Background
Benign prostatic hyperplasia (BPH) is the most common benign tumor in men. Epidemiological data have indicated that BPH may be associated with the metabolic syndrome (MetS) [
1] which can substantially increase the risk of BPH and low urinary tract symptoms (LUTS) [
2].
When complicated with diabetes mellitus (DM), the mechanisms that regulate reactive stroma biology in BPH can be altered anatomically, pathologically, and biochemically [
3]. Prostatic volume and the anterior-posterior diameter are positively associated with the component number of MetS [
1]. The possible pathophysiologic mechanisms needed to explain these relations include an increased sympathetic tone, the alterations in sex steroid hormone expression, and the induction of systemic inflammation and oxidative stress [
4]. The levels of insulin-like growth factor (IGF) and IGF-binding proteins (IGFBPs) in prostate tissue and blood are associated with the risk of developing BPH, which also regulate the circulating androgen and growth hormones [
2].
Regular exercise (EX) is associated with low levels of interleukin-6 (IL-6), tumor necrosis factor-α (TNF-
α), and simultaneously, with increases in antiinflammatory substances, such as adiponectin, IL-4, and IL-10 [
5]. Hence, moderate EX training can exert antioxidant and antiinflammatory systemic protective effects [
5]. Much of the literature supports a clinically relevant, independent, and strong inverse relationship between EX and the development of BPH and LUTS [
6‐
8]. Furthermore, running considerable distances per week may lower the BPH risk, independent of the BMI and diet [
9].
EX has been shown to have beneficially improved the Type 2 DM (T2DM) [
10,
11] and BPH [
9]. Moreover, amounting evidences also have revealed that a close association between BPH and T2DM through a common pathogenic mechanism is possible [
12]. Parsons et al. in a chort report indicated that obesity, elevated fasting plasma glucose level, and DM are risk factors for BPH [
13]. Previous document even substantially pointed out that diabetic vascular damage may cause hypoxia which in turn may contribute to pathogenesis of BPH [
14]. Recently, we have showed EX beneficially alleviated BPH [
7], however, the documented effect of EX on patients concomitantly affiliated with BPH plus Type 1 DM (BPH + T1DM) is still lacking. We hypothesized that EX could be beneficial to BHP + T1DM subjects. In this present study we developed a BPH + DM rat model to verify whether EX could improve such a metabolic syndrome.
Methods
Chemicals
T-Pro Western Blot Stripping Reagent was obtained from BioPioneer (San Diego, CA, USA); streptozotocin (STZ), Sirius Red, bovine serum albumin (BSA), and sodium dodecyl sulfate-polyacrylamide were purchased from Sigma-Aldrich (St. Louis, MO, USA); Coomassie Brillant Blue R-250, Coomassie Brilliant Blue-G, glycine, and Tris base were obtained from US Biological (USA). Bis-acrylamide solution was purchased from Serva (Germany). Sustanon® was provided by the Schering-Plough Company (Kenilworth, NJ, USA) which is an injectable testosterone medication containing four testosterone esters at concentrations: 30 mg/mL of testosterone propionate, 60 mg/mL of testosterone phenylpropionate, 60 mg/mL of testosterone isocaproate, and 100 mg/mL of testosterone decanoate. The overall androgenic potency per mL of Sustanon® is equivalent to 176 mg of testosterone. Tris (hydroxymethyl) aminomethane hydrochloride (Tris-HCl) and hydrogen peroxide were purchased from Panreac (Spain). PageRuler™ Prestained Protein Ladder was supplied by Fermentas (Canada). TEMED, ammonium persulfate (APS), and mineral oil were products of Bio-Rad (USA).
The sources of various kits were: Rat insulin ELISA kit (Mercodia, Sweden), AssayMax mouse insulin-like growth factor-1 (IGF-1 ELISA Kit; AssayPro, USA), rat Interleukin-6 (IL-6) ELISA kit (PeproTech, USA). TBARS ELISA kit (Cayman Chemical, USA), hydrogen peroxide (H2O2) assay kit (BioVision, USA) testosterone EIA (Cayman Chemical, USA), and dihydrotestosterone ELISA kit (Alpha Diagnostic, USA).
While the suppliers of antibodies were: antirabbit IgG (eBioscience, USA), antimouse IgG (Jackson ImmunoResearch, USA), β-actin antibody (Novus Biologicals, USA), antigoat IgG, 5α-reductase antibody, androgen receptor antibody (Santa Cruz Co., USA), and prostatic-specific antigen (PSA) antibody (Bioss, Scotland).
Animals
This experiment was approved by the Institutional Animal Care and Ethics Committee of Taipei Medical University (Taipei, Taiwan), and adhered to the animal care standards of the American College of Sports Medicine. In brief, 64 male Sprague-Dawley rats, aged 6 weeks, weighing 250–265 g were purchased from Biolasco Co. (Taipei, Taiwan). The rats were housed in an animal room conditioned at 24 ± 2 °C, RH 70–75 %, with a 12 h/12 h light/night cycle. The access of water and chow was ad libitum. The animals were acclimated in the animal room during the first week and then divided into eight groups, with eight rats in each group: Group 1, normal control; Group 2, BPH control; Group 3, DM control; Group 4, BPH + DM; Group 5, EX control; Group 6, BPH + EX; Group 7, DM + EX; and Group 8, BPH + DM + EX. The animals were separately caged, with 2 rats in each cage. In Week 2, DM groups were induced with a single intraperitoneal (ip) injection of streptozotocin (65 mg/kg) The BPH groups were induced in Week 3 by daily ip injection with Sustanon® (testosterone, 3.5 mg/head) and estradiol (0.1 mg/head), consecutively for 8 weeks. Exercise training was conducted from Week 12 until Week 17 on a rat exercise treadmill (Fortelice, International Co., Ltd., Taiwan). according to the program: rats were allowed to sprint at 20 m/min, 60 min per time, three courses per week, this program was continued successively for a total period of 6 weeks.
Blood collection and analysis of the lipid profiles
The control biochemical data were established at the end of the first week before experiment. The control data of the DM-control was established 1 week after STZ-induction, and those of the BPH-control group was collected 8 weeks after Sustanon®-induction. Blood collection was performed at the end of Week 2 and Week 17. In Week 17, the rats were bled from the abdominal arteriole immediately before euthanized with CO2 anesthesia. The blood obtained was centrifuged at 4 °C at 3000 × g for 10 min using a freezer-type centrifuge (1580 MGR, Gyrozen, Korea), and the serum high-density lipoprotein (HDL), serum low-density lipoprotein (LDL), serum cholesterol (CHOL), and triglyceride (TG) were determined using respective kits by following the manufacturer’s instructions.
Collection of tissue specimens
After euthanized, the prostates with seminiferous vesicles and testis were excised, photoed and weighed. Half of each organ was immersed in a 10 % formalin fixation solution, and the other half was rapidly immersed into liquid nitrogen, and stored at −80 °C for further use.
To 200 mg prostate tissues lysis buffer (1.6 mL) was added, homogenized (microquantity-type homogenizer, T10 Basic, IKA, Germany) on ice and left to react for 30 min. The homogenate was centrifuged using the freezer-type centrifuge (1580 MGR) at 12000 × g at 4 °C for 20 min. The supernatant (protein lysate, PLS) was separated and stored at −80 °C for further use.
Western blot analysis of 5α-reductase
The PLS was assayed for total protein content. To PSL, a two-fold volume of Western sample loading dye (WSLD) solution was loaded. The mixture (named herein PSL-WSLD) was heated in a dry heating bath (100 °C) for 10 min, and treated as follows. PSL-WSLD containing 30 μg of protein was loaded onto 10 % SDS-PAGE and the electrophoresis was conducted in the SDS-PAGE electrophoresis chamber (Mini-Protean Tetra Cell, Bio-Rad, USA), using the SDS-PAGE electrophoresis buffer (running buffer of pH 8.3, containing 25 mM Tri-HCl, 192 mM glycine, 0.1 % SDS, and deionized water to adjust to 1 L) at 75 V for 30 min. The protein spots were electrotransferred onto the PDVF membrane using a Mini Trans-Blot (Bio-Rad, USA) at 4 °C and 75 V for 20 h. The PVDF membrane was removed and marked with the obtained molecular weight. The marked membrane was sliced according to the molecular weight, immersed in a blocking buffer, and agitated at 4 °C overnight. The membrane was rinsed with a TBST solution thrice and left to stand for 10 min. The primary antibodies were applied and left to react for 1 h at ambient temperature, and then rinsed with the TBST solution. The secondary antibodies were applied, and left to react at ambient temperature for 1 h. After rinsed with the TBST solution, enhanced chemiluminescence (ECL) was applied and left to react completely. Protein expression was imaged using a luminescent image analyzer (LAS-4000; Fujifilm, Tokyo, Japan).
Enzyme linked immunosorbent analysis for determining serum insulin, IGF, TBARS, H2O2, testosterone, DHT and prostate IL-6
The blood obtained was immediately centrifuged using the freezer-type centrifuge (1580 MGR) at 3000 × g and 4 °C for 10 min. The supernatant serum was separated and stored at −80 °C if not used immediately. The sera were used for determining insulin, IGF, TBARS, H2O2, testosterone and DHT. Prostatic tissues (100 mg) were minced into chops having size <3 mm3 and extensively washed with PBS containing heparin to prevent potential peripheral blood contamination. The mince was incubated with 200 U/mL type I collagenase and 100 mg/mL DNase type I (Sigma Chemical Company, St. Louis, MO) in RPMI 1640 medium plus 10 % fetal calf serum and 6 % penicillin/streptomycin solution (Gibco BRL Life Technologies, Gaithersburg). The tissues were dissociated overnight at 37 °C and used for determination of IL-6. The following protocol for assay was performed following the instructions given by the manufacturers.
Pathological examination and Sirius Red staining
After CO2-euthanized, prostate, testes, seminal vesicle, bladder, pancreas, kidneys, heart, liver, and muscles were excised, photoed and weighed. Half of each organ was fixed in 10 % formalin, paraffin-embedded, and sliced with a microtome. The specimens were forwarded to the National Laboratory Animal Center (NLAC, Taipei) to receive pathological examination.
The paraffin-embedded specimens were dewaxed with xylene, and rehydrated successively with gradient ethanol solutions (100, 95, 80, and 70 %). These specimens were first stained with the Fouchet dying agent to attain a clear contrasting background, then with Weigert’s haematoxylin to stain the nuclei (to a bluish-black color), and finally with Sirius Red to stain the collagen (to red). The semifinished specimens were immediately dehydrated, mounted, and examined using an optical microscope (BX41M-ESD, Olympus, Japan).
Immunohistochemical stain for androgen receptor (AR) and prostatic specific antigen (PSA)
The paraffin-embedded tissue specimens were placed in an incubator held at 37 °C overnight, immersed in xylene for 10 min to remove the residual embedding paraffin, and successively rehydrated with gradient ethanol solutions (100, 95, 80, and 70 %). Citric acid (10 mM, pH 6.0) was added to the rehydrated specimens. After 15 min, the specimens were treated with 3 % H2O2 for 15 min and then rinsed twice with PBS. The primary antibodies were applied and left to react for 2 h. After rinsed twice with PBS, the secondary antibodies were applied, left to react for 30 min, and peroxidase-conjugated streptavidin was added to react for 1 h and the specimens were rinsed with PBS twice. Finally, the specimens were reacted with the coloring agent diaminobenzidine (DAB) for 30 min, rinsed twice with PBS, dehydrated with gradient ethanol solutions, and mounted.
Determination of NO
Griess reagent (20 μL) was added to 20 μL of serum (or tissue homogenate) and mixed well. Double-distilled water (160 μL) was added to the mixture to make up to a total volume 200 μL. The optical density was read at 550 nm against the blank. A calibration curve was established using 20 μL of a standard sodium nitrite solution, similarly treated with 20 μL of Griess reagent and 160 μL of double-distilled water, and finally the optical density was read at 550 nm. The nitric oxide content of the samples was calculated from the reference curve.
Statistical analysis
Data obtained in the same group were analyzed with Duncan's multiple range test, using the Statistical Analysis System software (SAS 9.0). Data were expressed as mean ± SD. Different letters indicated significant differences at a confidence level of p < 0.05.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
KCC, SYS and CCP contributed to this work by designing the study, obtaining data, performing the statistical analysis, writing the manuscript and interpreted the data. CLH and KHS participated in the conception and design of the study and acquisition of data. RYP and YTL participated in the conception and design of the study and interpretation of the data and reviewed and edited the manuscript. All authors read and approved the final manuscript.