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Melatonin Treatment Ameliorates Hyperhomocysteinemia-Induced Impairment of Erectile Function in a Rat Model
ORIGINAL RESEARCH & REVIEWS
Melatonin Treatment Ameliorates Hyperhomocysteinemia-Induced Impairment of Erectile Function in a Rat Model Zhe Tang, MS,1,2 Jingyu Song, MS,1,2 Zhe Yu, MS,1,2 Kai Cui, MS,1,2 Yajun Ruan, MD, PhD,1,2 Tao Wang, MD, PhD,1,2 Jun Yang, MD, PhD,1,2 Shaogang Wang, MD, PhD,1,2 and Jihong Liu, MD, PhD1,2
ABSTRACT
Background: Hyperhomocysteinemia (HHcy) has been reported to be strongly correlated with the occurrence of erectile dysfunction (ED), but the mechanisms are not fully understood. Moreover, whether melatonin could be a potential treatment of HHcy-induced ED needs to be elucidated. Aim: The aim of this study was to investigate the effects of melatonin on HHcy-induced ED and the potential mechanisms via modulating oxidative stress and apoptosis. Methods: The Sprague-Dawley (SD) rat model of HHcy was induced by 7% methionine (Met)-rich diets. 36 male SD rats were randomly distributed into 3 groups (n ¼ 12 per group): control group, 7% Met group, and 7% Met þ melatonin (Mel; 10 mg/kg, intraperitoneal injection) treatment group. After 4 weeks, the erectile function of all rats was evaluated by electrical stimulation of the cavernous nerve. Histologic and molecular alterations of the corpus cavernosum were also analyzed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay, Western blotting, and polymerase chain reaction. Outcomes: HHcy-induced ED rat models were successfully established, and Mel could preserve erectile function mainly through inhibiting oxidative stress via the Erk1/2/Nrf2/HO-1 signaling pathway and suppression of apoptosis. Results: Erectile function was significantly reduced in the rats with HHcy compared with that in the control group and was ameliorated in the HHcy rats treated with Mel. Compared with the control group, the rats in the HHcy group showed the following: (1) higher levels of total plasma homocysteine; (2) fewer neuronal nitric oxide synthase-positive cells in the corpus cavernous; (3) higher levels of reactive oxygen species and malondialdehyde, higher expression levels of nicotinamide adenine dinucleotide phosphate oxidase, and lower activities of superoxide dismutase, indicating an overactivated oxidative stress; (4) lower expression levels of Erk1/2/Nrf2/ HO-1 signaling pathway components; and (5) higher levels of apoptosis, as determined by the expression levels of Bax, Bcl-2, and caspase 3. Mel treatment improved the erectile response, as well as histologic and molecular alterations. Clinical Translation: Our study on a rodent model of HHcy provided evidence that Mel could be a potential therapeutic method for HHcy-related ED. Conclusions: Mel treatment improves erectile function in rats with HHcy probably by potential antioxidative stress activity. This finding provides evidence for a potential new therapy for HHcy-induced ED. Tang Z, Song J, Yu, Z, et al. Melatonin Treatment Ameliorates Hyperhomocysteinemia-Induced Impairment of Erectile Function in a Rat Model. J Sex Med 2019;XX:XXXeXXX. Copyright 2019, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.
Key Words: Melatonin; Hyperhomocysteinemia; Erectile Dysfunction; Oxidative Stress; Apoptosis
INTRODUCTION Received May 8, 2019. Accepted July 1, 2019. 1
Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China;
2
Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Copyright ª 2019, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jsxm.2019.07.003
J Sex Med 2019;-:1e12
Erectile dysfunction (ED) is defined as the inability to attain or maintain an erection sufficient for satisfactory sexual intercourse,1 is an evolving health problem with a growing incidence in the male population worldwide, and is predicted to affect up to 322 million cases by the year 2025.2 ED shares many common cardiovascular risk factors, including hypercholesterolemia, hypertension, hyperlipidemia, metabolic disorders, diabetes mellitus (DM), and 1
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smoking.3,4 Among all the factors, hyperhomocysteinemia (HHcy), defined as a pathological condition characterized by elevations of total plasma homocysteine (Hcy), has gained rising attention for the correlation with an increased risk of cardiovascular disorders.58 Since Khan et al9 first reported that HHcy could exert an inhibitory effect on endothelium-dependent relaxation and nitric oxide (NO) formation in vitro, establishing a preliminary correlation between HHcy and cavernosal dysfunction, several animal studies and clinical trials have indicated further that HHcy may be a determinant for ED.1013 Several reports have demonstrated that HHcy impairs cell function mainly by enhancing oxidative stress, as well as cell apoptosis,1416 which have been reported to play a significant role in the occurrence and development of ED.17,18 However, the underlying mechanisms of HHcy-related ED were not fully understood and could partially explain why oral phosphodiesterase 5 (PDE5) inhibitor therapy still had limitations. Therefore, it is required to identify novel treatment methods for men responding poorly to traditional PDE5 inhibitors, as well as to have a better understanding of the molecular mechanisms. Melatonin (Mel) is a tryptophan-derived chemical messenger that is mainly secreted by the pineal gland.19 It possesses various biological functions, including antioxidative stress, antiinflammation, anti-apoptosis, and antineurotoxicity.20 In past decades, a close relationship was discovered between Mel and HHcy. Several reports have provided evidence that Mel could regulate the total plasma Hcy level21,22 and protect the cells against HHcyinduced toxicities.16,23 Mel is well known for its antioxidant properties, which are derived from its ability to scavenge free radicals as an exogenous antioxidant.24 Based on these antioxidant, antiapoptosis and vascular protective functions of melatonin, we speculated whether it could be applied as a potential treatment method for ED. Previously, it was found that Mel could exert a protective effect in ED induced by chronic lower body ischemia,25 spinal cord injury,26 and DM.27,28 Moreover, Bozkurt et al29 demonstrated an important relationship between ED occurrence and low serum Mel levels. Given the emerging evidence, we have been studying whether Mel treatment could be used as a protective agent in HHcy-induced ED.30 31
In a previous study, we have established successfully a rat model of HHcy-related ED in male Sprague-Dawley (SD) rats induced by a methionine (Met)-rich diet method. Here, we used the rat model to explore further the effect of HHcy on erectile function and tried to determine whether Mel could protect against HHcy-induced ED in the rat model, as well as determine the underlying mechanisms.
MATERIALS AND METHODS Animals and Treatment All animal experiments were conducted in accordance with the accepted standards of humane animal care approved by the Academic Administration Committee of Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology, Wuhan, China. All animal experiments were conducted based on the guideline of Academic Animal Care and Use Committee. In total, 36 male SD rats obtained from the Laboratory Animal Center of Tongji Medical College, 8 weeks of age and weighing 280e300 g, were randomly distributed into 3 groups (n ¼ 12 per group): control group, 7% methionine group (7% Met), and 7% methionine plus melatonin treatment group (7% Met þ Mel). The Met-enriched diets were fed orally to the rats daily for 4 weeks. The rats in the 7% Met þ Mel group were administered with daily intraperitoneal injections of Mel (Sigma Aldrich, St Louis, MO, USA) at 10 mg/kg for 4 weeks. The body weights were measured on day 0 and prior to euthanasia.
Evaluation of Erectile Function After 4 weeks, the erectile function of all rats was evaluated according to our previous experimental methods.32 Briefly, the rats were anesthetized by pentobarbital sodium (40 mg/kg, intraperitoneally). A PE-50 tube was intubated into the carotid artery for the continuous monitoring of the mean arterial pressure (MAP), and a 25-gauge needle was inserted into the right crura to monitor the intracavernous pressure (ICP). Next, cavernous nerves (CNs) were isolated and stimulated continuously for 1 minute at a frequency of 15 Hz and voltages of 2.5 V and 5 V, with a 3-minute interval before the next stimulation to avoid nerve fatigue. The ratio of the maximal ratio of ICP to MAP and area under the ICP curve (AUC) were calculated and analyzed to evaluate erectile function in vivo. After the measurement, the corpus cavernous (CC) of each rat was cut into 5 pieces, and the piece pierced by the needle was discarded. The remaining pieces of CC were immediately frozen in liquid nitrogen and stored at 80 C or fixed overnight in 4% paraformaldehyde (Beyotime Biotechnology, China) and then were embedded in paraffin for further experiments. Additionally, the blood samples of all rats were obtained from the carotid artery to detect plasma Hcy levels.
Measurement of the Antioxidant Activity
CC tissues stored at 80 C were processed and assayed for antioxidant activity. Commercial kits to detect malondialdehyde (MDA) and superoxide dismutase (SOD) were used according to the manufacturer’s protocol (Beyotime Biotechnology). The penile MDA levels and SOD activities were normalized to the wet weight of the penile tissue samples.
Detection of the intracellular reactive oxygen species in the CC Intracellular reactive oxygen species (ROS) in the CC were detected using an oxidation-sensitive fluorescent probe (DCFHDA; Beyotime Biotechnology). The sections were processed and then incubated with 10 mmol/L of DCFH-DA at 37 C for 20 minutes according to the manufacturer’s protocols. Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Beyotime J Sex Med 2019;-:1e12
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Melatonin Treatment Improved Hyperhomocysteinemia-Induced Erectile Dysfunction
Table 1. Metabolic and physiological variables Variables
Control (n ¼ 12)
Initial weight (g) Final weight (g) NO (mmol$g1 protein) cGMP (pmol$mg1 protein)
283.74 389.14 3.21 5.83
± ± ± ±
8.6 24.3 0.072 0.039
7% Met (n ¼ 12) 285.11 267.23 1.08 3.64
± ± ± ±
9.39 21.34* 0.05* 0.096*
7% Met þ Mel (n ¼ 12) 281.43 270.33 2.37 4.25
± ± ± ±
11.26 22.19* 0.043*,#,& 0.041*,#,&
cGMP ¼ cyclic guanosine monophosphate; Mel ¼ melatonin; Met ¼ methionine; NO ¼ nitric oxide. *,&P < .05 compared with the control group. # P < .05 compared with the 7% Met group.
Biotechnology). Fluorescence images were acquired using an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan).
was used to evaluate the relative mRNA expression compared with that of the controls. The primer sequences are listed in Supplementary Table 2.
Immunofluorescence
Protein Immunoblot Analysis
Immunofluorescence and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL) double staining were used to localize Bim and Puma in hair follicles and to colocalize them with apoptotic cells. The sections were deparaffinized, rehydrated, pretreated in citrate buffer, pH 6.0, for 5 minutes at 98 C for antigen retrieval, and incubated with 20 mg/ mL of proteinase K for 15 minutes at room temperature. The primary antibodies for a-SMA were incubated overnight at 4 C. A TUNEL apoptosis detection kit (Beyotime Biotechnology) was used to detect apoptotic cells on the same sections. The TUNEL reaction mixture was prepared according to the manufacturer’s instructions. Nuclei were stained with DAPI (Beyotime Biotechnology). Fluorescence images were acquired using an Olympus BX51 fluorescence microscope (Olympus Corporation).
Total proteins in cells were extracted by NP-40 lysis buffer with proteinase inhibitor cocktail (Roche, Branchburg, NJ, USA) and phosphatase inhibitor cocktail (EMD Millipore, Burlington, MA, USA), and Western blotting analysis was performed as follows. Protein samples (20 mg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes. After blocking in Tris-buffered saline-Tween 20 with 5% bovine serum albumin, the membranes were incubated overnight at 4 C with primary antibodies; the antibodies used are listed in Supplementary Table 1. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; Proteintech) and were analyzed using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Rockford, IL, USA).
Immunofluorescence of neuronal nitric oxide synthase (nNOS) was performed using the same method. The details of the antibodies used are listed in Supplementary Table 1.
Immunohistochemistry Primary antibodies to HO-1 (ab68477; Sigma Aldrich) was used for immunostaining on formalin-fixed, paraffin-embedded tissue slides from the rat models. Briefly, after tissue sections were deparaffinized and rehydrated through a graded alcohol series, they were heated by microwave in 0.01 mol/L of citrate buffer at pH 6.0 for 20 minutes to retrieve antigens. After a 30-minute incubation for protein blocking, the tissue sections were incubated in primary antibodies, followed by incubation in horseradish peroxidase polymer-conjugated secondary antibody (cat #K4061, Dako).
Quantitative Real-Time Polymerase Chain Reaction Analysis Total RNA in cells was extracted by Trizol reagent (ThermoFisher; cat #15596026) and was reversely transcribed to cDNA using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific). The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using the fast SYBR green master mix (Thermo Fisher Scientific). The 2-DDCt method J Sex Med 2019;-:1e12
Statistical Analysis The data were presented as the mean± SEM and were analyzed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance followed by the Tukey-Kramer test for post hoc comparisons was performed to
Figure 1. Plasma tHcy level in all rats. The plasma tHCy level determined by the enzyme-linked immunosorbent assay method was increased in the 7% Met group and decreased in the melatonin-treated group (n ¼ 12 per group). *,&P < .05 compared with the control group. #P < .05 compared with the 7% Met group. Mel ¼ melatonin; Met ¼ methionine; tHcy ¼ total plasma homocysteine.
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analyze the differences between any 2 groups, and P values < .05 were considered to indicate statistically significant differences.
RESULTS Metabolic Parameters The body weights of each group are shown in Table 1, and the plasma Hcy levels are shown in Figure 1. The body weights of the 7% Met group were lower than those of the controls after 4 weeks (P < .05). The Hcy levels of the 7% Met group were statistically higher than those of the control group (P < .05), demonstrating that the rat model with HHcy was successfully established. Mel treatment did not change the body weight. However, Mel decreased the plasma Hcy levels significantly compared with that of the 7% Met group, which was consistent with those of previous studies.31
Melatonin Protects Against ED Induced by HHcy CN electrical stimulation with different voltages (2.5 V and 5 V) was administered to evaluate the erectile function of rats. The maximum ICP/MAP and AUC were measured and recorded. The ratio of maximum ICP/MAP in the 7% Met group was sharply attenuated compared with that of the control group (P < .05). However, erectile function was partially improved in the Mel treatment group, although it was still lower than that in the control group (P < .05). The AUCs at
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2.5 and 5 V in all 3 groups exhibited the same trend as the maximum ICP/MAP values (Figure 2). Moreover, as shown in Table 1, decreased NO and cyclic guanosine monophosphate (cGMP) levels of CC were observed in the 7% Met group (P < 0.05), whereas four weeks of melatonin treatment increased NO and cGMP levels significantly compared with those in rats fed Met-rich diets. Together, these results suggested that HHcy impaired the erectile function of rats, whereas Mel could protect against ED induced by HHcy.
Melatonin Partially Restores the Number of nNOS-Positive Cells in the CC Immunofluorescence and Western blotting were used to analyze the content and expression of nNOS in the CC. Our results in Figure 3 show that nNOS expression in the CC was lower than that of the control group. Mel treatment could increase the contents, although not to those in the control group. Western blotting also showed the same trend (both P < .05).
Melatonin Inhibits Oxidative Stress in the CC of Rats With HHcy A fluorescent probe was used to detect the intracellular ROS of CC. As shown in Figure 4A, the levels of ROS were increased significantly in the CC of the 7% Met group and were reduced by Mel administration (both P < .05). The amount of MDA and activities of SOD were detected by enzyme-linked immunosorbent
Figure 2. Melatonin partially restores erectile function in rats with hyperhomocysteinemia (HHcy). (A, B) Representative tracing of ICP using electric stimulations at 2.5 V and 5 V for age-matched normal control, HHcy induced by 7% Met and Mel-treated HHcy groups (n ¼ 12 per group). Scale bars represent 1 minute. (C, D) The maximum ICP/MAP ratio and total ICP of the different stimulations are presented as bar graphs for max ICP/MAP and total ICP (AUC). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. AUC ¼ area under the curve; ICP ¼ intracavernous pressure; MAP ¼ mean arterial pressure; Mel ¼ melatonin; Met: methionine. Figure 2 is available in color online at www.jsm.jsexmed.org. J Sex Med 2019;-:1e12
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Figure 3. The nNOS expression in the corpus cavernous of all rats. (A) Representative images of nNOS content in the corpus cavernous of all rats at 200 magnification. The scale bar represents 100 mm. (B) Representative Western blot results for nNOS in the corpus cavernous (CC) of all rats. (C) The expression levels of nNOS, with b-actin as the loading control, in the CC of the 3 groups are presented as bar graphs (n ¼ 6e8 rats per group). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. DAPI ¼ 40 ,6diamidino-2-phenylindole; Mel ¼ melatonin; Met ¼ methionine; nNOS ¼ neuronal nitric oxide synthase. Figure 3 is available in color online at www.jsm.jsexmed.org. assay. The MDA levels were higher in the 7% Met group than that in the control group and the Mel treatment group (both P < .05; Figure 4B). The SOD activities in the 7% Met group were lower than those in the control group; however, in the Mel þ 7% Met group, the SOD activities were slightly higher than those in the 7% Met group (both P < .05; Figure 4C). We also detected the expression levels of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) subunits, including p22phox, p47phox, and gp91phox, whose expression levels in the 3 groups showed a similar trend as those of MDA in each group (all P < .05; Figure 5).
Melatonin Improves Oxidative Stress Via Activating the Erk1/2/Nrf2/HO-1 Signaling Pathway Western blotting analysis showed that the expression levels of the Erk1/2/Nrf2/HO-1 signaling pathway components, J Sex Med 2019;-:1e12
which exert a protective effect against overactivated oxidative stress, were significantly lower in the CC of the 7% Met groups than that of the control group (P < .05; Figure 6). Mel administration activated the signaling pathway, inducing the expression levels of p-Erk1/2, Nrf2, and HO-1, which were significantly higher than those in the 7% Met groups (P < .05; Figure 6). The RT-PCR results of Nrf2 and HO-1 were consistent with the Western blotting results (all P < .05; Figure 6).
Melatonin Inhibits Apoptosis in the CC of Rats With HHcy TUNEL staining was used to determine whether apoptosis was induced by HHcy in vivo. Additionally, a-SMA staining was performed on the same tissue slide to determine the expression of a-SMA. As shown in Figure 7, more apoptosis
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Figure 4. Melatonin can inhibit oxidative stress in the corpus cavernous of rats with HHcy. (A) Representative images of the ROS fluorescent probe in the corpus cavernous of all rats at 400 magnification. The scale bar represents 50 mm. (B) MDA levels determined by enzyme-linked immunosorbent assay (ELISA) in the corpus cavernous (CC) of all rats. (C) SOD activities determined by ELISA in the corpus cavernous of all rats. *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. DAPI ¼ 40 ,6-diamidino2-phenylindole; MDA ¼ malondialdehyde; Mel ¼ melatonin; Met ¼ methionine; ROS ¼ reactive oxygen species; SOD ¼ superoxide dismutase. Figure 4 is available in color online at www.jsm.jsexmed.org.
was observed in the CC of the 7% Met group than that of the control group, and Mel treatment could inhibit apoptosis. Additionally, the expression levels of a-SMA were lower in the CC of rats with HHcy. Western blotting analysis showed that the Bax/Bcl-2 ratio, expression levels of caspase 3 and cleaved caspase 3, which also reflect apoptosis, showed the same trend as TUNEL staining (all P < .05; Figure 7).
DISCUSSION In this study, we demonstrated the protective role of Mel in erectile function in a rat model of HHcy, as well as the underlying mechanisms. Our data indicated that HHcy-related ED was characterized by a decreased NO/cGMP signaling pathway, oxidative stress, and apoptosis. However, Mel treatment could
partially restore erectile function by activating NO/cGMP, inhibiting oxidative stress via the Erk1/2/Nrf2/HO-1 signaling pathway, and suppressing cell apoptosis. Hcy is a non-protein-forming sulfur amino acid and is formed during the metabolism of methionine. HHcy has been reported to be strongly correlated with the occurrence of ED,10,33,34 even in the presence of other risk factors for ED, such as DM,12,35 and could be an early predictor of ED.36 Furthermore, HHcy may be linked to nonresponsiveness in PDE5 inhibitor therapy.12,37 Although several drugs, such as folic acid, vitamin B6, and B12, have already been reported to decrease circulating Hcy concentrations,38,39 their effects on ED were still not fully elucidated. Exploring the mechanisms underlying how HHcy affects erectile function will be conducive to finding a novel therapy method for ED. J Sex Med 2019;-:1e12
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Figure 5. Melatonin can inhibit nicotinamide adenine dinucleotide phosphate oxidase subunits in the corpus cavernous (CC) of rats with hyperhomocysteinemia. (A) Representative Western blot results of p22phox, p47phox, and gp91phox in the CC of all rats. Expressions of p22phox, p47phox, and gp91phox with b-actin as the loading control in all rats are presented as bar graphs: (B) for p22phox, (C) for p47phox, and (D) for gp91phox (n ¼ 6 rats per group). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. Met: methionine; Mel: melatonin. Normal erectile function is dependent on the normal function of penile components and underlying molecular biological procedures. The risk factors of ED are mainly linked to the attenuated bioavailability of NO, partially due to the augmented generation of ROS.40,41 There have been several studies elucidating the relationship between ED and ROS, which reflect the levels of oxidative stress in a DM or aged condition.42,43 Endogenous ROS are produced mainly by NADPH oxidase, a multisubunit enzyme including p47phox, p67phox, gp91phox, p22phox, and p40phox in cell membranes, mitochondria, peroxisomes, and endoplasmic reticulum.44 It was reported that excessive ROS in CC could lead to ED via attenuating NO bioavailability, decreasing the cGMP level, and inducing apoptosis-mediated loss of smooth muscle and endothelial cells.45 Excessive ROS can be found in metabolic disorders, such as DM, chronic ethanol consumption, smoking, and, more recently, HHcy. Additionally, we previously reported that some NADPH oxidase subunits were greatly activated in the CC of rats with ED induced by type I DM or androgen deficiency.46,47 Moreover, a recent study by Jiang et al48 reported that oxidative stress was the main contributor to ED induced by HHcy. In our study, we observed that HHcy induced oxidative stress, the existence of which was verified via an increased level of ROS, activation of NADPH oxidase subunit expression, increased MDA levels, and reduced SOD activities, thus impairing normal erectile function in the rat model. Moreover, weight loss was J Sex Med 2019;-:1e12
observed during the process of breeding, reflecting the status of metabolic disorder, which was reported in our previous study.31 Interestingly, the ED rats also showed neurodegenerative changes characterized by the decrease in nNOS-positive nerve fibers in the CC, suggesting that HHcy could induce ED because of its detrimental effects on neurological function. Cavernous smooth muscle cells comprise the predominant structure of CC and play a significant role in achieving and maintaining sufficient erection via controlling the blood flow into the CC. Loss of cavernous smooth muscle cells was observed in our study, in line with previous reports in humans49 and various rat ED models, including diabetes,50 hyperlipidemia,51 and age-related ED.42 Moreover, numerous studies have confirmed that HHcy could increase apoptosis in various cells.15 In this study, we showed that the level of apoptosis was increased significantly in the corresponding area where cavernous smooth muscle cells were decreased, and HHcy triggered the apoptosis of CC, as evident by the elevated levels of the pro-apoptotic marker Bax and reduced levels of the anti-apoptotic marker Bcl-2, and activated effector caspases. Mel is derived from the pinealocytes of the pineal gland and exerts numerous physiological effects through its 3 receptors. Baydas et al22 first reported that the level of Mel had a causal relationship with the plasma concentration of Hcy. Our results showed that Mel treatment could decrease significantly the
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Figure 6. Melatonin inhibits oxidative stress via the Erk1/2/Nrf2/HO1 signaling pathway. (A) Representative Western blot results for Erk1/ 2, p-Erk1/2, Nrf2, and HO1 in the corpus cavernous (CC) of all rats. (B) The expression levels of Erk1/2, p-Erk1/2, Nrf2, and HO1, with b-actin as the loading control, in the CC of the 3 groups are presented as bar graphs (n ¼ 3 rats per group). (C) and (D) represent the relative mRNA expression of Nrf2 and HO1 genes, respectively, to GAPDH in the CC of all rats by quantitative real-time polymerase chain reaction. (E) Representative immunohistochemistry images showing the suppression of HO1 by hyperhomocysteinemia and activation by melatonin. The scale bar represents 100 mm. *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. GAPDH ¼ glyceraldehyde 3-phosphate dehydrogenase; Mel ¼ melatonin; Met ¼ methionine. Figure 6 is available in color online at www.jsm.jsexmed. org. plasma Hcy levels induced by a Met-rich diet, consistent with previous reports.21 Given Mel’s antioxidant properties, it was not surprising that it significantly decreased the ROS level, deactivated the expression of the NADPH oxidase subunit, decreased the MDA level, and increased SOD activity, accompanied by the inhibition of apoptosis. Moreover, we found that melatonin could restore the number of nNOS-positive cells in the CC, consistent with a previous study on ED in streptozotocin-induced diabetic rats.27
To explore how Mel decreased the levels of oxidative stress, we analyzed a critical signaling pathway against oxidative stressinduced injury (ie, Erk1/2/Nrf2/HO-1 signaling pathway). HO1 is one of the most important endogenous antioxidant enzymes and is thought to play a key role in maintaining the antioxidant/ oxidant imbalance.52 NRF2 is a basic region leucine-zipper transcription factor and has been reported to be involved in the transcriptional regulation of the HO-1 gene.53,54 It was reported that the phosphorylation of several signal transduction pathways, J Sex Med 2019;-:1e12
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Figure 7. Melatonin can reduce apoptosis in the corpus cavernous (CC) of rats with hyperhomocysteinemia. (A, B) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling and the a-SMA content in the CC of all rats at 400 magnification. The scale bar represents 50 mm. (C) Representative Western blot results of Bax, Bcl-2, caspase 3, and cleaved caspase 3 in the CC of all rats. (D) The expression levels of Bax, Bcl-2, caspase 3, and cleaved caspase 3, with b-actin as the loading control, in the CC of the 3 groups are presented as bar graphs (n ¼ 6 rats per group). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. DAPI ¼ 40 ,6-diamidino-2-phenylindole; Mel ¼ melatonin; Met ¼ methionine. Figure 7 is available in color online at www.jsm.jsexmed.org. including ERK1/2 and PI3K/AKT, could activate NRF2, leading to the translocation of NRF2.55,56 Here, we observed that HHcy could decrease the expression level of the Erk1/2/Nrf2/HO-1 signaling pathway and confirmed that Mel increased Nrf2 expression and HO-1 protein levels via the phosphorylation of ERK1/2 in the CC of rats. Taken together, the data showed that the roles in antioxidative stress and anti-apoptosis, together with the normalization of elevated Hcy and neuroprotective effects, validated Mel as a potential treatment to protect cells against Hcyinduced excitotoxicity and cell death, thus improving ED.
research is that our rat ED models induced by a Met-rich diet reflects a metabolic disorder characterized with HHcy but may not necessarily represent the real pathologic processes that occur in patients with ED induced by HHcy and other risk factors. Moreover, the absence of cell-based experiments was another limitation. Therefore, in future studies, we should examine the effect of Mel in vitro to more thoroughly investigate the mechanisms involved in the benefits of melatonin for HHcyinduced ED.
Although our study provides evidence for potential novel therapy for ED induced by HHcy and demonstrated several underlying mechanisms, a major limitation of the current
Strengths and Limitations
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Our study provided evidence, we believe for the first time, that Mel was effective in partially restoring erectile function in rats
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with HHcy. However, our HHcy-induced rat ED models may not necessarily represent the pathologic processes that occur in ED induced by other risk factors.
CONCLUSIONS This study revealed that oxidative stress, together with cell apoptosis, was involved in ED induced by HHcy in rats, whereas Mel preserved erectile function through decreasing the plasma Hcy level, inhibiting oxidative stress via the Erk1/2/ Nrf2/HO-1 signaling pathway, suppressing apoptosis, activating NO/cGMP, and partially restoring the content of cavernous smooth muscle cells. Thus, Mel could be a promising preventive factor for ED induced by HHcy because of its numerous beneficial effects. Corresponding Author: Jihong Liu, MD, PhD, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel: þ86 027 83663460; Fax: þ86 027 83663460; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest. Funding: This work was supported by a grant from the National Natural Sciences Foundation of China (No. 81270690 and No. 81501246).
STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Zhe Tang; Jun Yang; Jihong Liu (b) Acquisition of Data Zhe Tang; Jingyu Song; Zhe Yu (c) Analysis and Interpretation of Data Zhe Tang; Kai Cui; Yajun Ruan; Tao Wang; Shaogang Wang Category 2 (a) Drafting of the Article Zhe Tang; Jun Yang (b) Revising the Article for Intellectual Content Zhe Tang; Jun Yang; Tao Wang; Jihong Liu Category 3 (a) Final Approval of the Completed Article Zhe Tang; Jingyu Song; Zhe Yu; Kai Cui; Yajun Ruan; Tao Wang; Jun Yang; Shaogang Wang; Jihong Liu
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Tang et al 3. Allen MS, Walter EE. Erectile dysfunction: An umbrella review of meta-analyses of risk-factors, treatment, and prevalence outcomes. J Sex Med 2019;16:531-541. 4. Capogrosso P, Colicchia M, Ventimiglia E, et al. One patient out of four with newly diagnosed erectile dysfunction is a young man–Worrisome picture from the everyday clinical practice. J Sex Med 2013;10:1833-1841. 5. Homocysteine Studies Collaboration. Homocysteine and risk of ischemic heart disease and stroke: A meta-analysis. JAMA 2002;288:2015-2022. 6. Wald DS, Law M, Morris JK. Homocysteine and cardiovascular disease: Evidence on causality from a meta-analysis. BMJ 2002;325:1202. 7. Gurda D, Handschuh L, Kotkowiak W, et al. Homocysteine thiolactone and N-homocysteinylated protein induce proatherogenic changes in gene expression in human vascular endothelial cells. Amino Acids 2015;47:1319-1339. 8. Selhub J. Homocysteine metabolism. Annu Rev Nutr 1999; 19:217-246. 9. Khan MA, Thompson CS, Emsley AM, et al. The interaction of homocysteine and copper markedly inhibits the relaxation of rabbit corpus cavernosum: New risk factors for angiopathic erectile dysfunction? BJU Int 1999;84:720-724. 10. Demir T, Comlekҫi A, Demir O, et al. Hyperhomocysteinemia: A novel risk factor for erectile dysfunction. Metabolism 2006; 55:1564-1568. 11. Demir T, Cömlekci A, Demir O, et al. A possible new risk factor in diabetic patients with erectile dysfunction: Homocysteinemia. J Diabetes Complications 2008;22:395-399. 12. Al-Hunayan A, Thalib L, Kehinde EO, et al. Hyperhomocysteinemia is a risk factor for erectile dysfunction in men with adult-onset diabetes mellitus. Urology 2008; 71:897-900. 13. Jones RW, Jeremy JY, Koupparis A, et al. Cavernosal dysfunction in a rabbit model of hyperhomocysteinaemia. BJU Int 2005;95:125-130. 14. Yilmaz N. Relationship between paraoxonase and homocysteine: Crossroads of oxidative diseases. Arch Med Sci 2012; 8:138-153. 15. Perła-Kaján J, Twardowski T, Jakubowski H. Mechanisms of homocysteine toxicity in humans. Amino Acids 2007; 32:561-572. 16. Baydas G, Koz ST, Tuzcu M, et al. Melatonin inhibits oxidative stress and apoptosis in fetal brains of hyperhomocysteinemic rat dams. J Pineal Res 2007;43:225-231. 17. D’Andrea S, Micillo A, Francavilla F, et al. Serum from patients with erectile dysfunction and vascular risk factors triggered an oxidative stress-dependent mitochondrial apoptotic pathway in ex vivo expanded circulating angiogenic cells of healthy men. J Sex Med 2016;13:1063-1070. 18. Wang Y, Meng XH, Zhang QJ, et al. Losartan improves erectile function through suppression of corporal apoptosis and oxidative stress in rats with cavernous nerve injury. Asian J Androl https://doi.org/10.4103/aja.aja_8_19; E-pub ahead of print. J Sex Med 2019;-:1e12
Melatonin Treatment Improved Hyperhomocysteinemia-Induced Erectile Dysfunction 19. Cipolla-Neto J, Amaral FGD. Melatonin as a hormone: New physiological and clinical insights. Endocr Rev 2018; 39:990-1028. 20. Paul R, Borah A. The potential physiological crosstalk and interrelationship between two sovereign endogenous amines, melatonin and homocysteine. Life Sci 2015;139:97-107.
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35. Hu Y, Xu Y, Wang G. Homocysteine levels are associated with endothelial function in newly diagnosed type 2 diabetes mellitus patients. Metab Syndr Relat Disord 2019. https://doi. org/10.1089/met.2019.0011; Epub ahead of print.
21. Murawska-Cialowicz E, Januszewska L, Zuwala-Jagiello J, et al. Melatonin decreases homocysteine level in blood of rats. J Physiol Pharmacol 2008;59:717-729.
36. Giovannone R, Busetto GM, Antonini G, et al. Hyperhomocysteinemia as an early predictor of erectile dysfunction: International Index of Erectile Function (IIEF) and penile Doppler ultrasound correlation with plasma levels of homocysteine. Medicine (Baltimore) 2015;94:e1556.
22. Baydas G, Gursu MF, Cikim G, et al. Homocysteine levels are increased due to lack of melatonin in pinealectomized rats: Is there a link between melatonin and homocysteine? J Pineal Res 2002;32:63-64.
37. Lombardo F, Tsamatropoulos P, Piroli E, et al. Treatment of erectile dysfunction due to C677T mutation of the MTHFR gene with vitamin B6 and folic acid in patients non responders to PDE5i. J Sex Med 2010;7(1 Pt 1):216-223.
23. Ortega-Gutiérrez S, Fuentes-Broto L, Garcia JJ, et al. Melatonin reduces protein and lipid oxidative damage induced by homocysteine in rat brain homogenates. J Cell Biochem 2007;102:729-735.
38. Sansone M, Sansone A, Romano M, et al. Folate: A possible role in erectile dysfunction? Aging Male 2018;21:116-120.
24. Reiter RJ, Tan DX, Osuna C, et al. Actions of melatonin in the reduction of oxidative stress. A review. J Biomed Sci 2000; 7:444-458. 25. Sawada N, Nomiya M, Zarifpour M, et al. Melatonin improves erectile function in rats with chronic lower body ischemia. J Sex Med 2016;13:179-186. 26. Tavukҫu HH, Sener TE, Tinay I, et al. Melatonin and tadalafil treatment improves erectile dysfunction after spinal cord injury in rats. Clin Exp Pharmacol Physiol 2014;41:309-316. 27. Zhang JL, Hui Y, Zhou F, et al. Neuroprotective effects of melatonin on erectile dysfunction in streptozotocin-induced diabetic rats. Int Urol Nephrol 2018;50:1981-1988. 28. Qiu XF, Li XX, Chen Y, et al. Mobilisation of endothelial progenitor cells: One of the possible mechanisms involved in the chronic administration of melatonin preventing erectile dysfunction in diabetic rats. Asian J Androl 2012;14:481-486. 29. Bozkurt A, Karabakan M, Aktas BK, et al. Low serum melatonin levels are associated with erectile dysfunction. Int Braz J Urol 2018;44:794-799. 30. Tang Z, Cui K, Ruan YJ, et al. Melatonin treatment improved erectile dysfunction via inhibiting oxidative stress and apoptosis in a hyperhomocysteinemia rat model. Abstract 278. Lisbon, Portugal: Presented at the World Meeting on Sexual Medicine; Friday, March 2, 2018. 31. Cui K, Luan Y, Tang Z, et al. Human tissue kallikrein-1 protects against the development of erectile dysfunction in a rat model of hyperhomocysteinemia. Asian J Androl. https://doi.org/1 0.4103/aja.aja_111_18; Epub ahead of print. 32. Yang J, Zhang Y, Zang G, et al. Adipose-derived stem cells improve erectile function partially through the secretion of IGF-1, bFGF, and VEGF in aged rats. Andrology 2018;6:498509. 33. Zhang Z, Xu Z, Dai Y, et al. Elevated serum homocysteine level as an independent risk factor for erectile dysfunction: A prospective pilot case-control study. Andrologia 2017;49. 34. Sansone A, Cignarelli A, Sansone M, et al. Serum homocysteine levels in men with and without erectile dysfunction: A systematic review and meta-analysis. Int J Endocrinol 2018; 2018:7424792. J Sex Med 2019;-:1e12
39. Katsiki N, Perez-Martinez P, Mikhailidis DP. Homocysteine and non-cardiac vascular disease. Curr Pharm Des 2017; 23:3224-3232. 40. Roumeguère T, Van Antwerpen P, Fathi H, et al. Relationship between oxidative stress and erectile function. Free Radic Res 2017;51:924-931. 41. Jeremy JY, Jones RA, Koupparis AJ, et al. Reactive oxygen species and erectile dysfunction: Possible role of NADPH oxidase. Int J Impot Res 2007;19:265-280. 42. Tang Z, Cui K, Luan Y, et al. Human tissue kallikrein 1 ameliorates erectile function via modulation of macroautophagy in aged transgenic rats. Andrology 2018;6:766-774. 43. Malavige LS, Levy JC. Erectile dysfunction in diabetes mellitus. J Sex Med 2009;6:1232-1247. 44. Han D, Williams E, Cadenas E. Mitochondrial respiratory chain-dependent generation of superoxide anion and its release into the intermembrane space. Biochem J 2001; 353(Pt 2):411-416. 45. Bivalacqua TJ, Usta MF, Kendirci M, et al. Superoxide anion production in the rat penis impairs erectile function in diabetes: Influence of in vivo extracellular superoxide dismutase gene therapy. J Sex Med 2005;2:187-197 [discussion: 197198]. 46. Li R, Meng X, Zhang Y, et al. Testosterone improves erectile function through inhibition of reactive oxygen species generation in castrated rats. PeerJ 2016;4:e2000. 47. Yang J, Wang T, Yang J, et al. S-allyl cysteine restores erectile function through inhibition of reactive oxygen species generation in diabetic rats. Andrology 2013;1:487-494. 48. Jiang W, Xiong L, Yang Bin, et al. Hyperhomocysteinaemia in rats is associated with erectile dysfunction by impairing endothelial nitric oxide synthase activity. Sci Rep 2016; 6:26647. 49. Melman A, Gingell JC. The epidemiology and pathophysiology of erectile dysfunction. J Urol 1999;161:5-11. 50. Ruan Y, Li M, Wang T, et al. Taurine supplementation improves erectile function in rats with streptozotocin-induced type 1 diabetes via amelioration of penile fibrosis and endothelial dysfunction. J Sex Med 2016;13:778-785.
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51. Li R, Cui K,Wang T, et al. Hyperlipidemia impairs erectile function in rats by causing cavernosal fibrosis. Andrologia 2017;49.
55. Zhao J, Cheng YY, Fan W, et al. Botanical drug puerarin co-
52. Maines MD. The heme oxygenase system: A regulator of second messenger gases. Annu Rev Pharmacol Toxicol 1997; 37:517-554.
neuronal survival and neuritogenesis via activating ERK1/2 and
53. Pi J, Zhang Q, Fu J, et al. ROS signaling, oxidative stress and Nrf2 in pancreatic beta-cell function. Toxicol Appl Pharmacol 2010;244:77-83. 54. Li W, Kong AN. Molecular mechanisms of Nrf2-mediated antioxidant response. Mol Carcinog 2009;48:91-104.
ordinates with nerve growth factor in the regulation of PI3K/Akt signaling pathways in the neurite extension process. CNS Neurosci Ther 2015;21:61-70. 56. Numazawa S, Ishikawa M, Yoshida A, et al. Atypical protein kinase C mediates activation of NF-E2-related factor 2 in response to oxidative stress. Am J Physiol Cell Physiol 2003; 285:C334-C342.
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Melatonin Treatment Ameliorates Hyperhomocysteinemia-Induced Impairment of Erectile Function in a Rat Model Zhe Tang, MS,1,2 Jingyu Song, MS,1,2 Zhe Yu, MS,1,2 Kai Cui, MS,1,2 Yajun Ruan, MD, PhD,1,2 Tao Wang, MD, PhD,1,2 Jun Yang, MD, PhD,1,2 Shaogang Wang, MD, PhD,1,2 and Jihong Liu, MD, PhD1,2
ABSTRACT
Background: Hyperhomocysteinemia (HHcy) has been reported to be strongly correlated with the occurrence of erectile dysfunction (ED), but the mechanisms are not fully understood. Moreover, whether melatonin could be a potential treatment of HHcy-induced ED needs to be elucidated. Aim: The aim of this study was to investigate the effects of melatonin on HHcy-induced ED and the potential mechanisms via modulating oxidative stress and apoptosis. Methods: The Sprague-Dawley (SD) rat model of HHcy was induced by 7% methionine (Met)-rich diets. 36 male SD rats were randomly distributed into 3 groups (n ¼ 12 per group): control group, 7% Met group, and 7% Met þ melatonin (Mel; 10 mg/kg, intraperitoneal injection) treatment group. After 4 weeks, the erectile function of all rats was evaluated by electrical stimulation of the cavernous nerve. Histologic and molecular alterations of the corpus cavernosum were also analyzed by immunofluorescence, immunohistochemistry, enzyme-linked immunosorbent assay, Western blotting, and polymerase chain reaction. Outcomes: HHcy-induced ED rat models were successfully established, and Mel could preserve erectile function mainly through inhibiting oxidative stress via the Erk1/2/Nrf2/HO-1 signaling pathway and suppression of apoptosis. Results: Erectile function was significantly reduced in the rats with HHcy compared with that in the control group and was ameliorated in the HHcy rats treated with Mel. Compared with the control group, the rats in the HHcy group showed the following: (1) higher levels of total plasma homocysteine; (2) fewer neuronal nitric oxide synthase-positive cells in the corpus cavernous; (3) higher levels of reactive oxygen species and malondialdehyde, higher expression levels of nicotinamide adenine dinucleotide phosphate oxidase, and lower activities of superoxide dismutase, indicating an overactivated oxidative stress; (4) lower expression levels of Erk1/2/Nrf2/ HO-1 signaling pathway components; and (5) higher levels of apoptosis, as determined by the expression levels of Bax, Bcl-2, and caspase 3. Mel treatment improved the erectile response, as well as histologic and molecular alterations. Clinical Translation: Our study on a rodent model of HHcy provided evidence that Mel could be a potential therapeutic method for HHcy-related ED. Conclusions: Mel treatment improves erectile function in rats with HHcy probably by potential antioxidative stress activity. This finding provides evidence for a potential new therapy for HHcy-induced ED. Tang Z, Song J, Yu, Z, et al. Melatonin Treatment Ameliorates Hyperhomocysteinemia-Induced Impairment of Erectile Function in a Rat Model. J Sex Med 2019;XX:XXXeXXX. Copyright 2019, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved.
Key Words: Melatonin; Hyperhomocysteinemia; Erectile Dysfunction; Oxidative Stress; Apoptosis
INTRODUCTION Received May 8, 2019. Accepted July 1, 2019. 1
Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China;
2
Institute of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei, China
Copyright ª 2019, International Society for Sexual Medicine. Published by Elsevier Inc. All rights reserved. https://doi.org/10.1016/j.jsxm.2019.07.003
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Erectile dysfunction (ED) is defined as the inability to attain or maintain an erection sufficient for satisfactory sexual intercourse,1 is an evolving health problem with a growing incidence in the male population worldwide, and is predicted to affect up to 322 million cases by the year 2025.2 ED shares many common cardiovascular risk factors, including hypercholesterolemia, hypertension, hyperlipidemia, metabolic disorders, diabetes mellitus (DM), and 1
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smoking.3,4 Among all the factors, hyperhomocysteinemia (HHcy), defined as a pathological condition characterized by elevations of total plasma homocysteine (Hcy), has gained rising attention for the correlation with an increased risk of cardiovascular disorders.58 Since Khan et al9 first reported that HHcy could exert an inhibitory effect on endothelium-dependent relaxation and nitric oxide (NO) formation in vitro, establishing a preliminary correlation between HHcy and cavernosal dysfunction, several animal studies and clinical trials have indicated further that HHcy may be a determinant for ED.1013 Several reports have demonstrated that HHcy impairs cell function mainly by enhancing oxidative stress, as well as cell apoptosis,1416 which have been reported to play a significant role in the occurrence and development of ED.17,18 However, the underlying mechanisms of HHcy-related ED were not fully understood and could partially explain why oral phosphodiesterase 5 (PDE5) inhibitor therapy still had limitations. Therefore, it is required to identify novel treatment methods for men responding poorly to traditional PDE5 inhibitors, as well as to have a better understanding of the molecular mechanisms. Melatonin (Mel) is a tryptophan-derived chemical messenger that is mainly secreted by the pineal gland.19 It possesses various biological functions, including antioxidative stress, antiinflammation, anti-apoptosis, and antineurotoxicity.20 In past decades, a close relationship was discovered between Mel and HHcy. Several reports have provided evidence that Mel could regulate the total plasma Hcy level21,22 and protect the cells against HHcyinduced toxicities.16,23 Mel is well known for its antioxidant properties, which are derived from its ability to scavenge free radicals as an exogenous antioxidant.24 Based on these antioxidant, antiapoptosis and vascular protective functions of melatonin, we speculated whether it could be applied as a potential treatment method for ED. Previously, it was found that Mel could exert a protective effect in ED induced by chronic lower body ischemia,25 spinal cord injury,26 and DM.27,28 Moreover, Bozkurt et al29 demonstrated an important relationship between ED occurrence and low serum Mel levels. Given the emerging evidence, we have been studying whether Mel treatment could be used as a protective agent in HHcy-induced ED.30 31
In a previous study, we have established successfully a rat model of HHcy-related ED in male Sprague-Dawley (SD) rats induced by a methionine (Met)-rich diet method. Here, we used the rat model to explore further the effect of HHcy on erectile function and tried to determine whether Mel could protect against HHcy-induced ED in the rat model, as well as determine the underlying mechanisms.
MATERIALS AND METHODS Animals and Treatment All animal experiments were conducted in accordance with the accepted standards of humane animal care approved by the Academic Administration Committee of Tongji Hospital, Tongji
Medical College, Huazhong University of Science and Technology, Wuhan, China. All animal experiments were conducted based on the guideline of Academic Animal Care and Use Committee. In total, 36 male SD rats obtained from the Laboratory Animal Center of Tongji Medical College, 8 weeks of age and weighing 280e300 g, were randomly distributed into 3 groups (n ¼ 12 per group): control group, 7% methionine group (7% Met), and 7% methionine plus melatonin treatment group (7% Met þ Mel). The Met-enriched diets were fed orally to the rats daily for 4 weeks. The rats in the 7% Met þ Mel group were administered with daily intraperitoneal injections of Mel (Sigma Aldrich, St Louis, MO, USA) at 10 mg/kg for 4 weeks. The body weights were measured on day 0 and prior to euthanasia.
Evaluation of Erectile Function After 4 weeks, the erectile function of all rats was evaluated according to our previous experimental methods.32 Briefly, the rats were anesthetized by pentobarbital sodium (40 mg/kg, intraperitoneally). A PE-50 tube was intubated into the carotid artery for the continuous monitoring of the mean arterial pressure (MAP), and a 25-gauge needle was inserted into the right crura to monitor the intracavernous pressure (ICP). Next, cavernous nerves (CNs) were isolated and stimulated continuously for 1 minute at a frequency of 15 Hz and voltages of 2.5 V and 5 V, with a 3-minute interval before the next stimulation to avoid nerve fatigue. The ratio of the maximal ratio of ICP to MAP and area under the ICP curve (AUC) were calculated and analyzed to evaluate erectile function in vivo. After the measurement, the corpus cavernous (CC) of each rat was cut into 5 pieces, and the piece pierced by the needle was discarded. The remaining pieces of CC were immediately frozen in liquid nitrogen and stored at 80 C or fixed overnight in 4% paraformaldehyde (Beyotime Biotechnology, China) and then were embedded in paraffin for further experiments. Additionally, the blood samples of all rats were obtained from the carotid artery to detect plasma Hcy levels.
Measurement of the Antioxidant Activity
CC tissues stored at 80 C were processed and assayed for antioxidant activity. Commercial kits to detect malondialdehyde (MDA) and superoxide dismutase (SOD) were used according to the manufacturer’s protocol (Beyotime Biotechnology). The penile MDA levels and SOD activities were normalized to the wet weight of the penile tissue samples.
Detection of the intracellular reactive oxygen species in the CC Intracellular reactive oxygen species (ROS) in the CC were detected using an oxidation-sensitive fluorescent probe (DCFHDA; Beyotime Biotechnology). The sections were processed and then incubated with 10 mmol/L of DCFH-DA at 37 C for 20 minutes according to the manufacturer’s protocols. Nuclei were stained with 40 ,6-diamidino-2-phenylindole (DAPI; Beyotime J Sex Med 2019;-:1e12
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Table 1. Metabolic and physiological variables Variables
Control (n ¼ 12)
Initial weight (g) Final weight (g) NO (mmol$g1 protein) cGMP (pmol$mg1 protein)
283.74 389.14 3.21 5.83
± ± ± ±
8.6 24.3 0.072 0.039
7% Met (n ¼ 12) 285.11 267.23 1.08 3.64
± ± ± ±
9.39 21.34* 0.05* 0.096*
7% Met þ Mel (n ¼ 12) 281.43 270.33 2.37 4.25
± ± ± ±
11.26 22.19* 0.043*,#,& 0.041*,#,&
cGMP ¼ cyclic guanosine monophosphate; Mel ¼ melatonin; Met ¼ methionine; NO ¼ nitric oxide. *,&P < .05 compared with the control group. # P < .05 compared with the 7% Met group.
Biotechnology). Fluorescence images were acquired using an Olympus BX51 fluorescence microscope (Olympus Corporation, Tokyo, Japan).
was used to evaluate the relative mRNA expression compared with that of the controls. The primer sequences are listed in Supplementary Table 2.
Immunofluorescence
Protein Immunoblot Analysis
Immunofluorescence and terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate nick-end labeling (TUNEL) double staining were used to localize Bim and Puma in hair follicles and to colocalize them with apoptotic cells. The sections were deparaffinized, rehydrated, pretreated in citrate buffer, pH 6.0, for 5 minutes at 98 C for antigen retrieval, and incubated with 20 mg/ mL of proteinase K for 15 minutes at room temperature. The primary antibodies for a-SMA were incubated overnight at 4 C. A TUNEL apoptosis detection kit (Beyotime Biotechnology) was used to detect apoptotic cells on the same sections. The TUNEL reaction mixture was prepared according to the manufacturer’s instructions. Nuclei were stained with DAPI (Beyotime Biotechnology). Fluorescence images were acquired using an Olympus BX51 fluorescence microscope (Olympus Corporation).
Total proteins in cells were extracted by NP-40 lysis buffer with proteinase inhibitor cocktail (Roche, Branchburg, NJ, USA) and phosphatase inhibitor cocktail (EMD Millipore, Burlington, MA, USA), and Western blotting analysis was performed as follows. Protein samples (20 mg per lane) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were transferred to polyvinylidene fluoride membranes. After blocking in Tris-buffered saline-Tween 20 with 5% bovine serum albumin, the membranes were incubated overnight at 4 C with primary antibodies; the antibodies used are listed in Supplementary Table 1. The membranes were then incubated with horseradish peroxidase-conjugated secondary antibodies (1:5,000; Proteintech) and were analyzed using an enhanced chemiluminescence detection system (Pierce; Thermo Fisher Scientific, Rockford, IL, USA).
Immunofluorescence of neuronal nitric oxide synthase (nNOS) was performed using the same method. The details of the antibodies used are listed in Supplementary Table 1.
Immunohistochemistry Primary antibodies to HO-1 (ab68477; Sigma Aldrich) was used for immunostaining on formalin-fixed, paraffin-embedded tissue slides from the rat models. Briefly, after tissue sections were deparaffinized and rehydrated through a graded alcohol series, they were heated by microwave in 0.01 mol/L of citrate buffer at pH 6.0 for 20 minutes to retrieve antigens. After a 30-minute incubation for protein blocking, the tissue sections were incubated in primary antibodies, followed by incubation in horseradish peroxidase polymer-conjugated secondary antibody (cat #K4061, Dako).
Quantitative Real-Time Polymerase Chain Reaction Analysis Total RNA in cells was extracted by Trizol reagent (ThermoFisher; cat #15596026) and was reversely transcribed to cDNA using a high-capacity cDNA reverse transcription kit (ThermoFisher Scientific). The quantitative real-time polymerase chain reaction (qRT-PCR) was conducted using the fast SYBR green master mix (Thermo Fisher Scientific). The 2-DDCt method J Sex Med 2019;-:1e12
Statistical Analysis The data were presented as the mean± SEM and were analyzed using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). One-way analysis of variance followed by the Tukey-Kramer test for post hoc comparisons was performed to
Figure 1. Plasma tHcy level in all rats. The plasma tHCy level determined by the enzyme-linked immunosorbent assay method was increased in the 7% Met group and decreased in the melatonin-treated group (n ¼ 12 per group). *,&P < .05 compared with the control group. #P < .05 compared with the 7% Met group. Mel ¼ melatonin; Met ¼ methionine; tHcy ¼ total plasma homocysteine.
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analyze the differences between any 2 groups, and P values < .05 were considered to indicate statistically significant differences.
RESULTS Metabolic Parameters The body weights of each group are shown in Table 1, and the plasma Hcy levels are shown in Figure 1. The body weights of the 7% Met group were lower than those of the controls after 4 weeks (P < .05). The Hcy levels of the 7% Met group were statistically higher than those of the control group (P < .05), demonstrating that the rat model with HHcy was successfully established. Mel treatment did not change the body weight. However, Mel decreased the plasma Hcy levels significantly compared with that of the 7% Met group, which was consistent with those of previous studies.31
Melatonin Protects Against ED Induced by HHcy CN electrical stimulation with different voltages (2.5 V and 5 V) was administered to evaluate the erectile function of rats. The maximum ICP/MAP and AUC were measured and recorded. The ratio of maximum ICP/MAP in the 7% Met group was sharply attenuated compared with that of the control group (P < .05). However, erectile function was partially improved in the Mel treatment group, although it was still lower than that in the control group (P < .05). The AUCs at
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2.5 and 5 V in all 3 groups exhibited the same trend as the maximum ICP/MAP values (Figure 2). Moreover, as shown in Table 1, decreased NO and cyclic guanosine monophosphate (cGMP) levels of CC were observed in the 7% Met group (P < 0.05), whereas four weeks of melatonin treatment increased NO and cGMP levels significantly compared with those in rats fed Met-rich diets. Together, these results suggested that HHcy impaired the erectile function of rats, whereas Mel could protect against ED induced by HHcy.
Melatonin Partially Restores the Number of nNOS-Positive Cells in the CC Immunofluorescence and Western blotting were used to analyze the content and expression of nNOS in the CC. Our results in Figure 3 show that nNOS expression in the CC was lower than that of the control group. Mel treatment could increase the contents, although not to those in the control group. Western blotting also showed the same trend (both P < .05).
Melatonin Inhibits Oxidative Stress in the CC of Rats With HHcy A fluorescent probe was used to detect the intracellular ROS of CC. As shown in Figure 4A, the levels of ROS were increased significantly in the CC of the 7% Met group and were reduced by Mel administration (both P < .05). The amount of MDA and activities of SOD were detected by enzyme-linked immunosorbent
Figure 2. Melatonin partially restores erectile function in rats with hyperhomocysteinemia (HHcy). (A, B) Representative tracing of ICP using electric stimulations at 2.5 V and 5 V for age-matched normal control, HHcy induced by 7% Met and Mel-treated HHcy groups (n ¼ 12 per group). Scale bars represent 1 minute. (C, D) The maximum ICP/MAP ratio and total ICP of the different stimulations are presented as bar graphs for max ICP/MAP and total ICP (AUC). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. AUC ¼ area under the curve; ICP ¼ intracavernous pressure; MAP ¼ mean arterial pressure; Mel ¼ melatonin; Met: methionine. Figure 2 is available in color online at www.jsm.jsexmed.org. J Sex Med 2019;-:1e12
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Figure 3. The nNOS expression in the corpus cavernous of all rats. (A) Representative images of nNOS content in the corpus cavernous of all rats at 200 magnification. The scale bar represents 100 mm. (B) Representative Western blot results for nNOS in the corpus cavernous (CC) of all rats. (C) The expression levels of nNOS, with b-actin as the loading control, in the CC of the 3 groups are presented as bar graphs (n ¼ 6e8 rats per group). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. DAPI ¼ 40 ,6diamidino-2-phenylindole; Mel ¼ melatonin; Met ¼ methionine; nNOS ¼ neuronal nitric oxide synthase. Figure 3 is available in color online at www.jsm.jsexmed.org. assay. The MDA levels were higher in the 7% Met group than that in the control group and the Mel treatment group (both P < .05; Figure 4B). The SOD activities in the 7% Met group were lower than those in the control group; however, in the Mel þ 7% Met group, the SOD activities were slightly higher than those in the 7% Met group (both P < .05; Figure 4C). We also detected the expression levels of nicotinamide adenine dinucleotide phosphate-oxidase (NADPH oxidase) subunits, including p22phox, p47phox, and gp91phox, whose expression levels in the 3 groups showed a similar trend as those of MDA in each group (all P < .05; Figure 5).
Melatonin Improves Oxidative Stress Via Activating the Erk1/2/Nrf2/HO-1 Signaling Pathway Western blotting analysis showed that the expression levels of the Erk1/2/Nrf2/HO-1 signaling pathway components, J Sex Med 2019;-:1e12
which exert a protective effect against overactivated oxidative stress, were significantly lower in the CC of the 7% Met groups than that of the control group (P < .05; Figure 6). Mel administration activated the signaling pathway, inducing the expression levels of p-Erk1/2, Nrf2, and HO-1, which were significantly higher than those in the 7% Met groups (P < .05; Figure 6). The RT-PCR results of Nrf2 and HO-1 were consistent with the Western blotting results (all P < .05; Figure 6).
Melatonin Inhibits Apoptosis in the CC of Rats With HHcy TUNEL staining was used to determine whether apoptosis was induced by HHcy in vivo. Additionally, a-SMA staining was performed on the same tissue slide to determine the expression of a-SMA. As shown in Figure 7, more apoptosis
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Figure 4. Melatonin can inhibit oxidative stress in the corpus cavernous of rats with HHcy. (A) Representative images of the ROS fluorescent probe in the corpus cavernous of all rats at 400 magnification. The scale bar represents 50 mm. (B) MDA levels determined by enzyme-linked immunosorbent assay (ELISA) in the corpus cavernous (CC) of all rats. (C) SOD activities determined by ELISA in the corpus cavernous of all rats. *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. DAPI ¼ 40 ,6-diamidino2-phenylindole; MDA ¼ malondialdehyde; Mel ¼ melatonin; Met ¼ methionine; ROS ¼ reactive oxygen species; SOD ¼ superoxide dismutase. Figure 4 is available in color online at www.jsm.jsexmed.org.
was observed in the CC of the 7% Met group than that of the control group, and Mel treatment could inhibit apoptosis. Additionally, the expression levels of a-SMA were lower in the CC of rats with HHcy. Western blotting analysis showed that the Bax/Bcl-2 ratio, expression levels of caspase 3 and cleaved caspase 3, which also reflect apoptosis, showed the same trend as TUNEL staining (all P < .05; Figure 7).
DISCUSSION In this study, we demonstrated the protective role of Mel in erectile function in a rat model of HHcy, as well as the underlying mechanisms. Our data indicated that HHcy-related ED was characterized by a decreased NO/cGMP signaling pathway, oxidative stress, and apoptosis. However, Mel treatment could
partially restore erectile function by activating NO/cGMP, inhibiting oxidative stress via the Erk1/2/Nrf2/HO-1 signaling pathway, and suppressing cell apoptosis. Hcy is a non-protein-forming sulfur amino acid and is formed during the metabolism of methionine. HHcy has been reported to be strongly correlated with the occurrence of ED,10,33,34 even in the presence of other risk factors for ED, such as DM,12,35 and could be an early predictor of ED.36 Furthermore, HHcy may be linked to nonresponsiveness in PDE5 inhibitor therapy.12,37 Although several drugs, such as folic acid, vitamin B6, and B12, have already been reported to decrease circulating Hcy concentrations,38,39 their effects on ED were still not fully elucidated. Exploring the mechanisms underlying how HHcy affects erectile function will be conducive to finding a novel therapy method for ED. J Sex Med 2019;-:1e12
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Figure 5. Melatonin can inhibit nicotinamide adenine dinucleotide phosphate oxidase subunits in the corpus cavernous (CC) of rats with hyperhomocysteinemia. (A) Representative Western blot results of p22phox, p47phox, and gp91phox in the CC of all rats. Expressions of p22phox, p47phox, and gp91phox with b-actin as the loading control in all rats are presented as bar graphs: (B) for p22phox, (C) for p47phox, and (D) for gp91phox (n ¼ 6 rats per group). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. Met: methionine; Mel: melatonin. Normal erectile function is dependent on the normal function of penile components and underlying molecular biological procedures. The risk factors of ED are mainly linked to the attenuated bioavailability of NO, partially due to the augmented generation of ROS.40,41 There have been several studies elucidating the relationship between ED and ROS, which reflect the levels of oxidative stress in a DM or aged condition.42,43 Endogenous ROS are produced mainly by NADPH oxidase, a multisubunit enzyme including p47phox, p67phox, gp91phox, p22phox, and p40phox in cell membranes, mitochondria, peroxisomes, and endoplasmic reticulum.44 It was reported that excessive ROS in CC could lead to ED via attenuating NO bioavailability, decreasing the cGMP level, and inducing apoptosis-mediated loss of smooth muscle and endothelial cells.45 Excessive ROS can be found in metabolic disorders, such as DM, chronic ethanol consumption, smoking, and, more recently, HHcy. Additionally, we previously reported that some NADPH oxidase subunits were greatly activated in the CC of rats with ED induced by type I DM or androgen deficiency.46,47 Moreover, a recent study by Jiang et al48 reported that oxidative stress was the main contributor to ED induced by HHcy. In our study, we observed that HHcy induced oxidative stress, the existence of which was verified via an increased level of ROS, activation of NADPH oxidase subunit expression, increased MDA levels, and reduced SOD activities, thus impairing normal erectile function in the rat model. Moreover, weight loss was J Sex Med 2019;-:1e12
observed during the process of breeding, reflecting the status of metabolic disorder, which was reported in our previous study.31 Interestingly, the ED rats also showed neurodegenerative changes characterized by the decrease in nNOS-positive nerve fibers in the CC, suggesting that HHcy could induce ED because of its detrimental effects on neurological function. Cavernous smooth muscle cells comprise the predominant structure of CC and play a significant role in achieving and maintaining sufficient erection via controlling the blood flow into the CC. Loss of cavernous smooth muscle cells was observed in our study, in line with previous reports in humans49 and various rat ED models, including diabetes,50 hyperlipidemia,51 and age-related ED.42 Moreover, numerous studies have confirmed that HHcy could increase apoptosis in various cells.15 In this study, we showed that the level of apoptosis was increased significantly in the corresponding area where cavernous smooth muscle cells were decreased, and HHcy triggered the apoptosis of CC, as evident by the elevated levels of the pro-apoptotic marker Bax and reduced levels of the anti-apoptotic marker Bcl-2, and activated effector caspases. Mel is derived from the pinealocytes of the pineal gland and exerts numerous physiological effects through its 3 receptors. Baydas et al22 first reported that the level of Mel had a causal relationship with the plasma concentration of Hcy. Our results showed that Mel treatment could decrease significantly the
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Figure 6. Melatonin inhibits oxidative stress via the Erk1/2/Nrf2/HO1 signaling pathway. (A) Representative Western blot results for Erk1/ 2, p-Erk1/2, Nrf2, and HO1 in the corpus cavernous (CC) of all rats. (B) The expression levels of Erk1/2, p-Erk1/2, Nrf2, and HO1, with b-actin as the loading control, in the CC of the 3 groups are presented as bar graphs (n ¼ 3 rats per group). (C) and (D) represent the relative mRNA expression of Nrf2 and HO1 genes, respectively, to GAPDH in the CC of all rats by quantitative real-time polymerase chain reaction. (E) Representative immunohistochemistry images showing the suppression of HO1 by hyperhomocysteinemia and activation by melatonin. The scale bar represents 100 mm. *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. GAPDH ¼ glyceraldehyde 3-phosphate dehydrogenase; Mel ¼ melatonin; Met ¼ methionine. Figure 6 is available in color online at www.jsm.jsexmed. org. plasma Hcy levels induced by a Met-rich diet, consistent with previous reports.21 Given Mel’s antioxidant properties, it was not surprising that it significantly decreased the ROS level, deactivated the expression of the NADPH oxidase subunit, decreased the MDA level, and increased SOD activity, accompanied by the inhibition of apoptosis. Moreover, we found that melatonin could restore the number of nNOS-positive cells in the CC, consistent with a previous study on ED in streptozotocin-induced diabetic rats.27
To explore how Mel decreased the levels of oxidative stress, we analyzed a critical signaling pathway against oxidative stressinduced injury (ie, Erk1/2/Nrf2/HO-1 signaling pathway). HO1 is one of the most important endogenous antioxidant enzymes and is thought to play a key role in maintaining the antioxidant/ oxidant imbalance.52 NRF2 is a basic region leucine-zipper transcription factor and has been reported to be involved in the transcriptional regulation of the HO-1 gene.53,54 It was reported that the phosphorylation of several signal transduction pathways, J Sex Med 2019;-:1e12
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Figure 7. Melatonin can reduce apoptosis in the corpus cavernous (CC) of rats with hyperhomocysteinemia. (A, B) Representative images of terminal deoxynucleotidyl transferase dUTP nick end labeling and the a-SMA content in the CC of all rats at 400 magnification. The scale bar represents 50 mm. (C) Representative Western blot results of Bax, Bcl-2, caspase 3, and cleaved caspase 3 in the CC of all rats. (D) The expression levels of Bax, Bcl-2, caspase 3, and cleaved caspase 3, with b-actin as the loading control, in the CC of the 3 groups are presented as bar graphs (n ¼ 6 rats per group). *P < .05 compared with the control group. #P < .05 compared with the 7% Met group. DAPI ¼ 40 ,6-diamidino-2-phenylindole; Mel ¼ melatonin; Met ¼ methionine. Figure 7 is available in color online at www.jsm.jsexmed.org. including ERK1/2 and PI3K/AKT, could activate NRF2, leading to the translocation of NRF2.55,56 Here, we observed that HHcy could decrease the expression level of the Erk1/2/Nrf2/HO-1 signaling pathway and confirmed that Mel increased Nrf2 expression and HO-1 protein levels via the phosphorylation of ERK1/2 in the CC of rats. Taken together, the data showed that the roles in antioxidative stress and anti-apoptosis, together with the normalization of elevated Hcy and neuroprotective effects, validated Mel as a potential treatment to protect cells against Hcyinduced excitotoxicity and cell death, thus improving ED.
research is that our rat ED models induced by a Met-rich diet reflects a metabolic disorder characterized with HHcy but may not necessarily represent the real pathologic processes that occur in patients with ED induced by HHcy and other risk factors. Moreover, the absence of cell-based experiments was another limitation. Therefore, in future studies, we should examine the effect of Mel in vitro to more thoroughly investigate the mechanisms involved in the benefits of melatonin for HHcyinduced ED.
Although our study provides evidence for potential novel therapy for ED induced by HHcy and demonstrated several underlying mechanisms, a major limitation of the current
Strengths and Limitations
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Our study provided evidence, we believe for the first time, that Mel was effective in partially restoring erectile function in rats
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with HHcy. However, our HHcy-induced rat ED models may not necessarily represent the pathologic processes that occur in ED induced by other risk factors.
CONCLUSIONS This study revealed that oxidative stress, together with cell apoptosis, was involved in ED induced by HHcy in rats, whereas Mel preserved erectile function through decreasing the plasma Hcy level, inhibiting oxidative stress via the Erk1/2/ Nrf2/HO-1 signaling pathway, suppressing apoptosis, activating NO/cGMP, and partially restoring the content of cavernous smooth muscle cells. Thus, Mel could be a promising preventive factor for ED induced by HHcy because of its numerous beneficial effects. Corresponding Author: Jihong Liu, MD, PhD, Department of Urology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430030, Hubei, China. Tel: þ86 027 83663460; Fax: þ86 027 83663460; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest. Funding: This work was supported by a grant from the National Natural Sciences Foundation of China (No. 81270690 and No. 81501246).
STATEMENT OF AUTHORSHIP Category 1 (a) Conception and Design Zhe Tang; Jun Yang; Jihong Liu (b) Acquisition of Data Zhe Tang; Jingyu Song; Zhe Yu (c) Analysis and Interpretation of Data Zhe Tang; Kai Cui; Yajun Ruan; Tao Wang; Shaogang Wang Category 2 (a) Drafting of the Article Zhe Tang; Jun Yang (b) Revising the Article for Intellectual Content Zhe Tang; Jun Yang; Tao Wang; Jihong Liu Category 3 (a) Final Approval of the Completed Article Zhe Tang; Jingyu Song; Zhe Yu; Kai Cui; Yajun Ruan; Tao Wang; Jun Yang; Shaogang Wang; Jihong Liu
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