All herbs used to prepare GGN were purchased from Omniherb (Dong Woo Dang Pharmacy Co. Ltd., Yeongcheon, Korea). Finasteride was obtained from Merck & Co., Inc. (Whitehouse Station, NJ, USA). Antibodies against inducible nitric oxide synthase (iNOS; M-19; sc-650), COX-2 (C-20; sc-1745), procaspase-3 (E-8; sc-7272), procaspase-8 (C-20; sc-6136), B-cell lymphoma-2 (Bcl-2; C-2; sc-7382), Bcl-extra large (Bcl-xL; H-5; sc-8392), Bcl-2-associated X protein (Bax; B-9; sc-7480), p53 (FL-393; sc-6243), Fas (A-20; sc-1023), Fas ligand (Fas-L; C-178; sc-6237) and β-actin (ACTBD11B7; sc-81178) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). An antibody against Fas-associated protein with death domain (FADD; ab24533) was purchased from Abcam (Cambridge, UK). The horeseadish peroxidase-conjugated secondary antibodies (goat anti-rabbit, 111-035-003; rabbit anti-mouse, 315-035-003; and donkey anti-goat, 705-035-003) were purchased from Jackson ImmunoResearch Laboratories, Inc. (West Grove, PA, USA). All other reagents were purchased from Sigma-Aldrich (Merck Millipore, Darmstadt, Germany).

GGN consists of nine different herbs; Morinda officinalis How (120 g), Cistanche deserticola Y. C. Ma. (120 g), Cornus officinalis Sieb. et. Zucc. (120 g), Cuscuta chinensis Lamark (120 g), Psoralea corylifolia L. (100 g), Dendrobium nobile Lindl. (80 g), Trigonella foenum-graecum L. (80 g), Foeniculum vulgare Mill. (40 g) and Aconitum carmichaeli Debx. (20 g). The herbs had a moisture content of <13% by weight and were air-dried. The combination of herbs was extracted with 50% (v/v) ethanol-water at 60°C for 8 h. The extracts were then filtered through 15-µm cartridge paper and ethanol was removed via vacuum rotary evaporation (Eyela; Tokyo Rikakikai Co., Ltd., Tokyo Japan). The concentrates were freeze-dried and the yield was 12%. Powders were dissolved in distilled water prior to experiments and residual powders were stored at −20°C.

Ten-week-old male Wistar rats (n=24; weight, 200±20 g) were purchased from Daehan Biolink Co., Ltd., (Republic of Korea). Rats were housed under constant conditions (temperature, 22±2°C; humidity, 55±9%; 12 h light/dark cycle; ad libitum access to food and water) in accordance with the Guide for the Care and Use of Laboratory Animals. All procedures were approved by the Ethics Committee for Animal Care and the Use of Laboratory Animals at Sang-ji University (Wonju, Korea; approval documents, 2013-03;) and conducted in accordance with University guidelines. Rats were randomly distributed into four groups (n=6 in each); the sham-operated group (Con; administered with 200 µl distilled water orally), the BPH model group (BPH), the BPH-induced group administrated with finasteride (Fina; 5 mg/kg/day; p.o. Merck & Co., Inc.) and the BPH-induced group administrated with GGN (GGN; 100 mg/kg/day; p.o.). BPH was induced in rats via castration and subsequent subcutaneous injections of 100 µl testosterone propionate (Waco Pure Chemical Industries, Ltd., Osaka, Japan; 10 mg/kg/day), as previously reported (23). At the end of the four-week period, body weight was recorded and rats were fasted for 12 h. The following day, all rats were sacrificed by cervical dislocation following anesthetization with Zoletil 50 (20 mg/kg intraperitoneally; Virbac, Carros, France), and blood samples were obtained via cardiac puncture. Prostatic tissue was excised, rinsed, weighed and stored at −70°C until use.

Prostatic tissues were harvested, rinsed, and weighed immediately following sacrifice. The prostate weight to body weight (PW/BW) ratio was calculated. The relative prostate weight ratio was calculated as follows: Relative prostate weight ratio=prostate weight of rats in the experimental groups (BPH, Fina, GGN)/prostate weight of rats in the Con group.

Serum concentrations of testosterone were determined using a testosterone enzyme immunoassay kit (582701; Cayman Chemical Co., Ann Arbor, MI, USA). Serum concentrations of dihydrotestosterone (DHT) were determined via enzymatic methods using a commercially available assay kit (11-DHTHU-E01; ALPCO, Salem, NH, USA) according to the manufacturer's protocol.

The prostatic tissue was fixed in 4% buffered formalin and embedded in paraffin, and 4-µm thick sections were cut. The sections were stained with hematoxylin and eosin for histological examination, and images were acquired using an SZX10 microscope (Olympus Corp., Tokyo, Japan). The thickness of the epithelium in the prostate tissue (TETP) was measured using the Leica Application Suite software (LAS ver. 3.3.0; Leica Microsystems, Inc., Buffalo Grove, IL, USA).

The prostatic tissue from each rat was homogenized in PRO-PREP lysis buffer (Intron Biotechnology Inc., Seongnam, Korea). Tissue extracts were centrifuged at 16,000 × g (4°C) for 20 min, and the resulting supernatant was transferred to a clean tube. The protein concentration was determined using a Bio-Rad protein assay (Bio-Rad Laboratories, Inc., Hercules, CA, USA) according to the manufacturer's protocol. Aliquots of each protein sample (30 µg) were separated by 10–12% SDS-PAGE and transferred onto a polyvinylidene fluoride membrane (Immobilion-P transfer membrane; Merck Millipore). The membranes were blocked with 2.5% skimmed milk at 4°C for 30 min and incubated overnight with 1:1,000 dilutions of the primary antibodies (anti-iNOS, anti-COX-2, anti-caspase-3, anti-caspase-8, anti-Bcl-xL, anti-Bax, anti-P-53, anti-Fas, anti-Fas-L, anti-FADD and anti-β-actin). The blots were washed three times with Tween-20/TBS and then incubated with 1:2,000 dillutions of corresponding secondary antibodies (Santa Cruz Biotechnology, Inc.) for 2 h at room temperature. Blots were again washed three times with Tween-20/TBS and the immunoreactive protein bands were visualized via enhanced chemiluminescence, and the developed blots were exposed to X-ray film (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Each blot was performed in triplicate and densitometric analysis was performed using Bio-rad Quantity One Software (version 4.6.3; Bio-Rad Laboratories, Inc.).

Values are expressed as the mean ± standard error of the mean for six rats. Data were analyzed using one-way analysis of variance with Dunnett's test and P<0.05 was considered to indicate a statistically significant difference. Statistical analysis was performed using SPSS statistical analysis software (version 19.0; International Business Machines, Armonk, NY, USA).

The mean prostate weight of rats in the BPH group was significantly higher than that of rats in all other groups (P<0.001). Compared with BPH rats, the prostate weight of rats in the Fina and GGN groups were significantly decreased (P<0.001; Fig. 1A). The relative prostate weight ratio in the BPH group was 2.66 times that in the Con group, which was a significant increase (P<0.001), whereas in the GGN group, prostate weight was only 1.64 times that in the Con group (Fig. 1B). In addition, the PW/BW ratio in the BPH group was also significantly higher than that in the other groups (P<0.001). Compared with the BPH group, the PW/BW ratio was significantly lower in the Fina and GGN groups (P<0.001), whereas no significant difference was observed between the PW/BW ratios of the GGN group (2.54) and the Fina group (2.71).

The serum testosterone and DHT levels of the rats in each group are displayed in Fig. 2. The concentrations of the testosterone and DHT in the BPH group were significantly higher than those in all other groups (P<0.001). In the Fina and GGN groups, serum testosterone and DHT levels were significantly lower than those in the BHP group (P<0.001). Of note, DHT levels in the GGN group were markedly lower than those in the Fina group.

The effect of GGN on prostate gland morphology was investigated by histological analysis (Fig. 3). Rats in the BPH group exhibited histological changes typical of prostatic hyperplasia: Thickened glandular epithelium, vacuolated cytoplasm pointing into the glandular lumen and a decreased glandular luminal area (Fig. 3A). Administration of GGN for four weeks suppressed these typical histological patterns. The TETP was significantly higher in the BPH-induced group than in the other groups (P<0.001; Fig. 3B). In brief, histologic examination revealed that administration of finasteride or GGN ameliorated the major features of prostatic hyperplasia observed in the BPH group, although certain minor features of prostatic hyperplasia remained.

Inflammation is associated with the increased proliferation of prostatic epithelial cells (24). In patients with BPH, iNOS, which provides a sustained release of reactive nitrogen species that may induce cell damage, is typically present (25). In addition, COX-2, which generates pro-inflammatory prostaglandins, is detected in inflammatory cells (4). In the present study, the levels of iNOS and COX-2 in the prostatic tissue of BPH-induced rats were analyzed by western blotting to investigate the effects of GGN on inflammation. Compared with the Con group, treatment with testosterone increased the levels of iNOS and COX-2 in the BPH-induced group. By contrast, the Fina and GGN groups showed markedly reduced levels of these inflammatory proteins compared with the BPH group (Fig. 4).

BPH arises from an imbalance between cell proliferation and apoptosis (3). The two main apoptotic pathways are the intrinsic and extrinsic pathways. In the extrinsic pathway, apoptosis is induced by death activators binding to receptors at the cell surface and accumulation of the adaptor molecule FADD, which leads to the activation of initiator caspase (26). In the present study, the expression levels of proteins in the extrinsic pathway were assessed (Fig. 5A), and the Fina and GGN-treated group exhibited higher protein levels of Fas-L, Fas, FADD and p53, and lower protein levels of procaspase-8 and procaspase-3 compared with the levels in the BPH group.

In the intrinsic pathway, apoptosis, including disruption of the mitochondrial membrane potential, is critically regulated by Bcl-2 family proteins (27). As demonstrated in Fig. 5B, the Fina and GGN group exhibited lower levels of the anti-apoptotic proteins Bcl-2 and Bcl-xL, and higher levels of the pro-apoptotic protein Bax than the BPH group. Therefore, although the ratio of Bcl-2 to Bax was significantly increased in the BPH group compared with the control group (P<0.05), the ratio decreased significantly following treatment with Fina and GGN (P<0.001), suggesting that GGN-induced apoptosis is regulated by the Bcl-2 family of proteins, which are important mediators of apoptosis (Fig. 5C).