Published online Dec 21, 2022.
https://doi.org/10.52361/fsbh.2022.2.e26
Effects of a Mixture of Three Extracts of Wolfberry, Chives and Graviola on the Erectile Dysfunction Induced by Bilateral Cavernous Nerve Injury in Rats
Abstract
Although some chemicals are available for erectile dysfunction (ED), efforts have been also made to seek health functional foods that can improve ED. Lycium barbarum (wolfberry) and Allium tuberosum (chives) were reported to enhance erectile function. And Annona muricata L is known to have anti-inflammatory and antioxidant actions. We assumed that if the three plants were used together, they may enhance erectile function synergistically. Hyunsung Vital Co. Ltd. prepared a mixture of water extracts of the three plants and named it Jikdaijangryeuk (JDJR). In the present study, JDJR was tested for the effect of improving ED in the rats subjected to bilateral corpus cavernous nerve injury (BCNI). BCNI decreased intra-cavernous pressure and also induced biological responses in the penile tissue that lead to ED, which were decreases in the expressions of endothelial nitric oxide (NO) synthase (eNOS) and nervous NO synthase (nNOS) that produce NO (a stimulator of 3′,5′-cyclic guanosine monophosphate [cGMP] synthesis), neurofilament-1 (a marker of nerve fibers) and an anti-apoptotic protein (Bcl2) and in the amounts of NO, cGMP (a blood vessel dilator) and smooth muscle as well as increases in the expression of inducible NO synthase (iNOS) (an inducer of microvasculature dysfunction), phosphodiesterase-5 (a cGMP destroyer), apoptotic molecules (caspase-3 and Bax) and transforming growth factor-β (a fibrosis inducer) and in the amount of asymmetric dimethylarginine (an endogenous NO synthases inhibitor). These data indicate that BCNI suppresses function of NO/cGMP axis and causes apoptosis and fibrosis of cavernous tissue. JDJR, however, reversed all these responses in a dose-dependent manner and the effects of JDJR were comparable to those of sildenafil (a positive control). The powder of Platycodon grandiflorum (a negative control) did not show the effects. These data support that JDJR has the action to enhance erectile function and thus, may be of help for ED.
INTRODUCTION
Erectile dysfunction (ED) is defined as the inability to achieve and maintain erectile penile sufficient for satisfactory sexual performance, affecting men over 40 years.1, 2 The worldwide prevalence of ED will reach 320 million men by 2025.3 ED is the most common complication of aging,4 diabetes mellitus,5, 6, 7 radical prostatectomy,8, 9 drugs such as antidepressants and antipsychotics,10 and psychogenic issues,11 etc., caused by collagen deposition, endothelial dysfunction, cavernous fibrosis and arterial insufficiency.12
Current effective therapies include oral phosphodiesterase-5 (PDE5) inhibitors including sildenafil, vardenafil, tadalafil and avanafil.13, 14 By enhancing the nitric oxide (NO) and 3′-5′-cyclic guanosine monophosphate (cGMP) pathway by inhibiting the breakdown of cGMP, they are generally considered to be effective for ED.2, 12, 13 However, the patients with ED caused by cavernous nerve (CN) injury due to the diabetes and radical prostatectomy respond poorly to these drugs,13, 14 and they should be precautious when given with alpha receptor blocking drugs and CYP3A inhibitors such as anti-retroviral protease inhibitors or macrolide antibiotics, etc.12 due to side effects such as hot flush, digestive disorder and headache.13 There are many reports that natural formulae15, 16, 17, 18, 19 may improve ED by modulating NO synthase (NOS) activity and cGMP amount in cavernous tissues after bilateral CN injury, and they may also improve the ED related to diabetes.5, 6, 7, 20, 21, 22
Lycium barbarum (wolfberry) is known to have various biological activities and has been widely used in the last decades as a popular functional food. The polysaccharides extracted from L. barbarum promote nerve regeneration and erectile function recovery in the CN injury in rats.23 Lycium chinense Mill extracts improve ED via antioxidant effects in rats24 and promote erectile function in patients with ED in a preliminary clinical study.25
Allium tuberosum (chives), which is edible green vegetable in Korea and China, has been used in traditional Asian medicine. Recently, this plant or its chemical components (steroids, saponins, polysaccharides, flavonoids, etc.) have an antioxidant activity,26 possess sexual enhancing properties in rats,27 and show the improvement of ED via upregulating the amount of NO and cGMP in penis.28
Annona muricata L. (Annonaceae family), which is known as graviola and widely distributed in India and Central America,29 has not been reported yet for the recovery of ED symptoms, although its bark, leaves and roots graviola have a variety of biological activities such as anti-inflammatory, anti-microbial, anti-tumoral, anti-oxidant and smooth muscle relaxant activities, and efficacy for drug-resistant cancerous cells.30 Its main ingredients are annonaceous acetogenins, and they almost are nontoxic,31 except the neuropathological abnormality possibly observed in the chronic use in rats.30
L. barbarum (wolfberry) and A. tuberosum (chives) are reported to have the effect to enhance erectile function. And Annona muricata L. is known to have anti-inflammatory and antioxidant actions. We assumed that if the three plants were used together, they may exert a more enhancing effect on ED. Hyunsung Vital Co. Ltd. (Seoul, Korea) prepared such a mixture of extracts of the three plants and named it Jikdaijangryeuk (JDJR). In the present study, JDJR was tested for the ED improving action in the rats subjected to bilateral corpus cavernous nerve injury (BCNI). We observed that JDJR increased intra-cavernous pressure (ICP) and up-/down-regulated the various biological molecules involved in erectile function, which all together lead to the improvement of the NO-cGMP axis, anti-apoptosis and anti-fibrosis and thus, support that JDJR is a health functional food that can be used for ED.
METHODS
Drugs and chemicals
JDJR, a mixture of the extracts of L. barbarum (wolfberry), A. tuberosum (chives) and Annona muricata L. (graviola) is provided by Hyunsung Vital Co. Ltd. In brief, each dried leaves (50 g) of L. barbarum, Annona muricata L or A. tuberosum were added in 1,000 mL distilled water, treated at 90°C for 24 hours, and then filtered. Each filtrate was evaporated to yield a powder. The three powders were mixed as the ratio of 5:3.5:1.5, respectively, and then it was named the JDJR. The root of 6-year-old platycodon grandiflorum (PG) known to be rich in saponins was also treated as above and a powder of the extract obtained was used as a negative control.
The ratio of 5:3.5:1.5 of each extract was determined by screening the inhibitory effect of the mixtures of various ratio on the apoptosis (expression of cleaved caspase-3) and increasing effect on expression of PDE5 in TM3 cells (mouse Leydig testicular cell line) (Korean Cell Line Bank, Seoul, Korea) treated with 100 mM EtOH for 3 hours.31 The mixture of 5:3.5:1.5 showed the highest inhibitory effect. Using the in vitro ethanol-treated TM cells, the concentrations of JDJR and sildenafil to be used in vivo were also determined.32
Animals
Male Sprague Dawley rats (300–350 g, 10 weeks old) were purchased from Daehan Bio Link Co. (Eumseong, Korea). The rats were housed at 23°C–25°C, 50%–60% (relative humidity) and a 12-hour light/dark cycle. They were provided access to food and water ad libitum, and maintained in specific-pathogen-free conditions at the laboratory Animal Research Center of Hyunsung Vital Co. Ltd. The rats were kept in accordance with the guidelines of the Ministry of Food and Drug Safety in Korea. All animal experiments were approved by the Laboratory Animal Research Center of Hyunsung Vital Co. Ltd., which had been approved by the Ministry of Food & Drug Safety (Approval number, Hyunsung-2016-3).
Experimental design
Rats were equally and randomly divided into 7 groups (8 rats/group): NC with no treatment, BCNI, BCNI + JDJR (50, 100, 300 mg/kg), BCNI + sildenafil (30 mg/kg), and BCNI + PG (300 mg/kg). The extracts and drug were administered orally once a day for 7 days.
BCNI and treatment
Animals were anesthetized for surgical procedures using an intraperitoneal injection of zoletil (10 mg/kg) and xylazine (30 mg/kg) (Bayer Korea, Seoul, Korea) and kept isotherm on a heated pad. After the animal was shaved, the prostate gland and the CNs were exposed via a lower midline abdominal incision, and major pelvic ganglia (MPG) were identified bilaterally. BCNI was induced by applying a microsurgical vascular clamp to the CN 2 to 3 mm distal to the MPG for 30 seconds, removing it for 30 seconds and reapplying it for another 30 seconds.33 Every BCNI operation was performed by the same trained surgeon in a random blind manner. JDJR (50, 100, 300 mg/kg) sildenafil (30 mg/kg), a positive or PG (300 mg/kg), a negative control dissolved in phosphate-buffered saline (PBS) and orally administrated once daily for 7 days starting from one day after BCNI operation. After 7 days, the rats were sacrificed, and corpus cavernous and penile tissues were isolated for analysis of histopathology, reverse transcription-polymerase chain reaction (RT-PCR), western blotting, and biological molecules (Fig. 1A).
Fig. 1
***P < 0.001 vs. NC group; ++P < 0.01; +++P < 0.001 vs. BCNI group.
Schematic diagram for experimental design and effects of JDJR on the ICP and MAP in the rats treated with BCNI. (A) Experimental schedule. (B) Representative maximum ICP. (C) Maximum values for ICP. (D) Values for ICP/MAP ratio. The results are expressed as the mean ± SEM (n = 8). Treatment doses were: sildenafil, 30 mg/kg; JDJR50/100/300, JDJR 50, 100, 300 mg/kg, respectively; PG300, PG 300 mg/kg. Experimental details were described in “METHODS.”
JDJR = Jikdaijangryeuk, ICP = intra-cavernous pressure, BCNI = bilateral corpus cavernous nerve injury, MAP = mean arterial pressure, SEM = standard error of the mean, PG = Platycodon grandiflorum, NC = negative control.
In vivo erection studies
On day 7 after BCNI, erectile function was assessed by measuring maximal intra-cavernous pressure (ICP) upon direct CN electrostimulation.33 In briefly, the CN was isolated via a midline abdominal incision, and the crura of the penis was identified. To assess erectile responses to CN electrical stimulation, a 24-gauge angiocatheter was introduced into the unilateral common carotid artery after making a medial incision in the neck to monitor mean arterial pressure (MAP) continuously. The corpus cavernosum was cannulated with a 26-gauge needle to permit continuous ICP monitoring. A platinum bipolar electrode was placed around the CN distal to the site of nerve injury. Polyethylene-50 tubing was used to connect the needle to an MP160 pressure transducer (Biopac Systems, Inc., Goleta, CA, USA). The following stimulus conditions were used: voltage, 4.0 V; frequency, 15 Hz; and pulse width, 0.4 millisecond square (ms2) wave. The measurement of stimulation was for 30 seconds with 10 minutes′ rest period between stimulations. Erectile function was evaluated based on baseline ICP, maximal ICP, the ratio of maximal ICP to MAP.
After evaluating the erectile function, the whole penile was isolated from each rat. The penile shaft was denuded of skin. The middle part of the shaft was maintained overnight in 4% formaldehyde solution, and then embedded in paraffin wax for histological studies. The remaining penile tissues were rapidly frozen in liquid nitrogen and stored at −80°C until use.
RT-PCR
Total cellular RNA was isolated from TM3 cells (1 × 106 cells) or penile tissues (50 mg/500 μL) using Trizol reagent (Invitrogen, Carlsbad, CA, USA). RT-PCR was performed in a final volume of 20 μL using an amfiRivert one-step RT-PCR kit (GenDEPOT, Barker, TX, USA) in an automated thermal cycler (Bio-Rad, Laboratories, Hercules, CA, USA). The PCR assays were performed for 35 cycles. Each cycle consisted of the following steps: denaturation at 94°C for 30 seconds, annealing at 51°C for 45 seconds, and extension at 72°C for 3 minutes. The results were expressed as a ratio to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The PCR products were analyzed using 1% agarose gel and visualized under UV light after staining with StaySafe nucleic acid gel stain (Real Biotech Corporation, Banqiao, Taiwan).34 Numbers below band images (#) show the mean values (n = 4) obtained from the ratio of each band density of each group versus those of the control and GAPDH mRNA from four independent experiments.
The rat primer sequences used were as follows: endothelial NOS (eNOS) sense, 5-CAG GCT GCC TGT GAA ACT TT-3 and antisense, 5-TTG CTG CTC TGT AGG TTC TC-3; nervous NOS (nNOS) sense, 5-TGG AGG AGT ATG ACA TCG TG-3 and antisense, 5-TTC GGG AGT CAG AAT AGG AG-3; inducible NOS (iNOS) sense, 5-AGT ACA AGC TCA CCC AGA GC-3 and antisense, 5-GGG TAG TGA TGT CCA GGA AG-3; PDE5 sense, 5-GAC AAC AGA TTT CCC TGG AC-3 and antisense, 5-GCT GAT GCG TGA TAA GAC AG-3; Caspase-3 sense, 5-TGC AGC TAA CCT CAG AGA GA-3 and antisense, 5-TCG TCA GTT CCA CTG TCT GT-3; Bax sense, 5-AAG AAG CTG AGC GAG TGT CT-3 and antisense, 5-CAA AGA TGG TCA CTG TCT GC-3; Bcl2 sense, 5-GTA TGA TAA CCG GGA GAT CG-3 and antisense, 5-CAG CTG ACT GGA CAT CTC TG-3; transforming growth factor (TGF)-β sense, 5-TGG CGT TAC CTT GGT AAC CG-3 and antisense, 5-TCA GCT GCA CTT GCA GGA GC-3; GAPDH sense, 5-AAC TTT GGC ATT GTG GAA GG-3 and antisense, 5-ACA CAT TGG GGG TAG GAA CA-3.
Western blot analysis
TM3 cells (1 × 106 cells) or penile tissues (50 mg/500 µL) were suspended in lysis buffer (1% Triton X-100, 20 mM Tris-HCl [pH 7.6], 150 mM NaCl, 1 mM Na3VO4, 1 mM PMSF, 1 µg/mL aprotinin, 1 µg/mL leupeptin), and allowed to swell on ice for 60 minutes. TM3 cells or tissues were then homogenized by up and down using a micropipette and Polytron (Kinematica, Littau, Swizerland), respectively. After centrifugation, supernatants were subjected to 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membrane (Amersham Biosciences, Buckinghamshire, UK). The membranes were washed with PBS containing 0.1% Tween 20 (PBST), and blocked for 1 hour in 5% skim milk in PBST. After the membranes were washed with PBST, they were incubated for overnight at 4°C with primary antibody against β-actin (Sigma-Aldrich, St louis, MO, USA), eNOS (Cell Signaling, Beverly, MA, USA), nNOS (MyBioSource, Inc., San Diego, CA, USA), iNOS (Abcam, Boston, MA, USA), PDE5 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), cleaved caspase-3, Bax, Bcl2, TGF-β (Cell Signaling) diluted with PBST (1:100). Membranes were washed with PBST and treated with horseradish peroxidase (HRP)-conjugated goat anti-mouse or HRP-conjugated rabbit anti-goat IgG (diluted to 1:5,000) (Bethyl Laboratories, Montgomery, TX, USA) in PBST for 60 minutes. After washing, the protein bands were visualized by enhanced chemiluminescence (ECL) solution using a chemiluminometer (Amersham Biosciences, Buckinghamshire, UK).34
Measurement of NO, TGF-β, cGMP, and asymmetric dimethylarginine (ADMA)
Amounts of NO, TGF-β, cGMP or ADMA in the lysate obtained from penile tissues were determined using NO colorimetric detection kit (Kamiya Biomedical Company, Seattle, WA, USA), Quantikine TGF-β1 ELISA kit (R&D Systems, Minneapolis, MN, USA), cGMP complete ELISA kit (Enzo Life Science, Inc., Farmingdale, NY, USA) and ADMA ELISA kit (MyBioSource, Inc.), respectively, following the protocol provided by the manufactures.
Briefly, penile tissues (50 mg/500 µL) were washed three times with PBS and placed in lysis buffer via proteinase inhibitor cocktail for 10 minutes. The tissues were homogenized using a Polytron (Kinematica, Littau, Swizerland). After centrifuge, 50 µL of the supernatant was taken in triplicates in a 96-well plate, which is pre-coated with an antibody specific to the antigen of each target. The assay buffer and biotinylated detection antibody were then added into each well. The plate was incubated for 1 hour at room temperature, and 100 µL of color or enzyme-substrate reagent was added into each well. Absorbance was read at 500 nm for NO, 450 nm for TGF-β and ADMA, and 405 nm for cGMP. Standard curves were made using a serial dilution of each molecule. The lowest detection limit for NO, TGF-β, cGMP or ADMA was 3.0 μM, 31.2 pg/mL, 0.8 pmol/mL and 15.6 ng/mL, respectively.
Collagen deposition
Sectioned corporal tissues of the rat penis were washed with PBS three times, fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned at 3 µm. After de-waxing and rehydration, sections were fixed in Bouin’s or Zenker’s liquor overnight and washed in running water until the yellow color disappeared. The sections were counterstained with Mayer’s hematoxylin and aniline blue (Masson trichrome staining). Washing between the steps in the procedures was performed for 2–3 minutes. After washing, the slides were mounted using a mounting medium, and examined the collagen deposition under a light microscope (magnification, 200×) (Nikon Instruments Inc., Melville, NY, USA). The collagen and smooth muscle fibers were stained to blue and red color, respectively. The color distribution of the muscle tissues was observed by using Adobe Photoshop CS 8.0 (Adobe Systems, Inc., San Jose, CA, USA).
Immunohistochemistry (TGF-β)
Experiment was performed according to the procedure reported previously. Sectioned corporal tissues (3 µm) of the rat penile were de-paraffinized in xylene and de-washed in ethanol. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in methanol for 10 minutes. The slides were treated with citrate buffer and microwaved for antigen retrieval, and then blocked with 10% bovine serum albumin (BSA) in PBS for 1 hour. Slides were incubated overnight at 4°C with a primary antibody against TGF-β (1:50 dilution; Abcam) and incubated with HRP-conjugated goat anti-rabbit (1:100 dilution; Zymed Laboratory Inc., San Francisco, CA, USA) for 1 hour, and then washed again with PBST, followed by staining with 3,3-diaminobenzidine (DAB) (Abcam). Slides were counterstained with hematoxylin and finally mounted using aqueous mounting medium (Thermo Fisher Scientific Inc., Waltham, MA, USA), and then examined under a light microscope (Nikon Instruments Inc.).35 For image analysis, five randomly selected fields per animal were photographed and recorded at ×400 magnification. Images were analyzed with Image-Pro Plus 4.1 software (Media Cybernetics, Bethesda, MD, USA).
Cryosection and immunofluorescence
Sectioned corporal tissues of the penis was fixed for 4 hours in cold 2% formaldehyde and 0.002% picric acid in 0.1 M phosphate buffer, followed by overnight immersion in the buffer solution containing 30% sucrose. After fixation, the tissues were transferred to cryomolds with one flat plane facedown. The cryomolds were then filled with optimal cutting temperature compound and frozen by floating in an ethanol/dry ice slurry or cold isopentane with liquid nitrogen. After solidification, cryomolds were removed from the slurry and stored at −80°C until cryosection. The cryosection was performed on the frozen blocks using a cryostat (Leica CM 1850) at −20°C. For each block, 3 μm cross-sections along the flat plane of the construct were collected on SuperFrost® Plus Gold slides (Thermo Fisher Scientific Inc.). The slides were air-dried for several hours before immunofluorescence.
The slides were rehydrated with PBS, treated with citrate buffer, and microwaved for antigen retrieval. The slides were blocked with 10% BSA in PBS for 1 hour and incubated overnight at 4°C with a primary antibody against neurofilament-1 (1:50 dilution; Abcam) and incubated with goat anti-rat secondary Alexa Fluor™ 488 antibody (1:100 dilution; Thermo Fisher Scientific Inc.) for 1 hour. After washing, the slides were mounted onto coverslips with an aqueous mounting medium (Thermo Fisher Scientific Inc.). Nerve-positive fibers in the dorsal penile nerves (DPN) were evaluated at 200× magnification using a fluorescence microscope (Nikon Instruments Inc.).
Statistical analysis
Experimental data are shown as means ± standard error of the mean (SEM) (n = 8). The unpaired Student′’s t-test was used to compare the 2 groups. Multiple group comparisons were performed using a one-way analysis of variance followed by Scheffe’s post hoc test, using the SPSS software (SPSS Inc., Chicago, IL, USA). P values < 0.05 were considered to indicate statistical significance. The densitometry analysis of PCR or western blot was performed with Quantity One (version 4.6.3; Bio-Rad, Hercules, CA, USA) and are indicated as means ± SEM (n = 4) obtained from the ratio of each band density versus those in the control and loading control (GAPDH or β-actin) from four independent experiments.
RESULTS
Effects of JDJR on the erectile function
The maximum ratio of ICP/MAP represents erectile function.36 We first examined whether the ICP responses are induced in the CN by electrical stimulation 1 week after BCNI. We observed that erectile function was remarkably decreased in the BCNI group (42 ± 6.8 mm Hg) versus NC group (100 ± 5.7 mmHg) (Fig. 1B and C). JDJR treatment significantly recovered the decreased erectile function (approximately 85% for high dose) in the dose-dependent manner. The values for MAP (110 ± 10.9 mm Hg) showed similar responses to the all experimental groups (data not shown). Thus, ICP/MAP ratio was significantly lower in the BCNI group versus the NC group, but JDJR increased ICP/MAP ratio in the dose-dependent manner versus BCNI group (Fig. 1D). The effect of JDJR at 300 mg/kg was similar to that of sildenafil (30 mg/kg), a positive control which is currently used for ED.
PG powder, which is known to have anti-oxidant and anti-inflammatory effects,37 but is not reported yet for erectile function, was used as a natural negative control. PG at 300 mg/kg did not show any effect.
Effects of JDJR on the 3 NOS or PDE5 related to the ED
NO-triggered increase in cGMP, which promotes cavernous trabecular smooth muscle relaxation, plays a key role in erectile function.38 We examined the effects of JDJR on the three NO synthases (eNOS, nNOS, and iNOS) in the penile tissues of BCNI group. BCNI group showed decreased mRNA and protein expression of eNOS and nNOS, which promote cGMP production, but increased mRNA and protein expression of iNOS, which induces microvascular dysfunction in penile tissues (Fig. 2A and B). BCNI group also increased mRNA and protein expression of PDE5, which catalyzes cGMP degradation (Fig. 2A and B). JDJR treatment increased mRNA and protein expression of eNOS and nNOS, but decreased mRNA and protein expression of iNOS in dose-dependent manner versus those in BCNI group.
Fig. 2
***P < 0.001 vs. NC group; ++P < 0.01 vs. BCNI group.
Effects of JDJR on the expressions of enzymatic molecules or amounts of NO in the BCNI-treated rats. (A, B) Expression of mRNAs and proteins. (C) Amounts of No. #, Numbers below the bands show the mean values (n = 4) obtained from the ratio of each band density versus those of the control and loading control (GAPDH mRNA or β-actin) from 4 independent experiments. Treatment doses were: sildenafil, 30 mg/kg; JDJR50/100/300, JDJR 50, 100, 300 mg/kg, respectively; PG300, PG 300 mg/kg.
JDJR = Jikdaijangryeuk, NO = nitric oxide, BCNI = bilateral corpus cavernous nerve injury, GAPDH = glyceraldehyde-3-phosphate dehydrogenase, NC = negative control, PG = Platycodon grandiflorum, eNOS = endothelial nitric oxide synthase, nNOS = nervous nitric oxide synthases, iNOS = inducible nitric oxide synthases, PDE5 = phosphodiesterase-5.
BCNI decreased the amounts of NO in the penile tissues of group (Fig. 2C) but JDJR treatment increased the amounts of NO in dose-dependent manner.
Effects of JDJR on the expression of apoptotic molecules or smooth muscle content in the corpus cavernous tissues
ED causes apoptosis of smooth muscle cells and endothelial cells in the corpus cavernosum.39 Thus, we examined whether JDJR affects the expression of apoptotic molecules in the penile tissues. BCNI group showed the increase of mRNA and protein expression of apoptotic molecules (cleaved caspase-3 and Bax), and decreased mRNA and protein expression of anti-apoptotic molecule (Bcl2) (Fig. 3A and B). However, JDJR restored the changed expressions of these molecules.
Fig. 3
Effects of JDJR on the expression of cell death signaling molecules or on the morphology of corpus cavernosa smooth muscle in BCNI-treated rats. (A, B) Expression of mRNAs or proteins of apoptotic and anti-apoptotic molecules. Numbers below bands show the mean values (n = 4) obtained as described in Fig. 2. (C) Morphology of corpus cavernosum smooth muscle and collagen deposition. Red color, smooth muscle; blue color, collagen. Bar in the pannel NC indicates 50 μm (magnification: ×200). Treatment doses were: sildenafil, 30 mg/kg; JDJR50/100/300, JDJR 50, 100, 300 mg/kg, respectively; PG300, PG 300 mg/kg.
JDJR = Jikdaijangryeuk, BCNI = bilateral corpus cavernous nerve injury, NC = negative control, PG = Platycodon grandiflorum, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
Next, we examined whether JDJR affects the collagen deposition in the cavernosa smooth muscle tissues BCNI significantly decreased ratio of the smooth muscle content to collagen deposition in the corpus cavernous tissues, compared with the NC group (Fig. 3C). JDJR treatment increased ratio of the smooth muscle content to collagen deposition in the dose-dependent manner.
Effects of JDJR on the expression or amounts of TGF-β in the corpus cavernous tissues
TGF-β, which is related to fibrosis, is up-regulated in the ED.40 Thus, effect of BCNI on the expression of TGF-β was examined immunohistochemically. As expected the expression was markedly increased (brown color) in the corpus cavernous tissues (Fig. 4A). But JDJR decreased the increased expression of TGF-β in dose-dependent manner. Especially, high dose of JDJR group decreased the expression of TGF-β to the control level. Effects on TGF-β expression was also studied by mRNA and protein levels. The same results were also obtained by 3 methods (Fig. 4B).
Fig. 4
***P < 0.001 vs. NC group; ++P < 0.01; +++P < 0.001 vs. BCNI group.
Effects of JDJR on the expression and amounts of TGF-β in the corpus cavernous tissues of BCNI-treated rats. (A) Immunohistochemistry image for TGF-β. Bar in the pannel NC indicates 50 μm (magnification: ×400). (B) Expression of TGF-β mRNA (upper lane) and protein (lower lane). (C) Amounts of TGF-β. Treatment doses were: sildenafil, 30 mg/kg; JDJR50/100/300, JDJR 50, 100, 300 mg/kg, respectively; PG300, PG 300 mg/kg.
JDJR = Jikdaijangryeuk, TGF = transforming growth factor, BCNI = bilateral corpus cavernous nerve injury, NC = negative control, PG = Platycodon grandiflorum, GAPDH = glyceraldehyde-3-phosphate dehydrogenase.
In Fig. 4C, Effects of BNCI and JDJR were evaluated by measuring the amount of TGF- β.
BCNI group showed an increase of total TGF- β amounts (146 ± 14.1 pg/mL) in penile tissues versus NC group (63 ± 6.1 pg/mL). However, JDJR group was remarkably decreased amounts of TGF-β (140 ± 14.6 pg/mL for 50 mg/kg; 90.7 ± 15.8 pg/mL for 100 mg/kg; and 63 ± 9.6 pg/mL for 300 mg/kg) in the penile tissues versus those in the BCNI group.
Effects of JDJR on the morphology of dorsal nerves in the penile tissues
As the dorsal penile nerves (DPNs) are crucial for normal erectile and ejaculatory function,41 Nerve fibers positive regeneration was assessed by the expression of neurofilament-1 (NF-1) in DPN by immunofluorescence staining. BCNI group showed the reduced of NF-1-positive nerve fibers versus those in NC group (Fig. 5A). JDJR group remarkably recovered the NF-1-positive-nerve fibers versus those in BCNI group.
Fig. 5
***P < 0.001 vs. NC group; ++P < 0.01; +++P < 0.001 vs. BCNI group.
Effects of JDJR on the morphology of dosal nerve, and amounts of cGMP or ADMA in the penile tissues of BCNI-treated rats. (A) NF-1-positive nerve fibers on the DPN tissues stained by immunofluorescence method. Bar in the pannel NC indicates 50 μm (magnification: ×200). Small squasres are enlarged in the boxws of right upper corners (×400). (B, C) Amounts of cGMP and ADMA, respectively. Treatment of compounds were: sildenafil, 30 mg/kg; JDJR50/100/300, JDJR 50, 100, 300 mg/kg, respectively; PG300, PG 300 mg/kg.
DNP = dorsal penile nerve, JDJR = Jikdaijangryeuk, cGMP = 3′,5′-cyclic guanosine monophosphate, ADMA = asymmetric dimethylarginine, BCNI = bilateral corpus cavernous nerve injury, NF-1 = neurofilament-1, NC = negative control, PG = Platycodon grandiflorum.
Effects of JDJR on the amounts of soluble cGMP or ADMA in the penile tissues
Finally, we examined the amounts of cGMP which directly stimulate penile erection38 and ADMA which is known as endogenous NOS competitive inhibitor42 in the cavernosa of penile tissues. The cGMP levels were significantly lowered in BCNI group versus those in NC group (Fig. 5B). JDJR recovered the lowered cGMP levels in dose-dependent manner versus those in BCNI group.
The levels of ADMA were remarkably increased in the penile tissues by BCNI (Fig. 5C). But JDJR decreased amounts of ADMA in dose-dependent manner.
DISCUSSION
We demonstrate that natural material mixture JDJR may improve ED via increasing the ratio of the ICP/MAP pressure and smooth muscle/collagen, via repairing dorsal nerve damage, via up-regulating the NO-cGMP signaling pathway and anti-apoptotic molecules, via down-regulating apoptosis, fibrosis and PDE5 in the BCNI.
Although PDE5 inhibitors, which were known as the class of novel agents, have been proven to manage the ED, and although the second generation of PDE5 inhibitors have the greater efficacy and safety compared to the first generation PDE5 inhibitors, these agents have still several side effects similar to the first generation PDE5 inhibitors. Therefore, it is necessary to seek novel alternative materials or therapeutic agents which have safety and efficacy in ED management.
There are a few reports that L. barbarum and A. tuberosum contained in JDJR, except to Annona muricata L, has the efficacy in the ED.23, 24, 25, 26, 27, 28 Thus, it can be inferred that JDJR, which is prepared by the optimal ratio of these three natural materials, and which has little side effects, may show potent efficacy in the BCNI model.
The pathogenesis of ED is multifunctional process and has not been fully elucidated. Electrical stimulation of the CN promotes nitrergic (non-adrenergic and non-cholinergic nerve, NANC nerve) discharge inducing relaxation of the corpus cavernosum with consequent elevation of peak ICP and ICP/MAP ratio, leading to an increase of cGMP level produced by guanylate cyclase activated via NO released from NANC nerve ending and endothelium in the corpus cavernosum.2, 13, 20, 41 Thus, our data suggest that JDJR may improve the ED through relaxation of corpus cavernosum via increasing the ICP/MAP ratio (Fig. 1).
It has been reported that causes of ED include intraoperative neurogenic and vasculogenic injury.39 CN injury, which causes irreversible defects of smooth muscle cells, induces the most severe ED and nerve damage. Several pathophysiological theories have been proposed to explain ED after CN injury, including nNOS-positive nerve fiber decrease, eNOS-positive endothelial cell decrease and cavernosal smooth muscle apoptosis and fibrosis.38, 39, 40, 43 NO, which is synthesized using three NOS isoforms, including nNOS, eNOS and iNOS, and which is known as a neurotransmitter, plays an important role in penile erection. Thus, nNOS and eNOS are major source of NO in corpus cavernosum. Deprivation of NO causes decrease of cGMP production and smooth muscle contraction, and then the ED is induced. These reports agree with our data for the decrease of nNOS and eNOS in penile tissues of BCNI model. Thus, the data can be inferred that JDJR may induce smooth muscle relaxation and endothelial cell activation via repairing the CN injury through up-regulating expression of nNOS and eNOS, leading to an increase of cGMP produced by NO bioactivity, and then may improve erectile function, as demonstrated by showing the data that JDJR increased NO-cGMP mediators via up-regulating the activity of nNOS and eNOS in the penile tissues (Figs. 2A-C and 5B).
There are contrary reports that up-regulation of iNOS leads to microvascular dysfunction in patients with ED,44, 45 but it decreases collagen expression/smooth muscle cell ratio in penile tissues injected by adipose-derived stem cells, and then decreases the penile fibrosis.46 Our data agree with the data reported that up-regulation of iNOS induces microvascular dysfunction of ED.44, 45 The data suggest that JDJR may improve ED via down-regulating the iNOS expression in CN of penile tissues (Fig. 2A and B).
The components of penile erection include the CN, the endothelium and corporal smooth muscle. Interactions among these three components are critical for optimal erectile function.47 ED is caused in apoptosis of smooth muscle cells in the corpus cavernosum. Defects in the corporal smooth muscle cells are irreversible after CN crush injury. Corporal smooth muscle fibrosis may cause persistent ED, although other neurological functions recover after operation.39 Thus, protection of corporal smooth muscle cells from apoptosis may be more important than nerve protection in patients with ED. Thus, the data suggest that JDJR may ameliorate the apoptosis of corporal smooth muscle via down-/up-regulating the apoptotic and anti-apoptotic molecules, respectively, and via increasing the smooth muscle and collagen ratio (Fig. 3A-C).
Fibrosis is the end result of various chronic diseases, including various chronic urogenital organs. The mechanism of fibrotic tissue formation is mainly caused via remodeling cytokine TGF-β.48 TGF-β plays important roles in cavernous fibrosis and in the ED of the mouse BCNI.49 The data can be inferred that JDJR may improve cavernous fibrosis via down-regulating the TGF-β expression in the cavernous smooth muscle of penile tissues (Fig. 4).
DPN, which plays important roles in the normal erectile function, are known as major criteria to diagnose the ED caused by CN injury. And, the regeneration of the DPN ameliorates the erectile function in BCNI in rats.41 Our data agree with the results reported by others laboratory that the regeneration of the DPN may improve the ED in the BCNI in rats. Our data suggest that JDJR may ameliorate the ED in rat BCNI model via regenerating the DPN (Fig. 5A).
ADMA, which is known as all endogenous NOS competitive inhibitors, is a regulator of NO production in the cavernosa of penile tissues, and induces endothelial dysfunction.42 The ADMA is elevated in ED with cardiovascular diseases.50 Thus, the data suggest that JDJR may have a beneficial effect for ED improvement through increasing NO bioactivity via reducing the ADMA levels (Fig. 5C).
The ED may be caused through down-regulating the NO release, which produce cGMP, which plays a key role in erectile function via soluble guanylate cyclase activated by the NO released in the NANC nerve ending (NO produced by nNOS) and endothelial cells (NO produced by eNOS). The ED may also be caused through increasing apoptotic molecules in the corpus cavernous smooth muscle by neurotransmitter, which presumes to be released in the adrenergic nerve ending, and through increasing cavernous smooth muscle fibrosis via TGF-β signaling pathways and DPN damage. Thus, the data suggest that JDJR may increase the NO-cGMP pathway via up-regulating the production of nNOS or eNOS in the NANC nerve ending and endothelial cells, respectively, and that it may down-regulate the production of PDE5, apoptotic molecules, remodeling molecule TGF-β and ADMA. Finally, JDJR may improve erectile function due to regulation of a variety of molecules related to ED (Fig. 6).
Fig. 6
Schematic diagram for signaling pathways associated with activating/inhibiting effects of JDJR in BCNI-treated rats. Molecules inhibited by JDJR are indicated by blue block arrows, and molecules activated by JDJR by red arrows. The lined arrows indicate “determined,” but the dotted arrows indicate “not determined” in this study (see “DISCUSSION” for the Fig. 6). ED may be caused by reducing the release of NO, which stimulate guanylate cyclase to produce cGMP, a smooth muscle dilator causing penile erection in the NANC nerve ending and endothelial cells. ED may also be caused through up-regulating apoptotic and fibrotic molecules such as Bax and TGF-β in cavernous smooth muscle. JDJR increased the NO production via up-regulating eNOS in the endothelial cells and nNOS in the NANC nerve ending and Bcl1 (an anti-apoptotic protein) and down-regulating the production of caspase-3 and Bax (apoptotic proteins), TGF-β (a fibrosis inducer), PDE5 (a cGMP destroyer) and ADMA (a NO competitive inhibitor). All these responses contribute to enhancing erectile function by stimulating functions of NO/cGMP axis and processes of anti-apoptosis and anti-fibrosis of cavernous tissue.
JDJR = Jikdaijangryeuk, BCNI = bilateral corpus cavernous nerve injury, ED = erectile dysfunction, NO = nitric oxide, cGMP = 3′,5′-cyclic guanosine monophosphate, NANC = non-adrenergic non-cholinergic nerve, TGF = transforming growth factor, nNOS = nervous nitric oxide synthase, PDE5 = phosphodiesterase-5, ADMA = asymmetric dimethylarginine, eNOS = endothelial nitric oxide synthase, iNOS = inducible nitric oxide synthase, NT = neurotrophin, GPCR = G protein-coupled receptor, PKC = protein kinase C, GTP = guanosine-5′-triphosphate, 5′-GMP = guanosine-5′-monophosphate.
Funding:This study was supported by a research grant from Life & Science Research Center, Hyunsung Vital Co., Ltd., Seoul, Korea.
Disclosure:The authors have no potential conflicts of interest to disclose.
Author Contributions:
Conceptualization: Ro, JY.
Data curation: Chung MH, Ro JY.
Methodology: Hong GU, Shin YH.
Investigation: Hong GU, Shin YH.
Writing - original draft: Ro JY.
Writing - review & editing: Chung MH, Ro JY.
ACKNOWLEDGMENTS
Thanks are extended to Ms. Hyeryun Cho for taking animal care.
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