-treated rats was slightly lower but not significant at 5, 15 and 20 Hz (Figure 2a).
PE-induced maximum contractile response was not altered in the groups. However, PE-induced contractile response at 1 μM was decreased, which was not returned by PJ treatment in diabetic rat (Figure 2b).
MDA levels in penile tissues
MDA levels in penile tissue from diabetic rats significantly increased (P<0.05), when compared with the control group. Treatment with PJ normalized to MDA levels in diabetic rats (Figure 3).
Smooth muscle collagen ratios
The Masson’s trichrome staining procedure was used to determine the relative area of collagen to muscle fibers in the cavernosal tissue. The staining method revealed decreased smooth muscle cell (SMC)/collagen ratio (Figure 4). In diabetic tissue samples as compared with controls, the SMC/collagen ratio decreased, which was reversed by PJ treatment as seen in Figure 4d. We noticed that treated samples showed partial improvement when compared with diabetic samples (Figure 4d).
Figure 5 indicates that eNOS, nNOS and iNOS proteins as detected by an antibody to the penile variant was differentially expressed in these three groups. At 10 weeks in the diabetic group, there was a significant decrease in the abundance of nNOS (middle panel) and eNOS (upper panel) staining. Treatment with PJ in diabetic rats did not reverse the intensity of nNOS and eNOS protein staining compared with penile tissues from diabetic rats. iNOS protein staining was increased in the diabetic group, which was not ameliorated by PJ treatment as seen in Figure 5 (lower panel).
In this study, we showed that (1) PJ treatment partially reversed the blood glucose levels and in vivo erectile activity by stimulation of the CN in diabetic rats, (2) EFS- and ACh-induced relaxation responses in diabetic CC were significantly reduced while PJ treatment completely reversed EFS except ACh-induced relaxations. However, sympathetic neurogenic and α1-receptor mediated contraction was not altered, (3) CC samples from diabetic rats showed increased MDA levels, (4) SMC/collagen ratio decreased in diabetic samples, which was reversed by treatment, (5) immunohistochemical analysis showed an increase in iNOS intensity in the CC of diabetic rats that was partially reversed by PJ, while nNOS and eNOS intensities were decreased in diabetic slides but not normalized by treatment.
The present result showing elevated blood glucose levels in the diabetic group is consistent with results of previous studies.22, 23 Blood glucose levels in PJ-treated rats were partially normalized. In a similar manner, Jafri et al.24 demonstrated that blood glucose levels in alloxan-induced diabetic rats were decreased by pomegranate extract.24 Previous clinical studies reported reduced fasting serum glucose level, increased β-cell function and decreased insulin resistance after PJ consumption.25, 26 On the contrary, Nekooeian et al.27 showed that diabetic rats had increased blood glucose levels, which was not returned by pomegranate seed oil treatment. It can be recommended that blood glucose levels could be changed by animal model of diabetes and type of consumption.
In our data, we found that decreased in vivo erectile responses were partially prevented, while ex vivo EFS relaxation responses were completely returned by PJ treatment. The conflicting data might be related to altered presence of oxidative stress and hyperglycemia in in vivo, not in ex vivo, condition. Liu et al.28 showed that oxidative stress decreased the ICP. Similarly, previous studies demonstrated a reduction of ICP and relaxation response to EFS in diabetes.29 In the present study, diminished nNOS levels in the diabetic CC was not reversed by treatment. Likewise, nNOS levels are known to significantly decrease in experimental diabetes.30 This is the first study to show the effect of PJ treatment on erectile response in diabetic rat model. Similarly, according to Azadzoi et al.,14 PJ did not have a significant effect on nNOS expression in the penile tissue of rabbit with arteriogenic ED.
Isolated CC strips from diabetic rats showed a reduced response to ACh, suggesting endothelial dysfunction, which was not improved by PJ treatment. A recent study by Nunes et al.29 noted impaired relaxation to ACh in a diabetic mice model, suggesting that oxidative stress injury to the endothelium might affect the NO/cyclic guanosine monophosphate pathway. Conversely, PJ supplementation significantly increased the relaxation response to ACh in a resistance artery ex vivo.31 Also, we found that PJ did not have a significant effect on eNOS expression. Similarly, Azadzoi et al.14 showed that eNOS and iNOS proteins did not change in PJ-treated rabbits with arteriogenic ED.
In the present data, endothelium-independent relaxation responses to SNP were not altered in CC from all the groups. Thus it seems that CC smooth muscle responses are normal in the diabetic model. We found similar results in relaxant response of SNP in neonatal diabetic rats.32 In addition, previous data for SNP were not altered in the coronary artery from hypercholesterolemic pigs33 and mesenteric artery from obese Zucker rats with PJ treatment.31
Sympathetic neurogenic contractile responses to EFS in the CC strips and the maximum contractile response to PE (α-1 adrenergic receptor agonist) were not altered in diabetic rats when compared with control rats. We suggest that there was no change in nerve-evoked noradrenaline release and postreceptor responses in diabetic CC. Similarly, Claudino et al.34 and Nangle et al.35 demonstrated that both EFS and PE responses in cavernosal strips were not modified by type 1 diabetes. Thus CC from diabetic rats display unaltered neural and receptor-mediated contractile responses that may not favor penile detumescence. Also, the generation of free radicals resulting in oxidative stress may not modify contractile responses in this mild diabetic model.
In the present study, MDA levels were significantly increased in the CC of diabetic rats that was reversed by PJ. Similarly, MDA levels were significantly increased in the STZ-diabetic rat penis.36, 37 In clinical trials, consumption of pomegranate polyphenolic extract has led to a significant decrease in serum MDA in diabetic patients.38, 39 In this study, we suggest that PJ treatment seems to control reactive oxygen species levels by diminishing MDA levels.
An important observation was the increased collagen deposition in the diabetic group that was reversed by PJ. This study may explain the decrease in penile elasticity and compliance that was protected by PJ. Also, decreased SMC/collagen ratio has been observed in the CC of diabetic animals.40, 41 Collagen accumulation is a major feature of fibrosis. Punica granatum flower extract diminishes cardiac fibrosis in Zucker diabetic fatty rats.42 PJ intake prevented erectile tissue fibrosis in the ED group.14
In conclusion, diabetes-induced ED includes enhanced oxidative stress caused by alterations of NOS isoform expression, free radicals and collagen density in penile structures, which can be controlled by PJ. We found a moderate prevention of the impairment caused by diabetes on erectile responses in vivo and a near complete preservation of EFS-induced relaxation. PJ administration failed to preserve endothelium-dependent relaxation and eNOS or eNOS expression levels but prevented cavernosal fibrosis. There is still a need for further studies that may reveal the beneficial effect of antioxidative contribution to PDE5 inhibitors.
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The authors declare no conflict of interest.
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Onal, E., Yilmaz, D., Kaya, E. et al. Pomegranate juice causes a partial improvement through lowering oxidative stress for erectile dysfunction in streptozotocin-diabetic rat. Int J Impot Res 28, 234–240 (2016). https://doi.org/10.1038/ijir.2016.34
Umbelliferone isolated from Zosima absinthifolia roots partially restored erectile dysfunction in streptozotocin-induced diabetic rats
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