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Probucol Improves Erectile Function by Restoring Endothelial Function and Preventing Cavernous Fibrosis in Streptozotocin-induced Diabetic Rats
Accepted Manuscript Title: Probucol Improves Erectile Function by Restoring Endothelial Function and Preventing Cavernous Fibrosis in Streptozotocin-Induced Diabetic Rats Author: Ke-Qin Zhang, Dong Chen, Ding-Qi Sun, Hui Zhang, Bo Li, Qiang FU PII: DOI: Reference:
S0090-4295(16)00133-3 http://dx.doi.org/doi: 10.1016/j.urology.2016.02.004 URL 19618
To appear in:
Urology
Received date: Accepted date:
9-12-2015 3-2-2016
Please cite this article as: Ke-Qin Zhang, Dong Chen, Ding-Qi Sun, Hui Zhang, Bo Li, Qiang FU, Probucol Improves Erectile Function by Restoring Endothelial Function and Preventing Cavernous Fibrosis in Streptozotocin-Induced Diabetic Rats, Urology (2016), http://dx.doi.org/doi: 10.1016/j.urology.2016.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Probucol Improves Erectile Function by Restoring Endothelial Function and Preventing Cavernous Fibrosis in Streptozotocin-Induced Diabetic Rats Ke-Qin Zhang, Dong Chen, Ding-Qi Sun, Hui Zhang, Bo Li, and Qiang FU
Ke-Qin Zhang and Dong Chen contributed equally to this article and share coauthorship.
Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwuweiqi Road, Jinan 250021, China.
Reprint requests: Qiang FU, M.D., Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwuweiqi Road, Jinan 250021, China. E-mail:[email protected]. Tel.: +86-531-68772912.Fax: +86-531-68772916.
Financial Disclosure: The authors declare that they have no relevant financial interests.
Key Words. ADMA; DDAH; Probucol; Erectile Dysfunction; Oxidative Stress; PRMT1.
1
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ABSTRACT： OBJECTIVE.
To
investigate
the
effects
of
probucol
on
erectile
function
in
streptozotocin-induced diabetic rats and explore the underlying mechanisms. METHODS. A total of thirty 12-week-old Sprague-Dawley male rats received a one-time intraperitoneal STZ (60 mg/kg) or vehicle injection after a 12-hour fast. Three days later, the STZ-induced diabetic rats were randomly divided into two groups and were treated with daily gavage feedings of probucol at doses of 0 and 500mg/kg for 12 weeks. A positive control group underwent intraperitoneal injection of saline followed by daily gavage of saline solution. Erectile function was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure measurement. After euthanasia, penile tissue was investigated using immunohistochemistry, Western blot, and ELISA to assess the PRMT1/DDAH/ADMA/NOS metabolism pathway. SOD activity and MDA levels were detected by colorimetry. We also evaluated penile histological changes such as smooth muscle contents and Masson’s trichrome stain. RESULTS. Significant recovery of erectile function was observed in the probucol-treated rats than the untreated diabetic rats. The protein expression of DDAH and NOS, cGMP concentrations and SOD activity in cavernous tissue of probucol-treated rats were significantly higher than the untreated diabetic rats. The protein expression of PRMT1, ADMA concentrations and MDA levels in cavernous tissue of probucol-treated rats were significantly lower than the untreated diabetic rats. In addition, probucol treatment markedly augments the ratio of smooth muscle cell/collagen fibril. CONCLUSION. Probucol treatment improves erectile function by restoring endothelial 2
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function and preventing cavernous fibrosis in streptozotocin-induced diabetic rats.
Erectile dysfunction (ED) is a common sexual disorder in males with diabetes mellitus (DM), which seriously affects their quality of life. ED occurs earlier (10 to 15 years)1 and shows a threefold probability in males with DM compared to those without DM2. Moreover, ED is a marker of significant increased risk of cardiovascular disease and stroke.3 Up to now, the mechanism of diabetic erectile dysfunction (DMED) is not fully understood. Current study suggests that endothelial dysfunction with reduced nitric oxide (NO) production and/or bioavailability is not only an early sign of diabetic angiopathy, but also plays an important role in the occurrence and development of DMED.4 Its main characteristic is that endothelium-dependent NO-mediated vasodilation reduces or disappears. NO is believed to be the principal neurotransmitter responsible for penile erection, which is synthetized from L-arginine by Nitric oxide Synthase (NOS). Asymmetric dimethylarginine (ADMA) is endogenous NOS inhibitor, which is synthetized by protein arginine methyltransferases
(PRMTs)
and
can
be
degraded
by
diamethylarginine
dimethylaminohydrolases (DDAH). Our previous study found that the alteration of DDAH/ADMA/NOS pathway was related with age-related ED.5 Recent studies showed that the expression of PRMT and DDAH were regulated by oxidative stress6,7, while increased formation of oxygen-derived radicals were found in DM. So it is inferred that the alernations of PRMT/DDAH/ADMA/NOS pathway caused by oxidative stress in DM may be one of the reasons of DMED. In addition to endothelial dysfunction, cavernous fibrosis is common in DMED, which may be another cause.8 At present, phosphodiesterase type 5 inhibitor (PDE5i) is considered the first-line treatment for 3
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ED. Although PDE5i has shown good results in treating ED in nondiabetic patients, its efficacy is significantly lower in diabetic ED ones.9 Many researchers are trying to find a new drug to treat DMED. Probucol as a lipid-lowering, has been widely used. Recent studies have found that it has a strong antioxidant effect10,11 and could prevent tissue fibrosis12. In this study, we attempted to give diabetic rat oral probucol in order to determine its effect on the treatment of DMED and further explore the possible mechanism.
MATERIAL AND METHODS Experimental animals All procedures were approved by the Institutional Animal Care and Use Committee at Shandong University (Jinan, China). A total of thirty 12-week-old Sprague-Dawley (SD) male rats weighing 220–240 g were purchased from the Animal Center of Shandong University. The rats were maintained in specific pathogen-free environment around 23 ± 1 °C with a 12 h light/dark cycle and supplied food and water ad libitum. All animals were adapted to the new environment for 1 week before the experiment. To establish diabetes, twenty rats were fasted for 12 hours, then received a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ， Sigma-Aldrich Chemical Co, St Louis, MO, USA). Ten rats were administered vehicle only (0.1 mol/L citrate phosphate buffer, pH 4.5) and were used as a sham group. Random Blood glucose levels were monitored 72 hours later after STZ or vehicle injection. Tail blood samples were obtained from the rats and blood glucose concentrations were measured using a blood glucometer (One Touch，Johnson & Johnson, New Jersey，USA). Only those STZ-treated rats with random blood glucose concentrations consistently greater than 16.7 mmol/L were 4
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accepted as being diabetic (18 rats in total). These rats were divided randomly into a diabetic control group that were administered daily with intragastric normal saline (DM group, n = 9) and an experimental group that was administered with intragastric probucol (500 mg/kg; Sigma-Aldrich, St Louis, MO, USA; Probucol group, n =9) daily for 12 weeks. Initial and final blood glucose levels and total body weight of all rats were recorded.
Evaluation of erectile function Measurements of the maximal intracavernous pressure (Max ICP) and the ratio of Max ICP /mean systemic arterial pressure (MAP) were used to assess erectile function. Following the induction of anesthesia with 5% sodium pentobarbital, the left carotid artery was cannulated with PE-50 tubing (Intramedic; Becton Dickinson & Co., Sparks, MD, USA) to facilitate continuous measurement of mean systemic artery pressure (MAP). Then, the major pelvic ganglion and cavernous nerve on either side of the prostate were exposed. The pressure was measured and recorded using a Windows computer program-controlled multiplying channel physiograph and analyzed using a BL-420V pressure transducer system (Chengdu Implement Company, Chengdu, China). The nerve was stimulated at a frequency of 15 Hz and using a pulse width of 5 ms. Stimulations were performed at 5 V for 60 s with resting periods of 3 min between subsequent stimulations. After assessing ICP, rats were euthanized using an overdose of pentobarbital. The middle regions of the skin-denuded penile shaft were maintained overnight in 4% paraformaldehyde and embedded in paraffin for histological analysis. The remaining penile tissues, which had been cleaned of the corpus spongiosum and dorsal vein, were snap frozen in liquid nitrogen and stored at liquid nitrogen for further processing. 5
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Measurement of SOD activity and MDA levels Approximately 20 mg of corpus cavernosum tissue was used for the experiment; the tissue was homogenized in 0.2 mL of normal saline. Subsequently, 0.05 mL of tissue homogenate was used for SOD determination and 0.1 mL of tissue homogenate was used for MDA determination. The total SOD activity was measured at 525 nm using spectrophotometric kits (Nanjing Jiancheng Biotechnology Institute, China). The MDA level was measured using spectrophotometric kits (Nanjing Jiancheng Biotechnology Institute, China).
Immunohistochemistry for α-SMA, DDAH1, DDAH2 and PRMT1 For immunohistochemistry, 3μm sections of formalin-fixed, paraffin-embedded (FFPE) tissues were deparaffinized in xylene (3 washes for 3 min each) and hydrated in graded ethanol to distilled water. After washing in phosphate-buffered saline (PBS) (3 washes for 5min each), the sections were blocked with 3% H2O2 for 20 minutes to quench endogenous peroxidase activity and with heat-induced epitope retrieval methods to perform the antigen unmasking (antigen retrieval solution: 0.01 M sodium citrate buffer, pH 6.0, 95°C). The tissue sections were incubated with primary antibody to alpha smooth muscle actin (a-SMA, rabbit polyclonal, 1:400; Abcam, Cambridge, UK), eNOS (rabbit polyclonal, 1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA), DDAH1 (goat rabbit polyclonal, 1:200; Abcam, Cambridge, UK), DDAH2 (goat rabbit polyclonal, 1:200; Abcam, Cambridge, UK), PRMT1 (rabbit polyclonal, 1:300; Abcam, Cambridge, UK), and then the sections were washed and incubated with secondary antibodies Zhong Shan Goldenbridge Biotechnology Co., Beijing, China (1:100 dilution; Zhong Shan Goldenbridge Biotechnology Co., Beijing, China).Thereafter, the sections were incubated with a 3, 3’-diaminobenzidine (DAB) and the cell nucleus was stained 6
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with hematoxylin staining. The primary antibodies were replaced with the normal serum of the host species with the secondary antibody as a negative control. Sections were examined under a light microscope. Slides were examined by an observer blinded to treatment group. α-SMA, DDAH1, DDAH2, and PRMT1 positivity was quantified by selection of four random fields of the corporal bodies. The computerized densitometric analyses of the expression of a-SMA, DDAH1, DDAH2, and PRMT1 in cavernous tissue in the images were performed by Image-Pro Plus version 5.0 software (Media Cybernetics Inc., Bethesda, MD, USA). The levels of these proteins expression were quantitated by measurement of integral optical density (IOD). The IOD was calculated for each image using the following equation: positive area × average density.
Western Blot assay for α-SMA, DDAH1, DDAH2, PRMT1, eNOS and nNOS Rat penile tissues were homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA USA) on ice for 10 min and the supernatant was collected after centrifugation at 12000 g for 15 min at 4°C. The protein concentration was assayed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA USA). Proteins were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gels, transferred to a polyvinylidene fluoride membrane (Millipore Corp, Bedford, Massachusetts) and blocked by 1 h of incubation at room temperature in TBS with 0.05% Tween 20 (TBST) plus 5% skimmed milk. The polyvinylidene fluoride membrane was then incubated overnight at 4°C with primary antibody against β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-SMA (1:1000; Abcam, Cambridge, UK), eNOS (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and nNOS (1:600, Santa Cruz Biotechnology, Santa Cruz, CA, USA), DDAH1 (1:1000; Abcam, 7
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Cambridge, UK), DDAH2 (1:500; Abcam, Cambridge, UK), PRMT1 (1:500; Abcam, Cambridge, UK). After washing in tris-buffered saline with 0.1% tween 20 (TBST) three times for 10 minutes, the membrane was incubated for 1h with the appropriate diluted horseradish peroxidase-conjugated secondary antibody. Subsequently, the membrane was washed three times again using TBST at intervals of 10 minutes and then the protein bands were detected using the enhanced chemiluminescence (ECL) system (Pierce Biotech Inc., Rockford, IL, USA). Signals were obtained in the linear range of detection and measured using a Fujifilm LAS-3000 imaging system (Fujifilm, Tokyo, Japan). Quantified by densitometry (Quantity One Analysis software; Bio-Rad) as the integrated optical density (IOD) after subtraction of background. The IOD was factored for β-actin to correct for any variations in total protein loading, and the amount of protein was represented as IOD/Std.
ELISA for ADMA and cGMP The concentration of total protein in the penile tissue was detected using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA USA). The levels of the ADMA and cGMP in penile tissue were assessed using a commercial cGMP ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) and a commercial ADMA ELISA kit (Enzo Life Sciences, Lorrach, Germany) following the protocol provided by the manufacturer.
Masson’s trichrome stain Masson’s trichrome stain was used to evaluate the smooth muscle cell/collagen fibril (SMC/CF) expression
in
cavernous
tissue.
Three-micrometer
sections
of
formalin-fixed,
paraffin-embedded (FFPE) tissues were deparaffinized in xylene (3 washes for 3 min each) and hydrated in graded ethanol to distilled water. The slides were then stained with Masson's 8
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trichrome stain kit (Dako Sciences, Glostrup, Denmark), followed by dehydration in graded ethanol to xylene. The areas of smooth muscle and collagen were analyzed using the Image Pro Plus 5.0 software package (Media Cybernetics, Inc., Bethesda, MD, USA).
Statistical analysis Data were expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed with one-way analysis of variance test with Bonferroni multiple comparison post-test. All statistical analyses were performed using the SPSS 14.0 (SPSS Inc., IL, USA) statistical software. The level of statistical significance was taken as P < .05.
RESULTS Body Weight and Blood Glucose Assessment The body weights and blood glucose levels are shown in Table. Compared with the normal control rats, the diabetic rats showed significantly higher blood glucose levels but significantly lower body weights (all P < .01). Probucol treatment did not improve these changes.
Erectile Function The Max ICP/MAP ratios are shown in Figure 1 and Table. When compared with sham rats, the Max ICP/MAP ratio in DM group rats decreased significantly (P < .01). Following the administration of probucol for 12 weeks, the Max ICP/MAP ratio in probucol group rats increased (P < .05).
SOD activity and MDA levels in the corpus cavernosum The SOD activity and MDA levels in the corpus cavernosum are shown in Table. Decreased 9
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SOD activity and increased MDA levels were found in the corpus cavernosum of DM group rats compared with shams (P < .01 for both). Following treatment with probucol, SOD activity were increased and MDA levels were decreased (P < .01 for both) compared with DM group rats.
Immunohistochemical Analysis of PRMT1, DDAH1 and DDAH2 expression The protein expressions of PRMT1, DDAH1 and DDAH2 in rat penile tissue by immunohistochemistry are demonstrated in Figure 2. Penile tissue from the DM group rats showed greater PRMT1 immunoreactivity relative to the shams (P < .01), and the probucol-treated diabetic rats had significantly lower corporal expression of PRMT1 relative to the DM group rats (P < .05). Immunohistochemical analysis revealed that the protein expression of DDAH1 and DDAH2 were significantly lower in the DM group rats compared with the shams (P < .01 for both), and 12 weeks of probucol administration increased significantly the protein immunoreactivity of DDAH1 and DDAH2 in the probucol group (P < .05 for both).
Western Blot Analysis of PRMT1, DDAH1, DDAH2, eNOS and nNOS Expression The protein expression of PRMT1, DDAH1, DDAH2, eNOS and nNOS in rat penile tissue by western blot were shown in Figure 2. Compared with the shams, the protein levels of PRMT1 were increased in the diabetic rats (P < .01). Probucol treatment significantly reduced protein levels of PRMT1 (P < .05). The protein levels of DDAH1, DDAH2, eNOS and nNOS were decreased in penile tissue of DM group rats compared to those of the shams (all P < .01). However, probucol treatment significantly elevated expression levels of DDAH1, DDAH2, 10
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eNOS and nNOS proteins (all P < .05) when compared with the diabetic rats that were not treated.
ADMA and cGMP levels in the corpus cavernosum The ADMA and cGMP levels in the corpus cavernosum are shown in Table. Increased ADMA levels and decreased cGMP levels were found in the corpus cavernosum of diabetic rats compared with shams (all P <.01). Following treatment with probucol, cGMP levels were increased and ADMA levels were decreased (P < .05 for both) in probucol group compared with DM group.
Effects of probucol treatment on the smooth muscle/collagen ratio in the cavernosum As demonstrated in Figure 3, the ratio between smooth muscle and collagen was significantly reduced in the cavernosum corpus of diabetic rats compared with the shams (P < .01). Probucol treatment significantly increased the ratio of smooth muscle to collagen (P < .05 vs. DM group). Western blot and immunohistochemical staining of α-SMA content are shown in Figure 3. The smooth muscle content decreased significantly in diabetic rats compared with shams (P < .01). Twelve weeks of probucol administration increased smooth muscle content significantly in probucol group compared with DM group (P < .05).
COMMENT The major findings of the present study are as follows: (1) the erectile function examiniation revealed the Max ICP/MAP ratio significantly decreased in DM group compared with control group, while this change was significantly attenuated by treatment with probucol. (2) DM 11
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decreased the production of SOD, DDHA1, DDAH2, eNOS, nNOS and cGMP, and enhanced the production of MDA, PRMT1 and ADMA in the corpus cavernosum tissue. Probucol had reverse effects on these changes. (3) The smooth muscle content and smooth muscle/collagen ratio significantly decreased in DM state, was significantly reduced by probucol. The STZ-induced diabetic rat has been widely used as an ED model for type I diabetes by several researches.13-15 The potential mechanisms underlying the development of ED in DM are not completely understood. Oxidative stress is thought to be a major factor contributing to the development and progression of diabetic complications. It is defined as a state in which reactive oxygen species (ROS) overproduction in vivo exceeds the buffering capacity of antioxidant enzymes and antioxidants thus resulting in a local imbalance between ROS production and destruction. Increased ROS and decreased antioxidant enzymes and antioxidants were reported in patients with DM and animal models with DM, which may be caused by hyperglycemia and metabolic abnormalities.16 In this study, we found that the SOD activtiy was significantly lower, while MDA content was simultaneously higher in DM group than control group, which was consitent with previous studies. After 12 weeks administration of probucol, significantly increased SOD activity and decreased MDA content were observed in probucol group than DM group, which indicated that probucol is helpful for alleviating the oxidative stress. What is the mechanism? Probucol has been recognized to have antioxidant properties, which can prevent oxidative injury to endothelial cells by inhibiting peroxidant generation, blocking the oxidative modification of LDL and paraoxonase-1.17 Meanwhile, probucol could increase the total antioxidant capacity such as SOD and glutathione peroxidase activity.18,19 12
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In current study, we found that PRMT1/DDAH/ADMA/NOS pathway was altered by probucol. As we know, PRMT1/DDAH/ADMA/NOS pathway is important in regulation of NO. ADMA is closely related with endothelial dysfunction, which is synthesized by PRMTs and degraded by DDAH. PRMTs consists two types, and PRMT1 is thought to be mainly involved in the generation of ADMA in vivo. There are two DDAH isoforms, DDAH1 and DDAH2. DDAH1 predominates in penile tissue expressing nNOS, and DDAH2 predominates in penile tissue expressing eNOS. Our study found that the expressions of PRMT1 are higher in DM group than control group, while lower than probucol group. On the contrary, the expression of DDAH1and DDAH2 are lower in DM group than control group, while higher in probucol group. These results indicate that probucol could influence the expression of PRMT1, DDAH1 and DDAH2. What is the mechanism? Firstly, the expression of PRMT1, DDAH1 and DDAH2 were regulated by oxidative stress.20 As above noted, probucol is an antioxidant, which can reduce the oxidative stress. Thus, probucol could decrease the expression of PRMT1 and increase the expression of DDAH1 and DDAH2 though alleviating oxidative stress in DM. Secondly, as an antioxidant, probucol can upregulate the nuclear factor erythroid 2–related factor 2 (Nrf2)21, which transcribe downstream genes including DDAH1 and DDAH2 to reduce ADMA level and PPAR-γ to increase the eNOS activity22. NO is unstable and not easily measured. Cyclic guanosine monophosphate (cGMP) is known as another vital component in penile erection, which is positively correlated with NO production. So in this study cGMP level was measured to stand for NO level. The cGMP level in our study decreased in DM group compared with control group, while this change was significantly improved by treatment with probucol. So probucol may increase the NO level to restore endothelial function 13
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via PRMT1/DDAH/ADMA/NOS axis. Our study found probucol could attenuate the corpus cavernosum fibrosis. ED is associated with loss of smooth muscle cells and an increase in fibrosis.23 Cavernous fibrosis plays a key role in the deterioration of ED, which decreases in penile elasticity and compliance. Previous study found the degree of cavernous fibrosis was significantly greater in diabetic rats.8 Our results were consistent with previous study: the smooth muscle/collagen ratio and smooth muscle content significantly decreased in DM group compared with control group. The pathological mechanisms of diabetes-related cavernous remodeling are not completely clear. Increasing evidences suggest the inflammation and oxidative stress may play important roles 16,24
. TNF-α and NF-κB are believed to be the key mediators of inducing fibrosis in DM.
Further study has suggested that oxidative stress has a major role in promoting tissue inflammation and fibrosis in DM.25 Increased expression of TNF-α, NF-κB and collagen factors was significantly inhibited by treatment of antioxidant. Our study confirmed that the smooth muscle/collagen ratio and smooth muscle content significantly increased after 12 weeks of probucol administration, which indicated that probucol may attenuate the cavernous fibrosis. What is the mechanism? Firstly, probucol could attenuate oxidative stress by decreasing the production of ROS and lipid peroxidation. Secondly, probucol has anti-inflammatory effect, which could alleviate structural remodeling by inhibiting NF-κB, TNF-α and TGF-β overexpression.11 Thirdly, probucol could prevent apoptosis in vitro and in vivo.26,27 More evidence for its optimized dosage and mechanism of effect on recovery of erectile function is needed.
14
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CONCLUSION In summary, we demonstrated that probucol restore erectile function in DMED rats. These effects appear to be mediated through attenuation of oxidative stress, regulation of PRMT1/DDAH/ADMA/NOS axis and alleviation of corpus cavernosum fibrosis.
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Angulo J, Cuevas P, Gabancho S, et al. Enhancement of both EDHF and NO/cGMP pathways is necessary to reverse erectile dysfunction in diabetic rats. J Sex Med. 2005; 2:341-346.
10．Zhu WB, Wang YH, Sun GF, et al. Protective effect and mechanism of probucol in the treatment of spinal cord injury. Genet Mol Res. 2015; 14:8029-8037. 11．Fu H, Li G, Liu C, et al. Probucol prevents atrial remodeling by inhibiting oxidative stress and TNF-alpha/NF-kappaB/TGF-beta signal transduction pathway in alloxan-induced diabetic rabbits. J Cardiovasc Electrophysiol. 2015; 26:211-222. 12．Su X, Wang Y, Zhou G, et al. Probucol attenuates ethanol-induced liver fibrosis in rats by inhibiting oxidative stress, extracellular matrix protein accumulation and cytokine production. Clin Exp Pharmacol Physiol. 2014; 41:73-80. 13．Yang Z, Zhou Z, Wang X, et al. Short hairpin ribonucleic acid constructs targeting 15
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insulin-like growth factor binding protein-3 ameliorates diabetes mellitus-related erectile dysfunction in rats. Urology. 2013; 81:464 e411-466. 14． Lei H, Xin H, Guan R, et al. Low-intensity Pulsed Ultrasound Improves Erectile Function in Streptozotocin-induced Type I Diabetic Rats. Urology. 2015; 86:1241 e1211-1248. 15．Mostafa ME, Senbel AM, Mostafa T. Effect of chronic low-dose tadalafil on penile cavernous tissues in diabetic rats. Urology. 2013; 81:1253-1259. 16．Kayama Y, Raaz U, Jagger A, et al. Diabetic Cardiovascular Disease Induced by Oxidative Stress. Int J Mol Sci. 2015; 16:25234-25263. 17．Guo YS, Wang CX, Cao J, et al. Antioxidant and lipid-regulating effects of probucol combined with atorvastatin in patients with acute coronary syndrome. J Thorac Dis. 2015; 7:368-375. 18．Gong YT, Li WM, Li Y, et al. Probucol attenuates atrial autonomic remodeling in a canine model of atrial fibrillation produced by prolonged atrial pacing. Chin Med J (Engl). 2009; 122:74-82. 19．Colle D, Santos DB, Moreira EL, et al. Probucol increases striatal glutathione peroxidase activity and protects against 3-nitropropionic acid-induced pro-oxidative damage in rats. PLoS One. 2013; 8:e67658. 20．El Assar M, Angulo J, Rodriguez-Manas L. Oxidative stress and vascular inflammation in aging. Free Radic Biol Med. 2013; 65:380-401. 21．Du Y, Zhang X, Ji H, et al. Probucol and atorvastatin in combination protect rat brains in MCAO model: upregulating Peroxiredoxin2, Foxo3a and Nrf2 expression. Neurosci Lett. 2012; 509:110-115. 22．Luo Z, Aslam S, Welch WJ, et al. Activation of nuclear factor erythroid 2-related factor 2 coordinates dimethylarginine dimethylaminohydrolase/PPAR-gamma/endothelial nitric oxide synthase pathways that enhance nitric oxide generation in human glomerular endothelial cells. Hypertension. 2015; 65:896-902. 23．El-Sakka AI, Yassin AA. Amelioration of penile fibrosis: myth or reality. J Androl. 2010; 31:324-335. 24．Duerrschmid C, Crawford JR, Reineke E, et al. TNF receptor 1 signaling is critically involved in mediating angiotensin-II-induced cardiac fibrosis. J Mol Cell Cardiol. 2013; 57:59-67. 25 ．Suzuki H, Kayama Y, Sakamoto M, et al. Arachidonate 12/15-lipoxygenase-induced inflammation and oxidative stress are involved in the development of diabetic cardiomyopathy. Diabetes. 2015; 64:618-630. 26.
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Figure 1 Erectile function evaluation. The ratio of maximal intracavernous pressure (Max ICP) and mean systemic arterial pressure (MAP) was shown to evaluate erectile function in each group. Representative ICP in response to electrical stimulation of the cavernous nerve at 5 V in (A) Sham group, (B) DM group (C) Probucol group. (D) Statistical chart of Max ICP/MAP ratio. Each bar depicted the mean values (± standard deviation) from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 vs. DM group.
Figure 2 Evaluation of DDAH1, DDAH2, PRMT1, eNOS and nNOS protein expression by immunohistochemistry and Western Blot. Representative immunohistochemistry of (A) DDAH1, (B) DDAH2 and (C) PRMT1 in corpus cavernosum among groups. Representative Western Blot analysis of (D) DDAH1, (E) DDAH2, (F) PRMT1, (G) eNOS and (H) nNOS in corpus cavernosum among groups. β-actin was used as a loading control. Each bar depicted the mean values (± standard deviation) from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 and ## P <.01 vs. DM group. Scale bar = 200 μm.
Figure 3 Masson’s trichrome stain and evaluation of α-SMA protein expression by immunohistochemistry and Western Blot. Masson’s trichrome stain was used to evaluate the cavernous tissue via smooth muscle cell (blue) /collagen fibril (red) (SMC/CF). (A) Masson’s trichrome staining in corporal tissue in different groups (original magnification x100). (B) Immunohistochemical staining of α-SMA protein in different groups (original magnification x100). Statistical chart of α-SMA integrated optical density among groups. (C) Western Blot analysis of α-SMA protein in different groups. Statistical chart of α-SMA relative to β-actin. 17
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Each bar depicted the mean values (± standard deviation) from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 vs. DM group.
Table Comparisons of body weight, blood glucose ADMA content, cGMP content, SOD activity and MDA content in experimental animals Sham DM Probucol Intial BW(g) 230.86 ± 6.43 BG (mmol/L) 5.87 ± 0.16 After 12 weeks BW(g) 571.41 ± 20.33 BG (mmol/L) 5.89 ± 0.11 Max ICP (mmHg) 90.87 ± 4.71 MAP (mmHg) 112.03 ± 3.25 Max ICP/ MAP 0.81 ± 0.05 -1 ADMA content (nmol mgpro ) 7.32 ± 0.10 -1 cGMP content (pmol mgpro ) 1.21 ± 0.06
227.29 ± 4.91 5.90 ± 0.12 202.87 ± 8.55 ** 28.54 ± 2.73 ** 53.16 ± 2.59 100.65 ± 6.73 0.44 ± 0.02** 12.58 ± 0.14** 0.57 ± 0.08**
233.12 ± 5.67 5.88 ± 0.15. 242.73 ± 11.69 ** 26.17 ± 1.53 ** 70.54 ± 2.12 110.34 ± 4.40 0.61 ± 0.03** , # 10.63 ± 0.17**, # 0.78 ± 0.03**,#
SOD activity (U mgpro-1 ) 158.36 ± 6.84 64.35 ± 9.77** 92.71 ± 10.41**, ## MDA content (nmol mgpro-1 ) 2.14 ± 0.18 6.59 ± 0.26** 4.95 ± 0.22**, ## Data are expressed as mean ± SD from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 and ## P < .01 vs. DM group. BW = body weight; BG = blood glucose.
18
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S0090-4295(16)00133-3 http://dx.doi.org/doi: 10.1016/j.urology.2016.02.004 URL 19618
To appear in:
Urology
Received date: Accepted date:
9-12-2015 3-2-2016
Please cite this article as: Ke-Qin Zhang, Dong Chen, Ding-Qi Sun, Hui Zhang, Bo Li, Qiang FU, Probucol Improves Erectile Function by Restoring Endothelial Function and Preventing Cavernous Fibrosis in Streptozotocin-Induced Diabetic Rats, Urology (2016), http://dx.doi.org/doi: 10.1016/j.urology.2016.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Probucol Improves Erectile Function by Restoring Endothelial Function and Preventing Cavernous Fibrosis in Streptozotocin-Induced Diabetic Rats Ke-Qin Zhang, Dong Chen, Ding-Qi Sun, Hui Zhang, Bo Li, and Qiang FU
Ke-Qin Zhang and Dong Chen contributed equally to this article and share coauthorship.
Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwuweiqi Road, Jinan 250021, China.
Reprint requests: Qiang FU, M.D., Department of Urology, Shandong Provincial Hospital Affiliated to Shandong University, 324 Jingwuweiqi Road, Jinan 250021, China. E-mail:[email protected]. Tel.: +86-531-68772912.Fax: +86-531-68772916.
Financial Disclosure: The authors declare that they have no relevant financial interests.
Key Words. ADMA; DDAH; Probucol; Erectile Dysfunction; Oxidative Stress; PRMT1.
1
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ABSTRACT： OBJECTIVE.
To
investigate
the
effects
of
probucol
on
erectile
function
in
streptozotocin-induced diabetic rats and explore the underlying mechanisms. METHODS. A total of thirty 12-week-old Sprague-Dawley male rats received a one-time intraperitoneal STZ (60 mg/kg) or vehicle injection after a 12-hour fast. Three days later, the STZ-induced diabetic rats were randomly divided into two groups and were treated with daily gavage feedings of probucol at doses of 0 and 500mg/kg for 12 weeks. A positive control group underwent intraperitoneal injection of saline followed by daily gavage of saline solution. Erectile function was assessed by electrical stimulation of the cavernous nerves with real-time intracavernous pressure measurement. After euthanasia, penile tissue was investigated using immunohistochemistry, Western blot, and ELISA to assess the PRMT1/DDAH/ADMA/NOS metabolism pathway. SOD activity and MDA levels were detected by colorimetry. We also evaluated penile histological changes such as smooth muscle contents and Masson’s trichrome stain. RESULTS. Significant recovery of erectile function was observed in the probucol-treated rats than the untreated diabetic rats. The protein expression of DDAH and NOS, cGMP concentrations and SOD activity in cavernous tissue of probucol-treated rats were significantly higher than the untreated diabetic rats. The protein expression of PRMT1, ADMA concentrations and MDA levels in cavernous tissue of probucol-treated rats were significantly lower than the untreated diabetic rats. In addition, probucol treatment markedly augments the ratio of smooth muscle cell/collagen fibril. CONCLUSION. Probucol treatment improves erectile function by restoring endothelial 2
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function and preventing cavernous fibrosis in streptozotocin-induced diabetic rats.
Erectile dysfunction (ED) is a common sexual disorder in males with diabetes mellitus (DM), which seriously affects their quality of life. ED occurs earlier (10 to 15 years)1 and shows a threefold probability in males with DM compared to those without DM2. Moreover, ED is a marker of significant increased risk of cardiovascular disease and stroke.3 Up to now, the mechanism of diabetic erectile dysfunction (DMED) is not fully understood. Current study suggests that endothelial dysfunction with reduced nitric oxide (NO) production and/or bioavailability is not only an early sign of diabetic angiopathy, but also plays an important role in the occurrence and development of DMED.4 Its main characteristic is that endothelium-dependent NO-mediated vasodilation reduces or disappears. NO is believed to be the principal neurotransmitter responsible for penile erection, which is synthetized from L-arginine by Nitric oxide Synthase (NOS). Asymmetric dimethylarginine (ADMA) is endogenous NOS inhibitor, which is synthetized by protein arginine methyltransferases
(PRMTs)
and
can
be
degraded
by
diamethylarginine
dimethylaminohydrolases (DDAH). Our previous study found that the alteration of DDAH/ADMA/NOS pathway was related with age-related ED.5 Recent studies showed that the expression of PRMT and DDAH were regulated by oxidative stress6,7, while increased formation of oxygen-derived radicals were found in DM. So it is inferred that the alernations of PRMT/DDAH/ADMA/NOS pathway caused by oxidative stress in DM may be one of the reasons of DMED. In addition to endothelial dysfunction, cavernous fibrosis is common in DMED, which may be another cause.8 At present, phosphodiesterase type 5 inhibitor (PDE5i) is considered the first-line treatment for 3
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ED. Although PDE5i has shown good results in treating ED in nondiabetic patients, its efficacy is significantly lower in diabetic ED ones.9 Many researchers are trying to find a new drug to treat DMED. Probucol as a lipid-lowering, has been widely used. Recent studies have found that it has a strong antioxidant effect10,11 and could prevent tissue fibrosis12. In this study, we attempted to give diabetic rat oral probucol in order to determine its effect on the treatment of DMED and further explore the possible mechanism.
MATERIAL AND METHODS Experimental animals All procedures were approved by the Institutional Animal Care and Use Committee at Shandong University (Jinan, China). A total of thirty 12-week-old Sprague-Dawley (SD) male rats weighing 220–240 g were purchased from the Animal Center of Shandong University. The rats were maintained in specific pathogen-free environment around 23 ± 1 °C with a 12 h light/dark cycle and supplied food and water ad libitum. All animals were adapted to the new environment for 1 week before the experiment. To establish diabetes, twenty rats were fasted for 12 hours, then received a single intraperitoneal injection of 60 mg/kg streptozotocin (STZ， Sigma-Aldrich Chemical Co, St Louis, MO, USA). Ten rats were administered vehicle only (0.1 mol/L citrate phosphate buffer, pH 4.5) and were used as a sham group. Random Blood glucose levels were monitored 72 hours later after STZ or vehicle injection. Tail blood samples were obtained from the rats and blood glucose concentrations were measured using a blood glucometer (One Touch，Johnson & Johnson, New Jersey，USA). Only those STZ-treated rats with random blood glucose concentrations consistently greater than 16.7 mmol/L were 4
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accepted as being diabetic (18 rats in total). These rats were divided randomly into a diabetic control group that were administered daily with intragastric normal saline (DM group, n = 9) and an experimental group that was administered with intragastric probucol (500 mg/kg; Sigma-Aldrich, St Louis, MO, USA; Probucol group, n =9) daily for 12 weeks. Initial and final blood glucose levels and total body weight of all rats were recorded.
Evaluation of erectile function Measurements of the maximal intracavernous pressure (Max ICP) and the ratio of Max ICP /mean systemic arterial pressure (MAP) were used to assess erectile function. Following the induction of anesthesia with 5% sodium pentobarbital, the left carotid artery was cannulated with PE-50 tubing (Intramedic; Becton Dickinson & Co., Sparks, MD, USA) to facilitate continuous measurement of mean systemic artery pressure (MAP). Then, the major pelvic ganglion and cavernous nerve on either side of the prostate were exposed. The pressure was measured and recorded using a Windows computer program-controlled multiplying channel physiograph and analyzed using a BL-420V pressure transducer system (Chengdu Implement Company, Chengdu, China). The nerve was stimulated at a frequency of 15 Hz and using a pulse width of 5 ms. Stimulations were performed at 5 V for 60 s with resting periods of 3 min between subsequent stimulations. After assessing ICP, rats were euthanized using an overdose of pentobarbital. The middle regions of the skin-denuded penile shaft were maintained overnight in 4% paraformaldehyde and embedded in paraffin for histological analysis. The remaining penile tissues, which had been cleaned of the corpus spongiosum and dorsal vein, were snap frozen in liquid nitrogen and stored at liquid nitrogen for further processing. 5
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Measurement of SOD activity and MDA levels Approximately 20 mg of corpus cavernosum tissue was used for the experiment; the tissue was homogenized in 0.2 mL of normal saline. Subsequently, 0.05 mL of tissue homogenate was used for SOD determination and 0.1 mL of tissue homogenate was used for MDA determination. The total SOD activity was measured at 525 nm using spectrophotometric kits (Nanjing Jiancheng Biotechnology Institute, China). The MDA level was measured using spectrophotometric kits (Nanjing Jiancheng Biotechnology Institute, China).
Immunohistochemistry for α-SMA, DDAH1, DDAH2 and PRMT1 For immunohistochemistry, 3μm sections of formalin-fixed, paraffin-embedded (FFPE) tissues were deparaffinized in xylene (3 washes for 3 min each) and hydrated in graded ethanol to distilled water. After washing in phosphate-buffered saline (PBS) (3 washes for 5min each), the sections were blocked with 3% H2O2 for 20 minutes to quench endogenous peroxidase activity and with heat-induced epitope retrieval methods to perform the antigen unmasking (antigen retrieval solution: 0.01 M sodium citrate buffer, pH 6.0, 95°C). The tissue sections were incubated with primary antibody to alpha smooth muscle actin (a-SMA, rabbit polyclonal, 1:400; Abcam, Cambridge, UK), eNOS (rabbit polyclonal, 1:300; Santa Cruz Biotechnology, Santa Cruz, CA, USA), DDAH1 (goat rabbit polyclonal, 1:200; Abcam, Cambridge, UK), DDAH2 (goat rabbit polyclonal, 1:200; Abcam, Cambridge, UK), PRMT1 (rabbit polyclonal, 1:300; Abcam, Cambridge, UK), and then the sections were washed and incubated with secondary antibodies Zhong Shan Goldenbridge Biotechnology Co., Beijing, China (1:100 dilution; Zhong Shan Goldenbridge Biotechnology Co., Beijing, China).Thereafter, the sections were incubated with a 3, 3’-diaminobenzidine (DAB) and the cell nucleus was stained 6
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with hematoxylin staining. The primary antibodies were replaced with the normal serum of the host species with the secondary antibody as a negative control. Sections were examined under a light microscope. Slides were examined by an observer blinded to treatment group. α-SMA, DDAH1, DDAH2, and PRMT1 positivity was quantified by selection of four random fields of the corporal bodies. The computerized densitometric analyses of the expression of a-SMA, DDAH1, DDAH2, and PRMT1 in cavernous tissue in the images were performed by Image-Pro Plus version 5.0 software (Media Cybernetics Inc., Bethesda, MD, USA). The levels of these proteins expression were quantitated by measurement of integral optical density (IOD). The IOD was calculated for each image using the following equation: positive area × average density.
Western Blot assay for α-SMA, DDAH1, DDAH2, PRMT1, eNOS and nNOS Rat penile tissues were homogenized in RIPA lysis buffer (Thermo Fisher Scientific, Waltham, MA USA) on ice for 10 min and the supernatant was collected after centrifugation at 12000 g for 15 min at 4°C. The protein concentration was assayed using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA USA). Proteins were electrophoresed on a 10% sodium dodecyl sulfate-polyacrylamide gels, transferred to a polyvinylidene fluoride membrane (Millipore Corp, Bedford, Massachusetts) and blocked by 1 h of incubation at room temperature in TBS with 0.05% Tween 20 (TBST) plus 5% skimmed milk. The polyvinylidene fluoride membrane was then incubated overnight at 4°C with primary antibody against β-actin (1:1000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), α-SMA (1:1000; Abcam, Cambridge, UK), eNOS (1:500; Santa Cruz Biotechnology, Santa Cruz, CA, USA), and nNOS (1:600, Santa Cruz Biotechnology, Santa Cruz, CA, USA), DDAH1 (1:1000; Abcam, 7
Page 7 of 18
Cambridge, UK), DDAH2 (1:500; Abcam, Cambridge, UK), PRMT1 (1:500; Abcam, Cambridge, UK). After washing in tris-buffered saline with 0.1% tween 20 (TBST) three times for 10 minutes, the membrane was incubated for 1h with the appropriate diluted horseradish peroxidase-conjugated secondary antibody. Subsequently, the membrane was washed three times again using TBST at intervals of 10 minutes and then the protein bands were detected using the enhanced chemiluminescence (ECL) system (Pierce Biotech Inc., Rockford, IL, USA). Signals were obtained in the linear range of detection and measured using a Fujifilm LAS-3000 imaging system (Fujifilm, Tokyo, Japan). Quantified by densitometry (Quantity One Analysis software; Bio-Rad) as the integrated optical density (IOD) after subtraction of background. The IOD was factored for β-actin to correct for any variations in total protein loading, and the amount of protein was represented as IOD/Std.
ELISA for ADMA and cGMP The concentration of total protein in the penile tissue was detected using the Pierce BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA USA). The levels of the ADMA and cGMP in penile tissue were assessed using a commercial cGMP ELISA kit (R&D Systems, Inc., Minneapolis, MN, USA) and a commercial ADMA ELISA kit (Enzo Life Sciences, Lorrach, Germany) following the protocol provided by the manufacturer.
Masson’s trichrome stain Masson’s trichrome stain was used to evaluate the smooth muscle cell/collagen fibril (SMC/CF) expression
in
cavernous
tissue.
Three-micrometer
sections
of
formalin-fixed,
paraffin-embedded (FFPE) tissues were deparaffinized in xylene (3 washes for 3 min each) and hydrated in graded ethanol to distilled water. The slides were then stained with Masson's 8
Page 8 of 18
trichrome stain kit (Dako Sciences, Glostrup, Denmark), followed by dehydration in graded ethanol to xylene. The areas of smooth muscle and collagen were analyzed using the Image Pro Plus 5.0 software package (Media Cybernetics, Inc., Bethesda, MD, USA).
Statistical analysis Data were expressed as mean ± standard deviation (mean ± SD). Statistical analysis was performed with one-way analysis of variance test with Bonferroni multiple comparison post-test. All statistical analyses were performed using the SPSS 14.0 (SPSS Inc., IL, USA) statistical software. The level of statistical significance was taken as P < .05.
RESULTS Body Weight and Blood Glucose Assessment The body weights and blood glucose levels are shown in Table. Compared with the normal control rats, the diabetic rats showed significantly higher blood glucose levels but significantly lower body weights (all P < .01). Probucol treatment did not improve these changes.
Erectile Function The Max ICP/MAP ratios are shown in Figure 1 and Table. When compared with sham rats, the Max ICP/MAP ratio in DM group rats decreased significantly (P < .01). Following the administration of probucol for 12 weeks, the Max ICP/MAP ratio in probucol group rats increased (P < .05).
SOD activity and MDA levels in the corpus cavernosum The SOD activity and MDA levels in the corpus cavernosum are shown in Table. Decreased 9
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SOD activity and increased MDA levels were found in the corpus cavernosum of DM group rats compared with shams (P < .01 for both). Following treatment with probucol, SOD activity were increased and MDA levels were decreased (P < .01 for both) compared with DM group rats.
Immunohistochemical Analysis of PRMT1, DDAH1 and DDAH2 expression The protein expressions of PRMT1, DDAH1 and DDAH2 in rat penile tissue by immunohistochemistry are demonstrated in Figure 2. Penile tissue from the DM group rats showed greater PRMT1 immunoreactivity relative to the shams (P < .01), and the probucol-treated diabetic rats had significantly lower corporal expression of PRMT1 relative to the DM group rats (P < .05). Immunohistochemical analysis revealed that the protein expression of DDAH1 and DDAH2 were significantly lower in the DM group rats compared with the shams (P < .01 for both), and 12 weeks of probucol administration increased significantly the protein immunoreactivity of DDAH1 and DDAH2 in the probucol group (P < .05 for both).
Western Blot Analysis of PRMT1, DDAH1, DDAH2, eNOS and nNOS Expression The protein expression of PRMT1, DDAH1, DDAH2, eNOS and nNOS in rat penile tissue by western blot were shown in Figure 2. Compared with the shams, the protein levels of PRMT1 were increased in the diabetic rats (P < .01). Probucol treatment significantly reduced protein levels of PRMT1 (P < .05). The protein levels of DDAH1, DDAH2, eNOS and nNOS were decreased in penile tissue of DM group rats compared to those of the shams (all P < .01). However, probucol treatment significantly elevated expression levels of DDAH1, DDAH2, 10
Page 10 of 18
eNOS and nNOS proteins (all P < .05) when compared with the diabetic rats that were not treated.
ADMA and cGMP levels in the corpus cavernosum The ADMA and cGMP levels in the corpus cavernosum are shown in Table. Increased ADMA levels and decreased cGMP levels were found in the corpus cavernosum of diabetic rats compared with shams (all P <.01). Following treatment with probucol, cGMP levels were increased and ADMA levels were decreased (P < .05 for both) in probucol group compared with DM group.
Effects of probucol treatment on the smooth muscle/collagen ratio in the cavernosum As demonstrated in Figure 3, the ratio between smooth muscle and collagen was significantly reduced in the cavernosum corpus of diabetic rats compared with the shams (P < .01). Probucol treatment significantly increased the ratio of smooth muscle to collagen (P < .05 vs. DM group). Western blot and immunohistochemical staining of α-SMA content are shown in Figure 3. The smooth muscle content decreased significantly in diabetic rats compared with shams (P < .01). Twelve weeks of probucol administration increased smooth muscle content significantly in probucol group compared with DM group (P < .05).
COMMENT The major findings of the present study are as follows: (1) the erectile function examiniation revealed the Max ICP/MAP ratio significantly decreased in DM group compared with control group, while this change was significantly attenuated by treatment with probucol. (2) DM 11
Page 11 of 18
decreased the production of SOD, DDHA1, DDAH2, eNOS, nNOS and cGMP, and enhanced the production of MDA, PRMT1 and ADMA in the corpus cavernosum tissue. Probucol had reverse effects on these changes. (3) The smooth muscle content and smooth muscle/collagen ratio significantly decreased in DM state, was significantly reduced by probucol. The STZ-induced diabetic rat has been widely used as an ED model for type I diabetes by several researches.13-15 The potential mechanisms underlying the development of ED in DM are not completely understood. Oxidative stress is thought to be a major factor contributing to the development and progression of diabetic complications. It is defined as a state in which reactive oxygen species (ROS) overproduction in vivo exceeds the buffering capacity of antioxidant enzymes and antioxidants thus resulting in a local imbalance between ROS production and destruction. Increased ROS and decreased antioxidant enzymes and antioxidants were reported in patients with DM and animal models with DM, which may be caused by hyperglycemia and metabolic abnormalities.16 In this study, we found that the SOD activtiy was significantly lower, while MDA content was simultaneously higher in DM group than control group, which was consitent with previous studies. After 12 weeks administration of probucol, significantly increased SOD activity and decreased MDA content were observed in probucol group than DM group, which indicated that probucol is helpful for alleviating the oxidative stress. What is the mechanism? Probucol has been recognized to have antioxidant properties, which can prevent oxidative injury to endothelial cells by inhibiting peroxidant generation, blocking the oxidative modification of LDL and paraoxonase-1.17 Meanwhile, probucol could increase the total antioxidant capacity such as SOD and glutathione peroxidase activity.18,19 12
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In current study, we found that PRMT1/DDAH/ADMA/NOS pathway was altered by probucol. As we know, PRMT1/DDAH/ADMA/NOS pathway is important in regulation of NO. ADMA is closely related with endothelial dysfunction, which is synthesized by PRMTs and degraded by DDAH. PRMTs consists two types, and PRMT1 is thought to be mainly involved in the generation of ADMA in vivo. There are two DDAH isoforms, DDAH1 and DDAH2. DDAH1 predominates in penile tissue expressing nNOS, and DDAH2 predominates in penile tissue expressing eNOS. Our study found that the expressions of PRMT1 are higher in DM group than control group, while lower than probucol group. On the contrary, the expression of DDAH1and DDAH2 are lower in DM group than control group, while higher in probucol group. These results indicate that probucol could influence the expression of PRMT1, DDAH1 and DDAH2. What is the mechanism? Firstly, the expression of PRMT1, DDAH1 and DDAH2 were regulated by oxidative stress.20 As above noted, probucol is an antioxidant, which can reduce the oxidative stress. Thus, probucol could decrease the expression of PRMT1 and increase the expression of DDAH1 and DDAH2 though alleviating oxidative stress in DM. Secondly, as an antioxidant, probucol can upregulate the nuclear factor erythroid 2–related factor 2 (Nrf2)21, which transcribe downstream genes including DDAH1 and DDAH2 to reduce ADMA level and PPAR-γ to increase the eNOS activity22. NO is unstable and not easily measured. Cyclic guanosine monophosphate (cGMP) is known as another vital component in penile erection, which is positively correlated with NO production. So in this study cGMP level was measured to stand for NO level. The cGMP level in our study decreased in DM group compared with control group, while this change was significantly improved by treatment with probucol. So probucol may increase the NO level to restore endothelial function 13
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via PRMT1/DDAH/ADMA/NOS axis. Our study found probucol could attenuate the corpus cavernosum fibrosis. ED is associated with loss of smooth muscle cells and an increase in fibrosis.23 Cavernous fibrosis plays a key role in the deterioration of ED, which decreases in penile elasticity and compliance. Previous study found the degree of cavernous fibrosis was significantly greater in diabetic rats.8 Our results were consistent with previous study: the smooth muscle/collagen ratio and smooth muscle content significantly decreased in DM group compared with control group. The pathological mechanisms of diabetes-related cavernous remodeling are not completely clear. Increasing evidences suggest the inflammation and oxidative stress may play important roles 16,24
. TNF-α and NF-κB are believed to be the key mediators of inducing fibrosis in DM.
Further study has suggested that oxidative stress has a major role in promoting tissue inflammation and fibrosis in DM.25 Increased expression of TNF-α, NF-κB and collagen factors was significantly inhibited by treatment of antioxidant. Our study confirmed that the smooth muscle/collagen ratio and smooth muscle content significantly increased after 12 weeks of probucol administration, which indicated that probucol may attenuate the cavernous fibrosis. What is the mechanism? Firstly, probucol could attenuate oxidative stress by decreasing the production of ROS and lipid peroxidation. Secondly, probucol has anti-inflammatory effect, which could alleviate structural remodeling by inhibiting NF-κB, TNF-α and TGF-β overexpression.11 Thirdly, probucol could prevent apoptosis in vitro and in vivo.26,27 More evidence for its optimized dosage and mechanism of effect on recovery of erectile function is needed.
14
Page 14 of 18
CONCLUSION In summary, we demonstrated that probucol restore erectile function in DMED rats. These effects appear to be mediated through attenuation of oxidative stress, regulation of PRMT1/DDAH/ADMA/NOS axis and alleviation of corpus cavernosum fibrosis.
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Angulo J, Cuevas P, Gabancho S, et al. Enhancement of both EDHF and NO/cGMP pathways is necessary to reverse erectile dysfunction in diabetic rats. J Sex Med. 2005; 2:341-346.
10．Zhu WB, Wang YH, Sun GF, et al. Protective effect and mechanism of probucol in the treatment of spinal cord injury. Genet Mol Res. 2015; 14:8029-8037. 11．Fu H, Li G, Liu C, et al. Probucol prevents atrial remodeling by inhibiting oxidative stress and TNF-alpha/NF-kappaB/TGF-beta signal transduction pathway in alloxan-induced diabetic rabbits. J Cardiovasc Electrophysiol. 2015; 26:211-222. 12．Su X, Wang Y, Zhou G, et al. Probucol attenuates ethanol-induced liver fibrosis in rats by inhibiting oxidative stress, extracellular matrix protein accumulation and cytokine production. Clin Exp Pharmacol Physiol. 2014; 41:73-80. 13．Yang Z, Zhou Z, Wang X, et al. Short hairpin ribonucleic acid constructs targeting 15
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insulin-like growth factor binding protein-3 ameliorates diabetes mellitus-related erectile dysfunction in rats. Urology. 2013; 81:464 e411-466. 14． Lei H, Xin H, Guan R, et al. Low-intensity Pulsed Ultrasound Improves Erectile Function in Streptozotocin-induced Type I Diabetic Rats. Urology. 2015; 86:1241 e1211-1248. 15．Mostafa ME, Senbel AM, Mostafa T. Effect of chronic low-dose tadalafil on penile cavernous tissues in diabetic rats. Urology. 2013; 81:1253-1259. 16．Kayama Y, Raaz U, Jagger A, et al. Diabetic Cardiovascular Disease Induced by Oxidative Stress. Int J Mol Sci. 2015; 16:25234-25263. 17．Guo YS, Wang CX, Cao J, et al. Antioxidant and lipid-regulating effects of probucol combined with atorvastatin in patients with acute coronary syndrome. J Thorac Dis. 2015; 7:368-375. 18．Gong YT, Li WM, Li Y, et al. Probucol attenuates atrial autonomic remodeling in a canine model of atrial fibrillation produced by prolonged atrial pacing. Chin Med J (Engl). 2009; 122:74-82. 19．Colle D, Santos DB, Moreira EL, et al. Probucol increases striatal glutathione peroxidase activity and protects against 3-nitropropionic acid-induced pro-oxidative damage in rats. PLoS One. 2013; 8:e67658. 20．El Assar M, Angulo J, Rodriguez-Manas L. Oxidative stress and vascular inflammation in aging. Free Radic Biol Med. 2013; 65:380-401. 21．Du Y, Zhang X, Ji H, et al. Probucol and atorvastatin in combination protect rat brains in MCAO model: upregulating Peroxiredoxin2, Foxo3a and Nrf2 expression. Neurosci Lett. 2012; 509:110-115. 22．Luo Z, Aslam S, Welch WJ, et al. Activation of nuclear factor erythroid 2-related factor 2 coordinates dimethylarginine dimethylaminohydrolase/PPAR-gamma/endothelial nitric oxide synthase pathways that enhance nitric oxide generation in human glomerular endothelial cells. Hypertension. 2015; 65:896-902. 23．El-Sakka AI, Yassin AA. Amelioration of penile fibrosis: myth or reality. J Androl. 2010; 31:324-335. 24．Duerrschmid C, Crawford JR, Reineke E, et al. TNF receptor 1 signaling is critically involved in mediating angiotensin-II-induced cardiac fibrosis. J Mol Cell Cardiol. 2013; 57:59-67. 25 ．Suzuki H, Kayama Y, Sakamoto M, et al. Arachidonate 12/15-lipoxygenase-induced inflammation and oxidative stress are involved in the development of diabetic cardiomyopathy. Diabetes. 2015; 64:618-630. 26.
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Figure 1 Erectile function evaluation. The ratio of maximal intracavernous pressure (Max ICP) and mean systemic arterial pressure (MAP) was shown to evaluate erectile function in each group. Representative ICP in response to electrical stimulation of the cavernous nerve at 5 V in (A) Sham group, (B) DM group (C) Probucol group. (D) Statistical chart of Max ICP/MAP ratio. Each bar depicted the mean values (± standard deviation) from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 vs. DM group.
Figure 2 Evaluation of DDAH1, DDAH2, PRMT1, eNOS and nNOS protein expression by immunohistochemistry and Western Blot. Representative immunohistochemistry of (A) DDAH1, (B) DDAH2 and (C) PRMT1 in corpus cavernosum among groups. Representative Western Blot analysis of (D) DDAH1, (E) DDAH2, (F) PRMT1, (G) eNOS and (H) nNOS in corpus cavernosum among groups. β-actin was used as a loading control. Each bar depicted the mean values (± standard deviation) from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 and ## P <.01 vs. DM group. Scale bar = 200 μm.
Figure 3 Masson’s trichrome stain and evaluation of α-SMA protein expression by immunohistochemistry and Western Blot. Masson’s trichrome stain was used to evaluate the cavernous tissue via smooth muscle cell (blue) /collagen fibril (red) (SMC/CF). (A) Masson’s trichrome staining in corporal tissue in different groups (original magnification x100). (B) Immunohistochemical staining of α-SMA protein in different groups (original magnification x100). Statistical chart of α-SMA integrated optical density among groups. (C) Western Blot analysis of α-SMA protein in different groups. Statistical chart of α-SMA relative to β-actin. 17
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Each bar depicted the mean values (± standard deviation) from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 vs. DM group.
Table Comparisons of body weight, blood glucose ADMA content, cGMP content, SOD activity and MDA content in experimental animals Sham DM Probucol Intial BW(g) 230.86 ± 6.43 BG (mmol/L) 5.87 ± 0.16 After 12 weeks BW(g) 571.41 ± 20.33 BG (mmol/L) 5.89 ± 0.11 Max ICP (mmHg) 90.87 ± 4.71 MAP (mmHg) 112.03 ± 3.25 Max ICP/ MAP 0.81 ± 0.05 -1 ADMA content (nmol mgpro ) 7.32 ± 0.10 -1 cGMP content (pmol mgpro ) 1.21 ± 0.06
227.29 ± 4.91 5.90 ± 0.12 202.87 ± 8.55 ** 28.54 ± 2.73 ** 53.16 ± 2.59 100.65 ± 6.73 0.44 ± 0.02** 12.58 ± 0.14** 0.57 ± 0.08**
233.12 ± 5.67 5.88 ± 0.15. 242.73 ± 11.69 ** 26.17 ± 1.53 ** 70.54 ± 2.12 110.34 ± 4.40 0.61 ± 0.03** , # 10.63 ± 0.17**, # 0.78 ± 0.03**,#
SOD activity (U mgpro-1 ) 158.36 ± 6.84 64.35 ± 9.77** 92.71 ± 10.41**, ## MDA content (nmol mgpro-1 ) 2.14 ± 0.18 6.59 ± 0.26** 4.95 ± 0.22**, ## Data are expressed as mean ± SD from N = 9 per group. ** P < .01 vs. Sham group. # P < .05 and ## P < .01 vs. DM group. BW = body weight; BG = blood glucose.
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