Protective effects of combination of Stauntonia hexaphylla and Cornus officinalis on testosterone-induced benign prostatic hyperplasia through inhibition of 5α- reductase type 2 and induced cell apoptosis

Benign prostatic hyperplasia (BPH) is a progressive pathological condition associated with proliferation of prostatic tissues, prostate enlargement, and lower-urinary tract symptoms. However, the mechanism underlying the pathogenesis of BPH is unclear. The aim of this study was to investigate the protective effects of a combination of Stauntonia hexaphylla and Cornus officinalis (SC extract) on a testosterone propionate (TP)-induced BPH model. The effect of SC extract was examined in a TP-induced human prostate adenocarcinoma cell line. Male Sprague-Dawley rats were randomly divided into 5 groups (n = 6) for in vivo experiments. To induce BPH, all rats, except those in the control group, were administered daily with subcutaneous injections of TP (5 mg/kg) and orally treated with appropriate phosphate buffered saline/drugs (finasteride/saw palmetto/SC extract) for 4 consecutive weeks. SC extract significantly downregulated the androgen receptor (AR), prostate specific antigen (PSA), and 5α-reductase type 2 in TP-induced BPH in vitro. In in vivo experiments, SC extract significantly reduced prostate weight, size, serum testosterone, and dihydrotestosterone (DHT) levels. Histologically, SC extract markedly recovered TP-induced abnormalities and reduced prostatic hyperplasia, thereby improving the histo-architecture of TP-induced BPH rats. SC extract also significantly downregulated AR and PSA expression, as assayed using immunoblotting. Immunostaining revealed that SC extract markedly reduced the 5α-reductase type 2 and significantly downregulated the expression of proliferating cell nuclear antigen. In addition, immunoblotting of B-cell lymphoma 2 (Bcl-2) family proteins indicated that SC extract significantly downregulated anti-apoptotic Bcl-2 and markedly upregulated pro-apoptotic B cell lymphoma-associated X (Bax) expression. Furthermore, SC treatment significantly decreased the Bcl-2/Bax ratio, indicating induced prostate cell apoptosis in TP-induced BPH rats. Thus, our findings demonstrated that SC extract protects against BPH by inhibiting 5α-reductase type 2 and inducing prostate cell apoptosis. Therefore, SC extract might be useful in the clinical treatment of BPH.


Animal experimental design
The experimental protocols were approved (CNU-01108) by the International Animal Ethics Committee at Chungnam National University. Six-week-old male Sprague-Dawley (SD) rats were purchased from Orient Bio (Gyeonggi-do, Korea) and acclimated in a specific-pathogenfree (SPF) animal facility under controlled temperature, humidity, and photoperiod (22 ± 2 o C, 55 ± 5%, and 12 h light/dark cycle, respectively) for 1 week before the experiment. All animals were fed standard chow and water ad libitum. During the experimental period, rats were visually inspected for their health and well-being daily and the in-house standards were maintained, including the removal of bedding with full cage changes twice a week. SD rats were then randomly divided into 5 groups (n = 6 per group). To induce BPH, all rats, except for the control group, were given subcutaneous (S.C.) injections of TP (5 mg/kg) for 4 weeks. To minimize the animal suffering and distress during injections, these were carried out into the loose skin on the back of the neck and the site of injection varied to reduce local skin reactions. In addition, based on in vitro studies, appropriate drugs (finasteride, saw palmetto, SC extract) were administered orally for 4 weeks as follows: control group (PBS, P.O.), BPH group (testosterone/TP 5 mg/kg, S.C. + PBS, P.O.), Fina (TP 5 mg/kg, S.C. + Finasteride 10 mg/kg, P.O.), Saw (TP 5 mg/kg, S.C. + saw palmetto extract 100 mg/kg, P.O.), SC 50 (TP 5 mg/kg, S.C. + SC extract 50 mg/kg, P.O.). At the end of the experimental period, rats were fasted overnight and euthanized by CO 2 asphyxiation in the euthanasia apparatus. Animals were left in the chamber for approximately 5 min until the visible movements ceased and euthanization was confirmed. Blood was drawn from the abdominal vein. Then, the blood samples were centrifuged at 3000 rpm at 4 o C for 15 min, and the serum was stored at -80 o C until further analysis. Serum testosterone and DHT levels were determined using a commercial enzyme-linked immunosorbent assay (ELISA) kit (ALPCO Diagnostics, NH, USA), according to the manufacturer's protocol. Dissected prostate glands were weighed and stored in -80 o C liquid nitrogen for further analysis. The body weight of rats was measured at the beginning and the end of the experimental period.

Histological study
Paraffin-embedded prostate tissues were cut into 5 μm sections. After deparaffinization and dehydration, sections were subjected to hematoxylin and eosin (H&E) staining and examined using a light microscope (Nikon eclipse 80i, Nikon Corporation, Tokyo, Japan) at a × 400 magnification. Images were taken from 10 randomly selected fields, and epithelial thickness and lumen area were measured using Image J software (Image J v46a; NIH, USA).

Statistical analysis
All data are expressed as mean ± standard deviation (SD). Statistical analysis was performed with one-way ANOVA, with Tukey's multiple comparison test for all pairwise comparisons of the mean value of different treatment groups using SigmaPlot 12 program. All the experimental data related to the SC extract-treated groups were compared with the normal control and the BPH groups. P-values < 0.05 were considered statistically significant. Graphs were constructed using GraphPad Prism 5.

HPLC analysis of hederacoside D and morroniside in the SC extract
First, we identified the phytochemical compounds in the SC extract using HPLC. Hederacoside D is a major bioactive saponin that plays a pivotal role in overall biological activity.
Morroniside is also an active phytochemical component. Hederacoside D showed a characteristic peak at 39.52 min, while morroniside, at 6.53 min. The concentrations of Hederacoside D and morroniside were 25 and 1.5 mg/g, respectively (S1 Fig).

Cell viability and BPH-related protein expression
To examine the cytotoxicity of SC extract, an MTT assay was performed in LNCaP cells. As shown in Fig 1A, there was no significant difference in cell viability between SC-treated cells and control cells. However, exposure to 100 μg/mL SC extract markedly decreased cell viability by up to 86.73%, demonstrating that a high concentration inhibits cell viability due to its toxicity. The highest cell viability was observed in cells treated with 50 μg/mL. We then investigated the effect of SC extract on AR, PSA, and 5α-reductase type 2 protein expression in TP-induced LNCaP cells by using western blotting ( Fig 1B). As shown in Fig 1C-1E, TP treatment significantly (p < 0.001) increased the expression of AR, PSA, and 5α-reductase type 2, relative to the control group. In contrast, the SC treated group showed a significantly (p < 0.001) decreased expression of AR, PSA, and 5α-reductase type 2, along with the Fina and Saw groups, compared to the BPH group. These results suggest that SC extract acts as a 5ARI, thereby suppressing androgen signaling in LNCaP cells.

Effect of SC extract on body and prostate weight
Based on the in vitro results, we performed animal experiments to evaluate the efficacy of SC extract as an anti-BPH treatment. Body weight (BW) and prostate weight (PW) are commonly used to evaluate the progression of experimentally induced BPH in vivo. As shown in Table 1, the BPH group showed significantly (p < 0.001) increased PW and relative prostatic weight compared to the control group, while the Fina and SC 50 groups exhibited significantly (p < 0.001) decreased expression of the aforementioned parameters, compared with the BPH group. However, no significant differences in BW were observed among the groups.

Morphological changes in prostate tissue in TP-induced BPH rats
As shown in Fig 2A, the BPH group exhibited a markedly increased prostate size along with the Saw group relative to the control group; however, SC treatment markedly reduced the prostate size of TP-induced BPH rats, compared with the BPH group. Our histopathological examination ( Fig 2B) demonstrated that the BPH group had a serious disruption in the histoarchitecture of the prostate tissue, indicating stromal and epithelial proliferations, thereby glandular hyperplasia, hypertrophy, and significant thickening (p < 0.001; Fig 2C), with papillary projections in the epithelium. The lumen area of BPH group significantly (p < 0.001; Fig  2D) decreased, as compared to the control group, which was not associated with histological changes. However, SC treatment showed significant improvements in the histo-architecture of the prostatic tissue, such as a decreased prostate epithelial thickness (p < 0.001) and an increased lumen area (p < 0.001), along with the Fina and Saw groups, compared to the BPH group. These results indicate that SC extract could be an alternative treatment for prostatic hyperplasia by attenuating the abnormal morphological changes in BPH rats.

SC mediated downregulation of serum androgens and BPH-related protein expression in TP-induced BPH rats
Next, we studied the expression of testosterone and DHT, which are factors in the progression of BPH in a TP-induced BPH model. As shown in Fig 3A, a significant (p < 0.01) increase in serum testosterone levels was observed in the BPH group compared with the control group. In contrast, the Fina, SC 50 (p < 0.01), and Saw (p < 0.05) groups showed significantly decreased testosterone levels relative to the BPH group. Similarly, serum DHT levels also significantly (p < 0.01) increased in the BPH group (Fig 2B) compared to the control, while SC treatment showed significantly (p < 0.001) decreased DHT levels, together with the Fina group (p < 0.05), relative to the BPH group. These results proved that SC extract is a potential treatment for reducing androgen concentrations in BPH rats. Then, we examined the expression of AR and PSA and BPH-related protein expression using western blotting (Fig 3C-3E) and BPH group showed a significantly (p < 0.001) increased expression of AR and PSA, compared with the control group. In contrast, as expected, the rats treated with finasteride and SC extract showed a significantly (p < 0.001) decreased expression of AR and PSA relative to the BPH group. In addition, the Saw group exhibited markedly decreased AR and significantly (p < 0.001) decreased PSA expression relative to the BPH group. To further confirm this result, immunostaining for 5α-reductase type 2 was carried out as it is the enzyme that catalyzes the conversion of testosterone into DHT, thereby causing abnormal growth of the prostate gland. As shown in Fig 4A and 4B, rats in the control group showed no abnormalities; however, those in the BPH group showed a significantly (p < 0.001) increased expression of 5α-reductase type 2, as indicated by the color reaction in the prostate epithelial cells (Fig 4A. b2: short arrows, color reaction in prostate epithelial cells). Notably, the SC extract along with finasteride and saw palmetto significantly (p < 0.001) reversed the effect of TP, compared with the BPH group. Together, these results indicate that SC extract has the ability to suppress androgen signaling in prostate cells through its 5α-reductase type 2 inhibitory effect and possible treatment for TP-induced BPH rats.

SC-mediated regulation of apoptosis and cell proliferation in TP-induced BPH rats
During the progression of BPH, cell proliferation may increase and PCNA is a hallmark of proliferating cells in the prostate [29]. As shown in Fig 4C, the control group had a low expression of PCNA; however, rats in the BPH group exhibited a significantly (p < 0.001) increased number of PCNA-positive cells, which was characterized by brown-stained nuclei (Fig 4C and  4D; short arrows, PCNA-positive cells). Finasteride and SC extract significantly (p < 0.001) reversed the effect of TP, as compared to the BPH group. Next, the expression of Bcl-2 and Bax was examined to study the effect of SC extract on apoptosis of BPH in vivo ( Fig 5A). As shown in Fig 5B and 5C, the expression of Bcl-2, which includes the anti-apoptotic factor upregulated and the pro-apoptotic factor Bax significantly (p < 0.001) downregulated in the BPH group, compared with the control group. SC extract significantly (p < 0.001) decreased the expression

Fig 3. Effects of SC extract on serum androgens and BPH-related proteins expression in TP-induced BPH rats.
To induce BPH, all rats were daily received TP injection (5 mg/kg, S.C.), except for the control group along with appropriate drugs (finasteride, saw palmetto, SC extract) orally for 4 weeks. Serum testosterone and DHT levels were evaluated using an ELISA assay. Western blotting was performed to assay AR and PSA, the BPH-  of Bcl-2, but increased the expression of Bax relative to the BPH group. In addition, both the Fina and Saw groups exhibited a significantly (p < 0.001) decreased expression of Bcl-2 compared to the BPH group. Further, the ratio of Bcl-2 to Bax significantly (p < 0.001) decreased in both the Fina and SC 50 groups, compared with the BPH group, where the ratio of Bcl-2 to Bax markedly increased compared to the control group ( Fig 5D). These results proved that SC extract is a possible treatment for the modulation of PCNA and Bcl-2 family proteins and induces apoptosis in a positive manner. Therefore, SC extract is a potential treatment to maintain the balance between cell proliferation and apoptosis in TP-induced BPH.

Discussion
Benign prostatic hyperplasia and associated LUTS are common urological problems among older men. Androgens, such as testosterone and DHT, play a fundamental role in the development and maintenance of BPH [15]. The activities of these androgens can be regulated by adding a 5ARI, such as finasteride. However, finasteride is a drug that has been associated with adverse effects, such as sexual dysfunction and allergic reactions. In addition, a previous clinical study has revealed that patients subjected to 5ARI treatment can be exposed to a higher risk of high-grade prostate cancer [30]. Therefore, to substitute for synthetic drugs and reduce side effects, this study investigated the therapeutic effects of plant-based medicine using SC extract on BPH in vitro and in vivo. First, we conducted in vitro investigations to study the effect of SC extract on TP-induced BPH in the LNCaP cell line. During the progression of BPH, testosterone is converted into DHT by 5α-reductase type 2. DHT then binds to the nuclear ARs, and this complex gives rise to PSA, which is a good predictor of BPH progression [31]. Furthermore, this complex is involved in the prostatic expression of vascular endothelial growth factor, epidermal growth factor, and basic fibroblast growth factor, and finally promotes angiogenesis and prostatic growth [32]. Therefore, we first assayed the expression of AR, PSA, and 5α-reductase type 2 in vitro. According to our results, SC extract significantly reduced the expression of AR, PSA, and type 2 5α-reductase. These findings suggest that SC extract acts as a 5ARI, thereby suppressing androgen signaling in LNCaP cells and a possible treatment for TP-induced BPH. Therefore, we performed in vivo investigations to further examine the therapeutic effect of SC extract on the TP-induced BPH rat model.
Examination of BW, PW, and serum androgens is currently used to evaluate the progression of BPH. In line with previous studies [16,33], our investigation also demonstrated that rats with experimentally induced BPH have higher PW and serum androgens, such as testosterone and DHT. Testosterone is an androgen produced by Leydig cells of the testis, while DHT is an important byproduct of testosterone and the most potent androgen in men, which is involved in development and aging. Our study demonstrated significantly increased testosterone and DHT levels in the BPH group. However, SC extract significantly reduced the expression of the aforementioned serum androgens. These data suggest that SC extract has the ability to regulate androgen and its byproducts, thereby attenuating BPH development and progression through its anti-androgenic activity. BPH is a precancerous prostate condition, which is caused by the overgrowth of prostatic epithelial and stromal cells, and arises due to the imbalance between cell proliferation and apoptosis [34]. Hyperplasia of these cells results in increased prostate weight and causes contraction of the urethral canal, which may obstruct the urine flow [17]. Histologically, BPH is characterized by the proliferation of stromal and glandular cells [35]. Our histological examination demonstrated that the BPH group had a serious interruption in the histo-architecture of prostate tissue. However, SC extract significantly recovered these abnormal morphological changes, thereby reducing the prostate size of TP-induced BPH rats.
Androgens affect gene expression in various kinds of tissues and cells by binding with AR, which has been linked to prostate cancer [36,37]. DHT has a higher affinity for AR than testosterone. In the prostate, the interaction between DHT and AR results in the production of proteins, such as PSA [38,39]. PSA, which is a glycoprotein in humans, is encoded by the kallikrein-related peptidase 3 (KLK3) gene and is secreted by the prostatic epithelial cells. As serum PSA level is frequently elevated in prostate disorders such as BPH and prostate cancer, it is used as a clinical marker for disease prognosis [40]. In the current study, immunoblotting revealed that SC extract significantly reduced the expression of AR and markedly decreased PSA expression, indicating that SC extract has the ability to suppress androgen signaling in prostate cells of BPH rats. This result was further examined by immunostaining of 5α-reductase type 2, as it is the convertor of testosterone to its active form, DHT, and is involved in BPH development and progression [41]. As expected, SC extract reduced type-2 5α-reductase expression and, together, these results proved that the SC extract acts as a 5ARI.
Progression of BPH is associated with several molecular mechanisms. As described above, AR-related 5α-reductase type 2 activity is considered a fundamental pathway of BPH. In addition, the balance between cell proliferation and apoptosis also plays a pivotal role in the BPH condition. Hyperplasia of prostatic epithelial and stromal cells is the major pathological feature of BPH, which is caused by an imbalance between cell proliferation and cell death [42]. In this study, the processes of cell proliferation and programed cell death were comparatively analyzed to determine the underlying mechanism of pathogenesis of BPH by using PCNA and Bcl-2 family proteins. Cell proliferation is related to the progression of the cell cycle, which is composed of four phases: G1, S, G2, and M. These phases are controlled by cell-cycle regulatory proteins [43]. PCNA is a nuclear protein that is elevated during the G1/S phase transition, thereby acting as a marker for proliferating cells and plays a crucial role in physiological conditions such as BPH [43]. To investigate the anti-proliferative effect of the SC extract, PCNApositive cells in both glandular epithelium and stroma cells were examined. Our IHC results of PCNA demonstrated that SC extract significantly downregulated the expression of PCNA-positive cells, which is characterized by brown-stained nuclei. The Bcl-2 family has been identified for its role in apoptosis due to its Bcl-2 homology domains and involvement in the regulation of apoptosis. These Bcl-2 homology domains encourage family members interactions with each other. It also indicates pro-or anti-apoptotic functions. [44,45]. The ratio of pro-to antiapoptotic subfamily members in a cell may vary according to the number of signaling pathways and efficiency of transmitting information upon cellular stress, such as available nutrients, DNA damage, and protein processing [46]. Intrinsic apoptosis is regulated by antiapoptotic Bcl-2, a protein localized in the mitochondrial outer membrane and suppresses the release of the pro-apoptotic factor cytochrome c [47]. The Bax gene is a pro-apoptotic protein of the Bcl-2 family and activates the apoptotic signal. The ratio of Bcl-2 to Bax determines the apoptotic reactivity [48]. Under normal conditions, prostate tissues exhibit relatively low levels of Bcl-2 expression, which are associated with low levels of apoptosis, while in BPH, the incidence of imbalance between apoptosis and proliferation is indicated by either apoptosis signals or by increasing PCNA expression [49]. In this study, SC extract significantly downregulated the anti-apoptotic Bcl-2 and markedly increased the level of pro-apoptotic Bax. Moreover, the Bcl-2/Bax ratio was significantly decreased by using SC extract treatment. Collectively, these findings suggest that SC extract is a possible treatment for inhibiting cell proliferative activity and inducing apoptosis in TP-induced BPH rat models.

Conclusions
In conclusion, the findings of our study revealed that SC treatment significantly reduced prostate hyperplasia and prostate size, causing marked degenerative changes in prostatic stromal and epithelial cells. SC extract also prevents the 5α-reductase type 2-AR pathway by acting as a 5ARI, thereby reducing the conversion of testosterone into DHT. Furthermore, SC extract downregulated cell proliferation-related protein expression and induced cell apoptosis under BPH conditions. Hence, SC extract is a potential phytotherapeutic agent for modulating the balance between cell proliferation and apoptosis, which underlies the pathogenesis of BPH. Therefore, this investigation demonstrated that the combination of S. hexaphylla and C. officinalis has a protective effect against testosterone-induced benign prostatic hyperplasia through inhibition of 5α-reductase type 2 and induced apoptosis. The use of SC extract as a therapeutic intervention for the treatment of BPH warrants further investigation.