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Ginsenoside Rg3 Improves Erectile Function in Streptozotocin-Induced Diabetic Rats Tao Liu, MD, Yi-Feng Peng, MD, Chao Jia, MB, Ben-Hai Yang, MM, Xia Tao, MM, Jing Li, MM, and Xiang Fang, MB Department of Sexual Medicine, Yijishan Hospital, Wannan Medical College, Wuhu, China DOI: 10.1111/jsm.12779
ABSTRACT
Introduction. Ginsenoside Rg3 is one of the active ingredients isolated from Panax ginseng C.A. Meyer. Previous studies demonstrated that Rg3 has antioxidant and neuroprotective abilities. Aim. The purpose of this study was to evaluate the protective effect of Rg3 on erectile function in streptozotocin (STZ)-induced diabetic rats. Methods. Two-month-old Sprague-Dawley male rats received a one-time intraperitoneal (IP) STZ (60 mg/kg) or vehicle injection after a 16-hour fast. Three days later, rats were randomly divided into four groups and were treated with daily gavage feedings of a mix of distilled saline water and 0.5% carboxymethylcellulose or Rg3 dissolved in the mix at doses of 10 mg/kg and 100 mg/kg for 3 months. A sham group underwent IP injection of saline followed by daily gavage of the above mix for 3 months. Main Outcome Measure. Erectile function was assessed by cavernosal nerve electrostimulation at 3 months. The penis was then harvested and deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. Western blot was performed to examine cleaved caspase-3, platelet endothelial cell adhesion molecule (PECAM)-1, and smooth muscle actin (SMA). Neural regeneration was measured by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected by colorimetry. Results. In the negative control group, the functional evaluation showed a lower mean intracavernosal pressure (ICP) with cavernosal nerve stimulation than in the sham group; there was a significant change in the expression of cleaved caspase-3, bcl-2, bcl-xl, PECAM-1, and SMA, as well as in the SOD and MDA production in the corpus cavernosum. Histological analysis of specimens stained for NADPH showed a significant change in the staining quality of the neurons in the dorsal nerves; TUNEL showed a greater apoptotic index in corpus cavernosum cells. With daily oral gavage with 100 mg/kg Rg3, the ICP/mean arterial pressure value was significantly higher than in the controls. The level of cleaved caspase-3, bcl-2, bcl-xl, PECAM-1, and SMA and the number of positively stained nerve fibers tended to revert to normal after Rg3 treatment. The apoptotic index in corpus cavernosum cells was lowered. Conclusion. Oral gavage with Rg3 appears to both prevent degeneration of neurons in the dorsal nerves and exert an antioxidant effect in the corpus cavernosum of rats. Liu T, Peng Y-F, Jia C, Yang B-H, Tao X, Li J, and Fang X. Ginsenoside Rg3 improves erectile function in streptozotocin-induced diabetic rats. J Sex Med **;**:**–**. Key Words. Diabetes; Erectile Dysfunction; Oxidative Stress; Apoptosis
Introduction
E
rectile dysfunction (ED) is one of the major complications following diabetes [1]. In recent years, the incidence of diabetic ED remains
© 2014 International Society for Sexual Medicine
high. Pathological changes in the corpus cavernosum might be involved in diabetic ED, including increased reactive oxygen species (ROS), cavernous hypoxia, up-regulation of profibric factors, such as transforming growth factor, and J Sex Med **;**:**–**
2 subsequent structural changes including a decrease in smooth muscle and endothelial content, increased cavernous fibrosis, and a reduction of nitric oxide synthase (NOS) nerve density [2,3]. Ginseng, the root of Panax ginseng C.A. Meyer, is one of the most renowned herbs and has been used for thousands of years to maintain body homeostasis and enhance vital energy [4]. Recent investigations showed that the majority of ginseng pharmacological activities are closely related to its antioxidant property [5,6]. The main active ingredients of ginseng are ginsenosides. Among them, ginsenoside Rg3 has received much attention. Structurally, ginsenosides contain a hydrophobic triterpenoid skeleton attached with hydrophilic sugar moieties or hydroxyl groups at carbon-3 and carbon-6. Ginsenosides 20(S)-Rg3 and 20(R)-Rg3 are an enantiomeric pair that differs in the spatial orientation of the hydroxyl group on the chiral center at carbon-20; the S form usually exhibits significantly higher antioxidant effects than the R form [7]. Previous studies have demonstrated Rg3 has neuroprotective effect through reducing lipid peroxides, scavenging free radicals, and improving energy metabolism [8]. This study was designed to investigate the protective effect of 20(S)-Rg3 on erectile function and structural changes in the corpus cavernosum in rats with diabetes. Materials and Methods
Drugs and Animals 20(S)-Ginsenoside Rg3 extracted from the root of P. ginseng was purchased from Tauto Biotech Co., Ltd. (Shanghai, China). The saponins were a white powder with a purity of 98% (as determined by high performance liquid chromatography), a molecular weight of 785.013, and a molecular formula of C42H72O13. All experiments were approved by Wannan Medical College Research Subject Review Board. A total of 40 male Sprague-Dawley rats weighing 200–250 g were used. Rats were fasted for 16 hours, then received a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ) (Sigma-Aldrich Chemical Co, St Louis, MO, USA) or vehicle (0.1 mol/L citrate phosphate buffer, pH 4.5) [9,10]. Blood glucose was monitored by tail prick using a blood glucose meter (B. Braun, Melsungen, Germany) 3 days after STZ or vehicle injection, at regular intervals throughout the study, and immediately prior to sacrifice. Only those STZ-treated rats with fasting glucose concentrations consistently at >300 mg/dL were included in the diabetic J Sex Med **;**:**–**
Liu et al. group. Rats were divided into four groups: sham group (n = 10), diabetic rats received a daily gavage feedings of a mix of distilled saline water and 0.5% carboxymethylcellulose (Sigma-Aldrich) in which Rg3 was dissolved at concentrations of 0 (negative control group, n = 10) and 10 and 100 mg/kg (Rg3treated groups, n = 10 each) for 3 months. After a 3-day washout period, we evaluated penile hemodynamics by cavernous nerve electrical stimulation with real-time intracavernosal pressure (ICP) measurement. Following ICP assessment, rats were euthanized and penises were harvested for Western blot, nicotinamide adenine dinucleotide phosphate (NADPH) and transferase dUTP nick end labeling (TUNEL) staining, superoxide dismutase (SOD), and malondialdehyde (MDA) measurement.
Measurement of Erectile Function Rats form each group were anesthetized with 5% sodium pentobarbital intraperitoneally. Then the major pelvic ganglion, cavernous nerves, and pelvic organs were exposed. The skin overlying the penis was removed, and the penile crus was exposed by removing part of the overlying ischiocavernous muscle. Two 23-gauge trocars connected to a PE-50 tube with heparinized saline (250 IU/mL) were carefully inserted into the crus and the left carotid artery, respectively. The other end of the PE-50 tube was connected to a Biopac Systems MP150 (Aero Camino, Goleta, CA, USA). The cavernous nerve was exposed and electrostimulation (12 Hz; pulse width 5 milliseconds; 2.5 V, 5 V, 7.5 V; duration of 60 seconds) of the cavernous nerve was applied with a stainless steel bipolar hook electrode. The mean arterial pressure (MAP) was also measured at the same time. The ratio of maximal ICP (mm Hg) to MAP (mm Hg) was calculated to normalize penile hemodynamic response to variations in systemic blood pressure. Western Blot Cellular protein samples were prepared by homogenization of penile tissue in a lysis buffer containing 1% IGEPAL CA-630 (Sigma, St. Louis, MO, USA). 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, aprotinin (10 mg/ mL), leupeptin (10 mg/mL), and phosphatebuffered saline. Cell lysates containing 40 μg of protein were electrophoresed in sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Bedford, MA, USA). The membrane was blocked with 5%
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Ginsenoside Rg3 Improves Erectile Function skimmed milk for 1 hour at room temperature and incubated overnight at 4°C with antibody against cleaved caspase-3 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), platelet endothelial cell adhesion molecule (PECAM)-1, a- smooth muscle actin (SMA), β-actin (1:1,000, Santa Cruz Biotechnology), Bcl-2, and Bcl-xl (1:1,000, Abcam Plc, Cambridge, UK). After washing in trisbuffered saline with 0.1% tween 20 three times for 10 minutes, the membrane was hybridized to horseradish peroxidase-labeled second antibodies. Resulting images were analyzed with ChemiImager 4000 (Alpha Innotech Corporation, San Leandro, CA, USA) to determine the integrated density.
NADPH-Diaphorase and TUNEL Staining Frozen tissue sections (8 μm) were air-dried for 5 minutes, then incubated with 0.1 M NADPH, 0.2 M nitroblue tetrazolium, and 0.2% Triton X-100 (Sigma-Aldrich) with constant microscopic monitoring for color development. The reaction was terminated when the medium became deep blue. Staining was assessed by counting the number of NADPH-diaphorase-positive nerves in the dorsal nerves at optical magnification of 400× with light microscopy. Positive endothelial staining was excluded from the count (n = 10 per group). For TUNEL staining (n = 10 per group), sections (5 μm) were deparaffinized and hydrated by sequential incubations in xylene and ethanol. The samples were then processed according to the instructions provided with the in situ cell death detection kit, peroxidase (Roche Diagnostic Corporation, Indianapolis, IN, USA). Measurement of SOD and MDA Levels Assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). About 20-mg corpus cavernosum tissue was used for an experiment; the tissue was homogenized in 0.2-mL normal saline, then 0.05-mL tissue homogenate was used for SOD determination and 0.1-mL tissue homogenate was used for MDA determination. SOD activity was measured through the inhibition of nitroblue tetrazolium reduction by O2, which was generated by the xanthine/xanthine oxidase system. The total reaction volume was 3.35 mL, and the absorbance was measured at 550 nm according to the manufacturer instructions. One SOD activity unit was defined as the enzyme amount causing 50% inhibition in a 1-mL reaction solution per milligram
of tissue protein; the result was expressed as unit per gram protein. The MDA concentration of the homogenate was measured using the thiobarbituric acid method. The total reaction volume was 4.2 mL. The amount of lipid peroxides was measured by the production of MDA, which, in combination with thiobarbituric acid, forms a pink chromogen compound with an absorbance of 532 nm. The result was expressed as nanomoles per milligram protein [11].
Statistical Analysis All data are expressed as mean ± standard deviation and were compared by one-way analysis of variance with post hoc Bonferroni’s correction (GraphPad Prism 5.0; GraphPad Software, San Diego, CA, USA). In all cases, P < 0.05 was considered statistical significance. Results
Metabolic Variables and ICP/MAP Assessment Three months after diabetes was induced, the fasting glucose concentrations in diabetic rats were significantly higher than that in the age-matched shams (P < 0.05). Body weights were significantly lower in the diabetic rats than that in sham animals (P < 0.05). No significant differences in body weight or glucose concentration were found between negative control rats and Rg3-treated diabetic rats (P > 0.05, Table 1). Three different voltages (2.5, 5.0, and 7.5 V) were used to measure the MAP and the ICP. The ratio of the maximal ICP/MAP after the electrostimulation of the rats in negative control group was 0.28 ± 0.04 at 5.0 V and 0.35 ± 0.051 at 7.5 V, which were significantly lower than in the sham group (0.86 ± 0.066 at 5 V and 0.88 ± 0.062 at 7.5 V) (P < 0.05, Figure 1). ICP/MAP in the 10 mg/kg Rg3 group was 0.41 ± 0.039 at 5.0 V and 0.49 ± 0.043 at 7.5 V, and there were no significant Table 1 Weight and blood glucose levels in sham and STZ-diabetic rats after being treated for 3 months Groups
Weight (g)
Fasting blood glucose (mg/dL)
Sham Diabetic control Diabetic + Rg3 (10 mg/kg) Diabetic + Rg3 (100 mg/kg)
478.88 ± 22.59 246.37 ± 19.86 250.75 ± 16.18a,* 254.38 ± 16.45b,*
87.49 ± 5.86 485.62 ± 40.35 441.74 ± 41.28c,* 446.26 ± 38.94d,*
*P > 0.05 Values are mean ± SD; n = 10 a,b,c,das compared with the diabetic control SD = standard deviation; STZ = streptozotocin
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Figure 1 Effect of Rg3 on erectile responses in diabetic rats. (A) Representative ICP in response to electrical stimulation of the cavernous nerve at 5 V in shams, negative controls, diabetic rats. The stimulus interval (60 seconds) was indicated by a solid bar. (B) Erectile function presented as ICP/MAP in each group at 2.5 V, 5 V, and 7.5 V. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; † P < 0.05 vs. 100 mg/kg Rg3 group; group *P < 0.05 vs. NC group. ICP = intracavernosal pressure; MAP = mean arterial pressure; NC = negative control.
differences between the negative control and 10 mg/kg Rg3 group (P > 0.05, Figure 1). Diabetic rats treated with 100 mg/kg Rg3 had significantly higher ICP/MAP (0.71 ± 0.068 at 5 V and 0.74 ± 0.069 at 7.5 V) when compared with the negative control group (P < 0.05), and there were no significant differences in ICP/MAP between the 100 mg/kg Rg3 group and the sham group. However, there were significant differences in ICP/ MAP between the 10 mg/kg and the 100 mg/kg Rg3 group at all levels of 5 V and 7.5 V (P < 0.05, Figure 1).
Increased Expression of PECAM-1 and SMA in Corpus Cavernosum Tissue of Diabetic Rats after Rg3 Treatment We evaluated the differential expression of PECAM-1 and SMA in cavernous tissue. The expression of PECAM-1 and SMA was lower in the negative control group than in the sham group (P < 0.05, Figure 2). However, the expression of PECAM-1 and SMA was significantly greater in J Sex Med **;**:**–**
the 100 mg/kg Rg3-treated groups relative to the negative control group (P < 0.05, Figure 2). There was no significant difference in PECAM-1 expression between the negative control and the 10 mg/kg Rg3 group (P > 0.05, Figure 2).
Effect of Rg3 on the NOS-Containing Neurons in the Dorsal Nerves of Diabetic Rats To localize neuronal NOS protein expression in the penis, we performed NADPH-diaphorase staining. The number of NADPH-diaphorasepositive nerves in the dorsal nerves in the sham group was significantly greater than in the negative control group (P < 0.05, Figure 3). However, 100 mg/kg Rg3 treatment partly restored the number of positive nerves in the dorsal nerves compared with the negative control group (P < 0.05, Figure 3), and there was a significant difference in the number of NOS-containing neurons in the dorsal nerves between the 10 and the 100 mg/kg Rg3 group (P < 0.05, Figure 3).
Ginsenoside Rg3 Improves Erectile Function
5 Figure 4A). Western blot was also used to evaluate the level of cleaved caspase-3, bcl-2, and bcl-xl in the corpus cavernosum. The level of cleaved caspase-3 was significantly lower, but the expression of bcl-2 and bcl-xl was significantly greater in the sham group than in the negative control group (P < 0.05, Figure 4B). However, both the 10 mg/kg and 100 mg/kg Rg3 treatments
Figure 2 Effect of Rg3 on the expression of PECAM-1 and SMA in corpus cavernosum from diabetic rats. (A) Representative Western blots for PECAM-1 and SMA in corpus cavernosum. β-actin was used as a loading control. (B) Relative optical density changes of PECAM-1 and SMA. Results of each group were adjusted to the loading control. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 compared with the negative control (NC) group. PECAM-1 = platelet endothelial cell adhesion molecule 1; SMA = smooth muscle actin.
Effect of Rg3 on the Apoptosis of Corpus Cavernosum Cells in Diabetic Rats Cell apoptosis in the corpus cavernosum was assessed by TUNEL staining. The apoptotic index was significantly greater in the negative control group relative to the sham group (P < 0.05, Figure 4A). However, 100 mg/kg Rg3 could significantly reduce the apoptotic index compared with the negative control group (P < 0.05,
Figure 3 Effect of Rg3 on nNOS-containing neurons in the dorsal nerves of diabetic rats. NADPH-diaphorase staining showed the content of nNOS in the dorsal nerve in corpus cavernosum. The bar chart presented the number of NADPH-positive nerve per high power field (400×). Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; †P < 0.05 vs. 100 mg/kg Rg3 group; *P < 0.05 vs. NC group. NADPH = nicotinamide adenine dinucleotide phosphate; NC = negative control; nNOS = neuronal nitric oxide synthase.
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Figure 4 Effect of Rg3 on the apoptosis of corpus cavernosum cells in diabetic rats. (A1) Representative photos for TUNEL staining of corpus cavernosum tissue (400×). (A2) Apoptotic index presented as the ratio of apoptotic nuclei to the total number of nuclei counted. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 vs. NC group. (B1) Representative Western blots for cleaved caspase-3, bcl-2, and bcl-xl in corpus cavernosum. β-actin was used as a loading control. (B2) Relative optical density changes of cleaved caspase-3, bcl-2, and bcl-xl. Results of each group were adjusted to the loading control. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 vs. NC group. NC = negative control; TUNEL = transferase dUTP nick end labeling.
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Figure 5 Effect of Rg3 on the SOD and MDA levels in corpus cavernosum from diabetic rats. The MDA level was strongly increased, but the SOD level was reduced in diabetic rats, which could be partially reversed by Rg3 treatment. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 vs. NC group. MDA = malondialdehyde; NC = negative control; SOD = superoxide dismutase.
significantly lowered the level of cleaved casepase3, but significantly improved the expression of bcl-xl when compared with the negative control group (P < 0.05, Figure 4B). The 100 mg/kg Rg3 treatment significantly improved the expression of bcl-2 relative to the negative control group (P < 0.05, Figure 4B).
Oxidative Stress Is Involved in Apoptosis of Corpus Cavernosum Cells Induced by High Blood Glucose To investigate the effect of Rg3 on oxidative stress in corpus cavernosum cells, the relative MDA and SOD levels were examined. High blood glucose enhanced the MDA level and reduced SOD level in corpus cavernosum significantly compared with the sham group (P < 0.05, Figure 5). However, both the 10 mg/kg and 100 mg/kg Rg3 treatments significantly lowered the MDA level relative to the negative control group (P < 0.05, Figure 5). Treatment with 100 mg/kg Rg3 enhanced the SOD level compared with the negative control group (P < 0.05, Figure 5). Discussion
Penile erection is a neurovascular event modulated by psychological and hormonal factors. Upon sexual stimulation, neurotransmitters are released from the cavernous nerve terminals and the enzyme NOS catalyzes the production of nitric oxide (NO) and citrulline from oxygen and l-arginine. Secondary messenger cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate ultimately leads to relaxation of the cavernous smooth muscle cells, thus increasing blood flow in the sinusoids and their supplying arteries. This results in an increase in the volume and pressure in the sinusoids, and the subsequent engorgement of the corpus cavernosum leads to
penile erection [12]. Thus, the integrity of endothelium, smooth muscle, and nerves in the penis is very important for erectile function. The diminished endothelium causes reduced endothelial NOS (eNOS) and the cavernous smooth muscle to lose contractility as the cells become synthetic [13,14]. Once there is a decrease in the amount of corporal smooth muscle cells, the remaining corporal smooth muscle mass is unable to achieve sufficient relaxation to attain the high intracorporeal pressure that can compress the subtunical veins as they egress from the tunica albuginea of the penis [15]. Previous research has found that diabetes causes lower erectile function in rats, with significantly reduced myelinated axons number in the cavernous nerve and dorsal penile nerve and a greater apoptotic index of cavernous endothelial and smooth muscle cells [16]. Oxidative stress may play an important role in the development of diabetic ED. Quercetin, a potent bioflavonoid, has been reported to have antioxidant effect and ameliorate ED in diabetic rats with improved SOD activity, nitrite levels, and eNOS expression in cavernosum tissue [17]. Antioxidant tempol significantly improves intracavernous pressure and cavernous smooth muscle compared with that in untreated diabetic rats, although the endothelial cell area is not restored after treatment [18]. AC3056 (2, 6-di-t-butyl-4-((dimethyl-4-methoxyphenylsilyl) methyloxy)phenol), a novel antioxidant, has been shown to reverse ED in diabetic rats and lower MDA level, and improved total NO derivatives in serum of diabetic rats were also observed after treatment [19]. During a long-standing hyperglycemic state in diabetes mellitus, glucose forms covalent adducts with the plasma proteins through a nonenzymatic process known as glycation. The interaction J Sex Med **;**:**–**
8 between advanced glycation end-products and receptor for advanced glycation end-products may initiate the generation process of ROS, largely via cytosolic NADPH oxidase-dependent mechanisms [20]. Excessive generation of ROS, including superoxide, hydroxyl radical (OH), and hydrogen peroxide, is an important cause of the oxidative stress reaction, and the interaction between NO and ROS is an important mechanism implicated in the pathophysiological process of ED. NO interacts with superoxide to form peroxynitrite. Peroxynitrite nitrates the critical tyrosine residue in the active site of manganese (Mn)-SOD, which inactivates Mn-SOD and leads to decreased removal of superoxide [21]. This further increases the formation of peroxynitrite and reduces the available NO concentration. Peroxynitrite causes smooth muscle relaxation but it is less potent than NO, which ultimately produces ED [22]. Peroxynitrite and superoxide also have been reported to increase the incidence of apoptosis in the endothelium, which leads to denudation of endothelium and further reduction of available NO [23]. Low concentrations of oxidative stress have been reported to have a more prominent proliferative effect on cavernosal smooth muscle than high concentrations, which inhibit cell growth [24]. Additionally, reduced NO concentration aggravates the adhesion of platelets and leukocytes to the endothelium and releases substances that cause vasoconstriction and ultimately worsen ED [25]. It has been reported that erectile function was improved in diabetic rats that received one intracavernosal injection of AdCMVEC-SOD, and the total SOD activity and cavernosal cGMP were increased in the penis of transfected rats [26]. In this study, we further investigated the erectile function of diabetic rats and found that there was a significant decrease in mean ICP/MAP with reduced NOS-containing neurons in the dorsal nerves and decreased expression of cavernous endothelial and smooth muscle cell markers, as well as enhanced oxidative stress reaction in penile tissues. However, Rg3 could partially reverse these changes in a dose-dependent manner. The present studies demonstrate that Rg3 has antioxidant activities. The OH-scavenging activity test using an electron spin resonance spectrometer has been suggested to be the most appropriate to measure the antioxidant activities of ginsenosides. The OH-scavenging activities of ginsenosides were related to the ferrous metal ion-chelating activities of their aglycone, 20(S)-protopanaxadiol. Ferrous metal ion-chelating activities of ginsenosides are J Sex Med **;**:**–**
Liu et al. believed to be influenced by their types of hydrophilic sugar moieties, which are closely related to the geometrical arrangement of the OH group, especially at carbon-20 [27]. In this study, we found that ROS was increased in the corpus cavernosum of diabetic rats and that Rg3 could decrease the ROS in the corpus cavernosum, which was consistent with prior studies. The level of cleaved caspase-3 was decreased while the expression of bcl-2 and bcl-xl was increased after Rg3 treatment. Previous research also showed that 20(S)-Rg3 could prevent human endothelial cell apoptosis via inhibition of a mitochondrial caspase pathway with increased bcl-2 and Bax [28]. In this study, we hypothesized that Rg3 could attenuate the oxidative stress reaction and reduce the apoptosis of endothelial and smooth muscle cells in corpus cavernosum to improve the erectile function of diabetic rats. The accumulation of glutamate in the extracellular space under these neurological disorders can induce neuronal death, and this glutamate toxicity has been clearly attributed to a massive influx of Ca2+ through non-N-methyl-D-aspartic acid (NMDA) and primarily NMDA receptors [29]. Rg3 can significantly attenuate the activation of NMDA receptors, which can subsequently lead to a neuroprotective effect of ginsenoside Rg3 in primary cultures of rat hippocampal neurons [30]. Here, we also found that Rg3 could restore the number of NOS-containing nerves in the dorsal nerves of diabetic rats, which might be helpful for the recovery of erectile function of diabetic rats. In conclusion, we have found that daily gavage feedings of higher dose (100 mg/kg) of Rg3 improves erectile function in diabetic rats via exerting neuroprotective and antioxidative effects in corpus cavernosum functional cells. However, if Rg3 would be used in human study according to this experimental dose (100 mg/kg), at least from the economic point of view, it is not realistic. In view of its biological activities, we are considering whether it could be made into a microemulsion transdermal preparation (Rg3 is fat soluble and its molecular weight is less than 800 Da) and applied to a localized area of the body. This is an issue that we must make a serious consideration and exploration in the future works. Acknowledgments
This study was funded by the National Natural Science Foundation of China Grant No. 81200436 and Introduced Talent Foundation of Yijishan Hospital Grant No. YR201201.
Ginsenoside Rg3 Improves Erectile Function Corresponding Author: Tao Liu, MD, Department of Sexual Medicine, Yijishan Hospital, Wannan Medical College, No. 2 Zheshanxi Road, Wuhu 241001, China. Tel: +86-553-5739327; Fax: +86-553-5739327; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest.
Statement of Authorship
Category 1 (a) Conception and Design Tao Liu; Yi-Feng Peng (b) Acquisition of Data Tao Liu; Chao Jia; Xiang Fang; Jing Li (c) Analysis and Interpretation of Data Xia Tao; Ben-Hai Yang; Jing Li; Xiang Fang
Category 2 (a) Drafting the Article Tao Liu (b) Revising It for Intellectual Content Tao Liu; Yi-Feng Peng
Category 3 (a) Final Approval of the Completed Article Tao Liu; Yi-Feng Peng References 1 Shamloul R, Ghanem H. Erectile dysfunction. Lancet 2013;381:153–65. 2 Francis SH, Corbin JD. PDE5 inhibitors: Targeting erectile dysfunction in diabetics. Curr Opin Pharmacol 2011;11:683–8. 3 Cameron NE, Cotter MA. Erectile dysfunction and diabetes mellitus: Mechanistic considerations from studies in experimental models. Curr Diabetes Rev 2007;3:149–58. 4 Choi KT. Botanical characteristics, pharmacological effects and medicinal components of Korean Panax ginseng C A Meyer. Acta Pharmacol Sin 2008;29:1109–18. 5 Wei X, Su F, Su X, Hu T, Hu S. Stereospecific antioxidant effects of ginsenoside Rg3 on oxidative stress induced by cyclophosphamide in mice. Fitoterapia 2012;83:636–42. 6 Park HM, Kim SJ, Mun AR, Go HK, Kim GB, Kim SZ, Jang SI, Lee SJ, Kim JS, Kang HS. Korean red ginseng and its primary ginsenosides inhibit ethanol-induced oxidative injury by suppression of the MAPK pathway in TIB-73 cells. J Ethnopharmacol 2012;141:1071–6. 7 Shin YM, Jung HJ, Choi WY, Lim CJ. Antioxidative, antiinflammatory, and matrix metalloproteinase inhibitory activities of 20(S)-ginsenoside Rg3 in cultured mammalian cell lines. Mol Biol Rep 2013;40:269–79. 8 Tian JW, Fu FH, Geng MY, Jiang YT, Yang JX, Jiang WL, Wang CY, Liu K. Neuroprotective effect of 20(S)-ginsenoside Rg3 on cerebral ischemia in rats. Neurosci Lett 2012;526:106– 11. 9 Usta MF, Kendirci M, Gur S, Foxwell NA, Bivalacqua TJ, Cellek S, Hellstrom WJ. The breakdown of preformed advanced glycation end products reverses erectile dysfunction in streptozotocin-induced diabetic rats: Preventive vs. curative treatment. J Sex Med 2006;3:242–50. discussion 250–2.
9 10 Chen Y, Yang R, Yao L, Sun Z, Wang R, Dai Y. Differential expression of neurotrophins in penises of streptozotocininduced diabetic rats. J Androl 2007;28:306–12. 11 Chen Y, Li XX, Lin HC, Qiu XF, Gao J, Dai YT, Wang R. The effects of long-term administration of tadalafil on STZinduced diabetic rats with erectile dysfunction via a local antioxidative mechanism. Asian J Androl 2012;14:616–20. 12 Carson CC, Lue TF. Phosphodiesterase type 5 inhibitors for erectile dysfunction. BJU Int 2005;96:257–80. 13 Musicki B, Liu T, Lagoda GA, Strong TD, Sezen SF, Johnson JM, Burnett AL. Hypercholesterolemia-induced erectile dysfunction: Endothelial nitric oxide synthase (eNOS) uncoupling in the mouse penis by NAD(P)H oxidase. J Sex Med 2010;7: 3023–32. 14 Qiu X, Fandel TM, Lin G, Huang YC, Dai YT, Lue TF, Lin CS. Cavernous smooth muscle hyperplasia in a rat model of hyperlipidaemia-associated erectile dysfunction. BJU Int 2011;108:1866–72. 15 Ferrini MG, Kovanecz I, Sanchez S, Umeh C, Rajfer J, Gonzalez-Cadavid NF. Fibrosis and loss of smooth muscle in the corpora cavernosa precede corporal veno-occlusive dysfunction (CVOD) induced by experimental cavernosal nerve damage in the rat. J Sex Med 2009;6:415–28. 16 Moore CR, Wang R. Pathophysiology and treatment of diabetic erectile dysfunction. Asian J Androl 2006;8:675–84. 17 Ratnam DV, Ankola DD, Bhardrawj V, Sahana DK, Kumar MN. Role of antioxidants in prophylaxis and therapy: A pharmaceutical perspective. J Control Release 2006;113:189– 207. 18 Hirata H, Kawamoto K, Kikuno N, Kawakami T, Kawakami K, Saini S, Yamamura S, Dahiya R. Restoring erectile function by antioxidant therapy in diabetic rats. J Urol 2009;182:2518– 25. 19 Angulo J, Peiró C, Cuevas P, Gabancho S, Fernández A, González-Corrochano R, La Fuente JM, Baron AD, Chen KS, de Tejada IS. The novel antioxidant, AC3056 (2,6-di-t-butyl4-((dimethyl-4-methoxyphenylsilyl)methyloxy)phenol), reverses erectile dysfunction in diabetic rats and improves NO-mediated responses in penile tissue from diabetic men. J Sex Med 2009;6:373–87. 20 Singh VP, Bali A, Singh N, Jaggi AS. Advanced glycation end products and diabetic complications. Korean J Physiol Pharmacol 2014;18:1–14. 21 Radi R. Peroxynitrite, a stealthy biological oxidant. J Biol Chem 2013;288:26464–72. 22 Khan MA, Thompson CS, Mumtaz FH, Mikhailidis DP, Morgan RJ, Bruckdorfer RK, Naseem KM. The effect of nitric oxide and peroxynitrite on rabbit cavernosal smooth muscle relaxation. World J Urol 2001;19:220–4. 23 Beckman JS, Koppenol WH. Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and ugly. Am J Physiol 1996;271:C1424–37. 24 Sikka S, Zeng X, Hellstrom WJ. Redox signaling mechanisms and apoptotic response in human cavernosa under oxidative stress. In the 30th Annual Meeting of American Society of Andrology, March 30–April 5, 2005. Seattle, Washington. Abstract 94. 25 Jeremy JY, Angelini GD, Khan M, Mikhailidis DP, Morgan RJ, Thompson CS, Bruckdorfer KR, Naseem KM. Platelets, oxidant stress and erectile dysfunction: An hypothesis. Cardiovasc Res 2000;46:50–4. 26 Bivalacqua TJ, Usta MF, Kendirci M, Pradhan L, Alvarez X, Champion HC, Kadowitz PJ, Hellstrom WJ. 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;2:187–97. 27 Kang KS, Yokozawa T, Yamabe N, Kim HY, Park JH. ESR study on the structure and hydroxyl radical-scavenging activity
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10 relationships of ginsenosides isolated from Panax ginseng C A Meyer. Biol Pharm Bull 2007;30:917–21. 28 Min JK, Kim JH, Cho YL, Maeng YS, Lee SJ, Pyun BJ, Kim YM, Park JH, Kwon YG. 20(S)-Ginsenoside Rg3 prevents endothelial cell apoptosis via inhibition of a mitochondrial caspase pathway. Biochem Biophys Res Commun 2006;349: 987–94. 29 Vergun O, Sobolevsky AI, Yelshansky MV, Keelan J, Khodorov BI, Duchen MR. Exploration of the role of reactive
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Ginsenoside Rg3 Improves Erectile Function in Streptozotocin-Induced Diabetic Rats Tao Liu, MD, Yi-Feng Peng, MD, Chao Jia, MB, Ben-Hai Yang, MM, Xia Tao, MM, Jing Li, MM, and Xiang Fang, MB Department of Sexual Medicine, Yijishan Hospital, Wannan Medical College, Wuhu, China DOI: 10.1111/jsm.12779
ABSTRACT
Introduction. Ginsenoside Rg3 is one of the active ingredients isolated from Panax ginseng C.A. Meyer. Previous studies demonstrated that Rg3 has antioxidant and neuroprotective abilities. Aim. The purpose of this study was to evaluate the protective effect of Rg3 on erectile function in streptozotocin (STZ)-induced diabetic rats. Methods. Two-month-old Sprague-Dawley male rats received a one-time intraperitoneal (IP) STZ (60 mg/kg) or vehicle injection after a 16-hour fast. Three days later, rats were randomly divided into four groups and were treated with daily gavage feedings of a mix of distilled saline water and 0.5% carboxymethylcellulose or Rg3 dissolved in the mix at doses of 10 mg/kg and 100 mg/kg for 3 months. A sham group underwent IP injection of saline followed by daily gavage of the above mix for 3 months. Main Outcome Measure. Erectile function was assessed by cavernosal nerve electrostimulation at 3 months. The penis was then harvested and deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) was performed. Western blot was performed to examine cleaved caspase-3, platelet endothelial cell adhesion molecule (PECAM)-1, and smooth muscle actin (SMA). Neural regeneration was measured by nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining. Superoxide dismutase (SOD) and malondialdehyde (MDA) levels were detected by colorimetry. Results. In the negative control group, the functional evaluation showed a lower mean intracavernosal pressure (ICP) with cavernosal nerve stimulation than in the sham group; there was a significant change in the expression of cleaved caspase-3, bcl-2, bcl-xl, PECAM-1, and SMA, as well as in the SOD and MDA production in the corpus cavernosum. Histological analysis of specimens stained for NADPH showed a significant change in the staining quality of the neurons in the dorsal nerves; TUNEL showed a greater apoptotic index in corpus cavernosum cells. With daily oral gavage with 100 mg/kg Rg3, the ICP/mean arterial pressure value was significantly higher than in the controls. The level of cleaved caspase-3, bcl-2, bcl-xl, PECAM-1, and SMA and the number of positively stained nerve fibers tended to revert to normal after Rg3 treatment. The apoptotic index in corpus cavernosum cells was lowered. Conclusion. Oral gavage with Rg3 appears to both prevent degeneration of neurons in the dorsal nerves and exert an antioxidant effect in the corpus cavernosum of rats. Liu T, Peng Y-F, Jia C, Yang B-H, Tao X, Li J, and Fang X. Ginsenoside Rg3 improves erectile function in streptozotocin-induced diabetic rats. J Sex Med **;**:**–**. Key Words. Diabetes; Erectile Dysfunction; Oxidative Stress; Apoptosis
Introduction
E
rectile dysfunction (ED) is one of the major complications following diabetes [1]. In recent years, the incidence of diabetic ED remains
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high. Pathological changes in the corpus cavernosum might be involved in diabetic ED, including increased reactive oxygen species (ROS), cavernous hypoxia, up-regulation of profibric factors, such as transforming growth factor, and J Sex Med **;**:**–**
2 subsequent structural changes including a decrease in smooth muscle and endothelial content, increased cavernous fibrosis, and a reduction of nitric oxide synthase (NOS) nerve density [2,3]. Ginseng, the root of Panax ginseng C.A. Meyer, is one of the most renowned herbs and has been used for thousands of years to maintain body homeostasis and enhance vital energy [4]. Recent investigations showed that the majority of ginseng pharmacological activities are closely related to its antioxidant property [5,6]. The main active ingredients of ginseng are ginsenosides. Among them, ginsenoside Rg3 has received much attention. Structurally, ginsenosides contain a hydrophobic triterpenoid skeleton attached with hydrophilic sugar moieties or hydroxyl groups at carbon-3 and carbon-6. Ginsenosides 20(S)-Rg3 and 20(R)-Rg3 are an enantiomeric pair that differs in the spatial orientation of the hydroxyl group on the chiral center at carbon-20; the S form usually exhibits significantly higher antioxidant effects than the R form [7]. Previous studies have demonstrated Rg3 has neuroprotective effect through reducing lipid peroxides, scavenging free radicals, and improving energy metabolism [8]. This study was designed to investigate the protective effect of 20(S)-Rg3 on erectile function and structural changes in the corpus cavernosum in rats with diabetes. Materials and Methods
Drugs and Animals 20(S)-Ginsenoside Rg3 extracted from the root of P. ginseng was purchased from Tauto Biotech Co., Ltd. (Shanghai, China). The saponins were a white powder with a purity of 98% (as determined by high performance liquid chromatography), a molecular weight of 785.013, and a molecular formula of C42H72O13. All experiments were approved by Wannan Medical College Research Subject Review Board. A total of 40 male Sprague-Dawley rats weighing 200–250 g were used. Rats were fasted for 16 hours, then received a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ) (Sigma-Aldrich Chemical Co, St Louis, MO, USA) or vehicle (0.1 mol/L citrate phosphate buffer, pH 4.5) [9,10]. Blood glucose was monitored by tail prick using a blood glucose meter (B. Braun, Melsungen, Germany) 3 days after STZ or vehicle injection, at regular intervals throughout the study, and immediately prior to sacrifice. Only those STZ-treated rats with fasting glucose concentrations consistently at >300 mg/dL were included in the diabetic J Sex Med **;**:**–**
Liu et al. group. Rats were divided into four groups: sham group (n = 10), diabetic rats received a daily gavage feedings of a mix of distilled saline water and 0.5% carboxymethylcellulose (Sigma-Aldrich) in which Rg3 was dissolved at concentrations of 0 (negative control group, n = 10) and 10 and 100 mg/kg (Rg3treated groups, n = 10 each) for 3 months. After a 3-day washout period, we evaluated penile hemodynamics by cavernous nerve electrical stimulation with real-time intracavernosal pressure (ICP) measurement. Following ICP assessment, rats were euthanized and penises were harvested for Western blot, nicotinamide adenine dinucleotide phosphate (NADPH) and transferase dUTP nick end labeling (TUNEL) staining, superoxide dismutase (SOD), and malondialdehyde (MDA) measurement.
Measurement of Erectile Function Rats form each group were anesthetized with 5% sodium pentobarbital intraperitoneally. Then the major pelvic ganglion, cavernous nerves, and pelvic organs were exposed. The skin overlying the penis was removed, and the penile crus was exposed by removing part of the overlying ischiocavernous muscle. Two 23-gauge trocars connected to a PE-50 tube with heparinized saline (250 IU/mL) were carefully inserted into the crus and the left carotid artery, respectively. The other end of the PE-50 tube was connected to a Biopac Systems MP150 (Aero Camino, Goleta, CA, USA). The cavernous nerve was exposed and electrostimulation (12 Hz; pulse width 5 milliseconds; 2.5 V, 5 V, 7.5 V; duration of 60 seconds) of the cavernous nerve was applied with a stainless steel bipolar hook electrode. The mean arterial pressure (MAP) was also measured at the same time. The ratio of maximal ICP (mm Hg) to MAP (mm Hg) was calculated to normalize penile hemodynamic response to variations in systemic blood pressure. Western Blot Cellular protein samples were prepared by homogenization of penile tissue in a lysis buffer containing 1% IGEPAL CA-630 (Sigma, St. Louis, MO, USA). 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, aprotinin (10 mg/ mL), leupeptin (10 mg/mL), and phosphatebuffered saline. Cell lysates containing 40 μg of protein were electrophoresed in sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Bedford, MA, USA). The membrane was blocked with 5%
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Ginsenoside Rg3 Improves Erectile Function skimmed milk for 1 hour at room temperature and incubated overnight at 4°C with antibody against cleaved caspase-3 (1:500, Santa Cruz Biotechnology, Santa Cruz, CA, USA), platelet endothelial cell adhesion molecule (PECAM)-1, a- smooth muscle actin (SMA), β-actin (1:1,000, Santa Cruz Biotechnology), Bcl-2, and Bcl-xl (1:1,000, Abcam Plc, Cambridge, UK). After washing in trisbuffered saline with 0.1% tween 20 three times for 10 minutes, the membrane was hybridized to horseradish peroxidase-labeled second antibodies. Resulting images were analyzed with ChemiImager 4000 (Alpha Innotech Corporation, San Leandro, CA, USA) to determine the integrated density.
NADPH-Diaphorase and TUNEL Staining Frozen tissue sections (8 μm) were air-dried for 5 minutes, then incubated with 0.1 M NADPH, 0.2 M nitroblue tetrazolium, and 0.2% Triton X-100 (Sigma-Aldrich) with constant microscopic monitoring for color development. The reaction was terminated when the medium became deep blue. Staining was assessed by counting the number of NADPH-diaphorase-positive nerves in the dorsal nerves at optical magnification of 400× with light microscopy. Positive endothelial staining was excluded from the count (n = 10 per group). For TUNEL staining (n = 10 per group), sections (5 μm) were deparaffinized and hydrated by sequential incubations in xylene and ethanol. The samples were then processed according to the instructions provided with the in situ cell death detection kit, peroxidase (Roche Diagnostic Corporation, Indianapolis, IN, USA). Measurement of SOD and MDA Levels Assay kits were purchased from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). About 20-mg corpus cavernosum tissue was used for an experiment; the tissue was homogenized in 0.2-mL normal saline, then 0.05-mL tissue homogenate was used for SOD determination and 0.1-mL tissue homogenate was used for MDA determination. SOD activity was measured through the inhibition of nitroblue tetrazolium reduction by O2, which was generated by the xanthine/xanthine oxidase system. The total reaction volume was 3.35 mL, and the absorbance was measured at 550 nm according to the manufacturer instructions. One SOD activity unit was defined as the enzyme amount causing 50% inhibition in a 1-mL reaction solution per milligram
of tissue protein; the result was expressed as unit per gram protein. The MDA concentration of the homogenate was measured using the thiobarbituric acid method. The total reaction volume was 4.2 mL. The amount of lipid peroxides was measured by the production of MDA, which, in combination with thiobarbituric acid, forms a pink chromogen compound with an absorbance of 532 nm. The result was expressed as nanomoles per milligram protein [11].
Statistical Analysis All data are expressed as mean ± standard deviation and were compared by one-way analysis of variance with post hoc Bonferroni’s correction (GraphPad Prism 5.0; GraphPad Software, San Diego, CA, USA). In all cases, P < 0.05 was considered statistical significance. Results
Metabolic Variables and ICP/MAP Assessment Three months after diabetes was induced, the fasting glucose concentrations in diabetic rats were significantly higher than that in the age-matched shams (P < 0.05). Body weights were significantly lower in the diabetic rats than that in sham animals (P < 0.05). No significant differences in body weight or glucose concentration were found between negative control rats and Rg3-treated diabetic rats (P > 0.05, Table 1). Three different voltages (2.5, 5.0, and 7.5 V) were used to measure the MAP and the ICP. The ratio of the maximal ICP/MAP after the electrostimulation of the rats in negative control group was 0.28 ± 0.04 at 5.0 V and 0.35 ± 0.051 at 7.5 V, which were significantly lower than in the sham group (0.86 ± 0.066 at 5 V and 0.88 ± 0.062 at 7.5 V) (P < 0.05, Figure 1). ICP/MAP in the 10 mg/kg Rg3 group was 0.41 ± 0.039 at 5.0 V and 0.49 ± 0.043 at 7.5 V, and there were no significant Table 1 Weight and blood glucose levels in sham and STZ-diabetic rats after being treated for 3 months Groups
Weight (g)
Fasting blood glucose (mg/dL)
Sham Diabetic control Diabetic + Rg3 (10 mg/kg) Diabetic + Rg3 (100 mg/kg)
478.88 ± 22.59 246.37 ± 19.86 250.75 ± 16.18a,* 254.38 ± 16.45b,*
87.49 ± 5.86 485.62 ± 40.35 441.74 ± 41.28c,* 446.26 ± 38.94d,*
*P > 0.05 Values are mean ± SD; n = 10 a,b,c,das compared with the diabetic control SD = standard deviation; STZ = streptozotocin
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Figure 1 Effect of Rg3 on erectile responses in diabetic rats. (A) Representative ICP in response to electrical stimulation of the cavernous nerve at 5 V in shams, negative controls, diabetic rats. The stimulus interval (60 seconds) was indicated by a solid bar. (B) Erectile function presented as ICP/MAP in each group at 2.5 V, 5 V, and 7.5 V. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; † P < 0.05 vs. 100 mg/kg Rg3 group; group *P < 0.05 vs. NC group. ICP = intracavernosal pressure; MAP = mean arterial pressure; NC = negative control.
differences between the negative control and 10 mg/kg Rg3 group (P > 0.05, Figure 1). Diabetic rats treated with 100 mg/kg Rg3 had significantly higher ICP/MAP (0.71 ± 0.068 at 5 V and 0.74 ± 0.069 at 7.5 V) when compared with the negative control group (P < 0.05), and there were no significant differences in ICP/MAP between the 100 mg/kg Rg3 group and the sham group. However, there were significant differences in ICP/ MAP between the 10 mg/kg and the 100 mg/kg Rg3 group at all levels of 5 V and 7.5 V (P < 0.05, Figure 1).
Increased Expression of PECAM-1 and SMA in Corpus Cavernosum Tissue of Diabetic Rats after Rg3 Treatment We evaluated the differential expression of PECAM-1 and SMA in cavernous tissue. The expression of PECAM-1 and SMA was lower in the negative control group than in the sham group (P < 0.05, Figure 2). However, the expression of PECAM-1 and SMA was significantly greater in J Sex Med **;**:**–**
the 100 mg/kg Rg3-treated groups relative to the negative control group (P < 0.05, Figure 2). There was no significant difference in PECAM-1 expression between the negative control and the 10 mg/kg Rg3 group (P > 0.05, Figure 2).
Effect of Rg3 on the NOS-Containing Neurons in the Dorsal Nerves of Diabetic Rats To localize neuronal NOS protein expression in the penis, we performed NADPH-diaphorase staining. The number of NADPH-diaphorasepositive nerves in the dorsal nerves in the sham group was significantly greater than in the negative control group (P < 0.05, Figure 3). However, 100 mg/kg Rg3 treatment partly restored the number of positive nerves in the dorsal nerves compared with the negative control group (P < 0.05, Figure 3), and there was a significant difference in the number of NOS-containing neurons in the dorsal nerves between the 10 and the 100 mg/kg Rg3 group (P < 0.05, Figure 3).
Ginsenoside Rg3 Improves Erectile Function
5 Figure 4A). Western blot was also used to evaluate the level of cleaved caspase-3, bcl-2, and bcl-xl in the corpus cavernosum. The level of cleaved caspase-3 was significantly lower, but the expression of bcl-2 and bcl-xl was significantly greater in the sham group than in the negative control group (P < 0.05, Figure 4B). However, both the 10 mg/kg and 100 mg/kg Rg3 treatments
Figure 2 Effect of Rg3 on the expression of PECAM-1 and SMA in corpus cavernosum from diabetic rats. (A) Representative Western blots for PECAM-1 and SMA in corpus cavernosum. β-actin was used as a loading control. (B) Relative optical density changes of PECAM-1 and SMA. Results of each group were adjusted to the loading control. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 compared with the negative control (NC) group. PECAM-1 = platelet endothelial cell adhesion molecule 1; SMA = smooth muscle actin.
Effect of Rg3 on the Apoptosis of Corpus Cavernosum Cells in Diabetic Rats Cell apoptosis in the corpus cavernosum was assessed by TUNEL staining. The apoptotic index was significantly greater in the negative control group relative to the sham group (P < 0.05, Figure 4A). However, 100 mg/kg Rg3 could significantly reduce the apoptotic index compared with the negative control group (P < 0.05,
Figure 3 Effect of Rg3 on nNOS-containing neurons in the dorsal nerves of diabetic rats. NADPH-diaphorase staining showed the content of nNOS in the dorsal nerve in corpus cavernosum. The bar chart presented the number of NADPH-positive nerve per high power field (400×). Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; †P < 0.05 vs. 100 mg/kg Rg3 group; *P < 0.05 vs. NC group. NADPH = nicotinamide adenine dinucleotide phosphate; NC = negative control; nNOS = neuronal nitric oxide synthase.
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Figure 4 Effect of Rg3 on the apoptosis of corpus cavernosum cells in diabetic rats. (A1) Representative photos for TUNEL staining of corpus cavernosum tissue (400×). (A2) Apoptotic index presented as the ratio of apoptotic nuclei to the total number of nuclei counted. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 vs. NC group. (B1) Representative Western blots for cleaved caspase-3, bcl-2, and bcl-xl in corpus cavernosum. β-actin was used as a loading control. (B2) Relative optical density changes of cleaved caspase-3, bcl-2, and bcl-xl. Results of each group were adjusted to the loading control. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 vs. NC group. NC = negative control; TUNEL = transferase dUTP nick end labeling.
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Ginsenoside Rg3 Improves Erectile Function
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Figure 5 Effect of Rg3 on the SOD and MDA levels in corpus cavernosum from diabetic rats. The MDA level was strongly increased, but the SOD level was reduced in diabetic rats, which could be partially reversed by Rg3 treatment. Each bar depicted the mean values (±standard deviation) from n = 10 animals per group. #P < 0.05 vs. sham group; *P < 0.05 vs. NC group. MDA = malondialdehyde; NC = negative control; SOD = superoxide dismutase.
significantly lowered the level of cleaved casepase3, but significantly improved the expression of bcl-xl when compared with the negative control group (P < 0.05, Figure 4B). The 100 mg/kg Rg3 treatment significantly improved the expression of bcl-2 relative to the negative control group (P < 0.05, Figure 4B).
Oxidative Stress Is Involved in Apoptosis of Corpus Cavernosum Cells Induced by High Blood Glucose To investigate the effect of Rg3 on oxidative stress in corpus cavernosum cells, the relative MDA and SOD levels were examined. High blood glucose enhanced the MDA level and reduced SOD level in corpus cavernosum significantly compared with the sham group (P < 0.05, Figure 5). However, both the 10 mg/kg and 100 mg/kg Rg3 treatments significantly lowered the MDA level relative to the negative control group (P < 0.05, Figure 5). Treatment with 100 mg/kg Rg3 enhanced the SOD level compared with the negative control group (P < 0.05, Figure 5). Discussion
Penile erection is a neurovascular event modulated by psychological and hormonal factors. Upon sexual stimulation, neurotransmitters are released from the cavernous nerve terminals and the enzyme NOS catalyzes the production of nitric oxide (NO) and citrulline from oxygen and l-arginine. Secondary messenger cyclic guanosine monophosphate (cGMP) or cyclic adenosine monophosphate ultimately leads to relaxation of the cavernous smooth muscle cells, thus increasing blood flow in the sinusoids and their supplying arteries. This results in an increase in the volume and pressure in the sinusoids, and the subsequent engorgement of the corpus cavernosum leads to
penile erection [12]. Thus, the integrity of endothelium, smooth muscle, and nerves in the penis is very important for erectile function. The diminished endothelium causes reduced endothelial NOS (eNOS) and the cavernous smooth muscle to lose contractility as the cells become synthetic [13,14]. Once there is a decrease in the amount of corporal smooth muscle cells, the remaining corporal smooth muscle mass is unable to achieve sufficient relaxation to attain the high intracorporeal pressure that can compress the subtunical veins as they egress from the tunica albuginea of the penis [15]. Previous research has found that diabetes causes lower erectile function in rats, with significantly reduced myelinated axons number in the cavernous nerve and dorsal penile nerve and a greater apoptotic index of cavernous endothelial and smooth muscle cells [16]. Oxidative stress may play an important role in the development of diabetic ED. Quercetin, a potent bioflavonoid, has been reported to have antioxidant effect and ameliorate ED in diabetic rats with improved SOD activity, nitrite levels, and eNOS expression in cavernosum tissue [17]. Antioxidant tempol significantly improves intracavernous pressure and cavernous smooth muscle compared with that in untreated diabetic rats, although the endothelial cell area is not restored after treatment [18]. AC3056 (2, 6-di-t-butyl-4-((dimethyl-4-methoxyphenylsilyl) methyloxy)phenol), a novel antioxidant, has been shown to reverse ED in diabetic rats and lower MDA level, and improved total NO derivatives in serum of diabetic rats were also observed after treatment [19]. During a long-standing hyperglycemic state in diabetes mellitus, glucose forms covalent adducts with the plasma proteins through a nonenzymatic process known as glycation. The interaction J Sex Med **;**:**–**
8 between advanced glycation end-products and receptor for advanced glycation end-products may initiate the generation process of ROS, largely via cytosolic NADPH oxidase-dependent mechanisms [20]. Excessive generation of ROS, including superoxide, hydroxyl radical (OH), and hydrogen peroxide, is an important cause of the oxidative stress reaction, and the interaction between NO and ROS is an important mechanism implicated in the pathophysiological process of ED. NO interacts with superoxide to form peroxynitrite. Peroxynitrite nitrates the critical tyrosine residue in the active site of manganese (Mn)-SOD, which inactivates Mn-SOD and leads to decreased removal of superoxide [21]. This further increases the formation of peroxynitrite and reduces the available NO concentration. Peroxynitrite causes smooth muscle relaxation but it is less potent than NO, which ultimately produces ED [22]. Peroxynitrite and superoxide also have been reported to increase the incidence of apoptosis in the endothelium, which leads to denudation of endothelium and further reduction of available NO [23]. Low concentrations of oxidative stress have been reported to have a more prominent proliferative effect on cavernosal smooth muscle than high concentrations, which inhibit cell growth [24]. Additionally, reduced NO concentration aggravates the adhesion of platelets and leukocytes to the endothelium and releases substances that cause vasoconstriction and ultimately worsen ED [25]. It has been reported that erectile function was improved in diabetic rats that received one intracavernosal injection of AdCMVEC-SOD, and the total SOD activity and cavernosal cGMP were increased in the penis of transfected rats [26]. In this study, we further investigated the erectile function of diabetic rats and found that there was a significant decrease in mean ICP/MAP with reduced NOS-containing neurons in the dorsal nerves and decreased expression of cavernous endothelial and smooth muscle cell markers, as well as enhanced oxidative stress reaction in penile tissues. However, Rg3 could partially reverse these changes in a dose-dependent manner. The present studies demonstrate that Rg3 has antioxidant activities. The OH-scavenging activity test using an electron spin resonance spectrometer has been suggested to be the most appropriate to measure the antioxidant activities of ginsenosides. The OH-scavenging activities of ginsenosides were related to the ferrous metal ion-chelating activities of their aglycone, 20(S)-protopanaxadiol. Ferrous metal ion-chelating activities of ginsenosides are J Sex Med **;**:**–**
Liu et al. believed to be influenced by their types of hydrophilic sugar moieties, which are closely related to the geometrical arrangement of the OH group, especially at carbon-20 [27]. In this study, we found that ROS was increased in the corpus cavernosum of diabetic rats and that Rg3 could decrease the ROS in the corpus cavernosum, which was consistent with prior studies. The level of cleaved caspase-3 was decreased while the expression of bcl-2 and bcl-xl was increased after Rg3 treatment. Previous research also showed that 20(S)-Rg3 could prevent human endothelial cell apoptosis via inhibition of a mitochondrial caspase pathway with increased bcl-2 and Bax [28]. In this study, we hypothesized that Rg3 could attenuate the oxidative stress reaction and reduce the apoptosis of endothelial and smooth muscle cells in corpus cavernosum to improve the erectile function of diabetic rats. The accumulation of glutamate in the extracellular space under these neurological disorders can induce neuronal death, and this glutamate toxicity has been clearly attributed to a massive influx of Ca2+ through non-N-methyl-D-aspartic acid (NMDA) and primarily NMDA receptors [29]. Rg3 can significantly attenuate the activation of NMDA receptors, which can subsequently lead to a neuroprotective effect of ginsenoside Rg3 in primary cultures of rat hippocampal neurons [30]. Here, we also found that Rg3 could restore the number of NOS-containing nerves in the dorsal nerves of diabetic rats, which might be helpful for the recovery of erectile function of diabetic rats. In conclusion, we have found that daily gavage feedings of higher dose (100 mg/kg) of Rg3 improves erectile function in diabetic rats via exerting neuroprotective and antioxidative effects in corpus cavernosum functional cells. However, if Rg3 would be used in human study according to this experimental dose (100 mg/kg), at least from the economic point of view, it is not realistic. In view of its biological activities, we are considering whether it could be made into a microemulsion transdermal preparation (Rg3 is fat soluble and its molecular weight is less than 800 Da) and applied to a localized area of the body. This is an issue that we must make a serious consideration and exploration in the future works. Acknowledgments
This study was funded by the National Natural Science Foundation of China Grant No. 81200436 and Introduced Talent Foundation of Yijishan Hospital Grant No. YR201201.
Ginsenoside Rg3 Improves Erectile Function Corresponding Author: Tao Liu, MD, Department of Sexual Medicine, Yijishan Hospital, Wannan Medical College, No. 2 Zheshanxi Road, Wuhu 241001, China. Tel: +86-553-5739327; Fax: +86-553-5739327; E-mail: [email protected] Conflict of Interest: The authors report no conflicts of interest.
Statement of Authorship
Category 1 (a) Conception and Design Tao Liu; Yi-Feng Peng (b) Acquisition of Data Tao Liu; Chao Jia; Xiang Fang; Jing Li (c) Analysis and Interpretation of Data Xia Tao; Ben-Hai Yang; Jing Li; Xiang Fang
Category 2 (a) Drafting the Article Tao Liu (b) Revising It for Intellectual Content Tao Liu; Yi-Feng Peng
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