Stimulation of cannabinoid receptors by using Rubus coreanus extracts to control osteoporosis in aged male rats

Abstract

A substantial proportion of men with prostatic disease have an increased risk of bone loss. In the present study, we investigated the effects of Rubus coreanus Miquel (RCM) extracts on osteoporosis that occurs with N-methyl-N-nitrosourea (MNU)-induced prostatic hyperplasia. The rats used in this study were categorized into groups of healthy controls, rats treated with MNU, and rats treated with MNU and RCM. The rats were sacrificed after 10 weeks of RCM treatment, after which ultrasonography, serum biochemical tests, histopathological examinations, immunohistochemical analysis, and semi-quantitative reverse-transcription polymerase chain reaction analysis were performed. There were no marked differences in body weight gain and the size and weight of the prostate gland between the MNU group and the MNU and RCM group. However, treatment with RCM inhibited osteoclastic osteolysis and reduced dysplastic progress in the prostate gland, as observed by histopathological evaluation and by analyzing changes in the levels of bone regulatory factors. In addition, the group treated with MNU and RCM had higher expression levels of cannabinoid receptors-1, -2, and osteoprotegerin. These results indicate that the anti-osteoporotic effect of RCM in prostatic hyperplasia is attributable to the cannabinoid receptor-related upregulation of osteoblastogenesis and inhibition of prostatic hyperplasia. The results of the present study suggest that treatment with RCM may benefit osteoporotic patients with prostatic disease by simultaneously altering the activation of osteoblasts and osteoclasts.

• Cannabinoid receptor
• male osteoporosis
• N-methyl-N-nitrosourea
• prostatic disease
• Rubus coreanus

Introduction

Osteoporosis is characterized by a reduction in bone mass and disruption of the microarchitectural structure of bone tissue, which results in a loss of mechanical strength and increases the risk of fracture [1]. Men, compared to women, are less likely to develop osteoporosis because of the greater skeletal mass accumulation and bone size in men [2]. Moreover, estrogen deficiency after menopause accelerates bone loss in women. Therefore, during the reproductive years, the prevalence of osteoporosis is higher in women than in men [2,3].

However, because of the increasing incidence of aging-related disorders, the differences in the prevalence of osteoporosis between men and women have reduced with age. Therefore, osteoporosis has become an increasingly important problem in men and accounts for increased morbidity rates associated with atraumatic hip fractures [2]. In addition to advanced age, the secondary risk factors for osteoporosis in men include hypogonadism, glucocorticoid excess, alcoholism, thyroid and parathyroid disorders, osteomalacia, and various cancers [2,4]. Prostatic cancer, which is one of the osteoporotic risk factors, is very common in elderly men [5–8]. The growth and development of a normal prostate gland depends on a functional androgen-signaling axis, the components of which include testosterone synthesis in the testis and adrenal gland, conversion of testosterone to dihydrotestosterone, transport of dihydrotestosterone to the target tissue, and binding of dihydrotestosterone to its target receptor with consequent gene modulation [5]. Therefore, changes in testosterone levels with advancing age are an important factor in the development of benign prostatic hypertrophy and prostate carcinogenesis, which are common conditions associated with pathological growth of the prostate gland [6,9]. Therefore, men with prostatic hyperplasia are treated with androgen deprivation therapy (ADT) to reduce prostate volume, arrest the disease process, and improve symptoms [6]. Several studies have shown a positive association between ADT and the loss of bone mineral density due to decreased levels of testosterone, which is important for normal bone growth and maintenance [2,9]. Furthermore, in men with prostate disease, factors other than ADT may also cause loss of bone mineral density. A high-grade prostatic cancer can also cause a state of high-bone turnover via pathological activation of osteoblasts and osteoclasts with bone metastasis [10]. Moreover, the results of a recent study revealed a high prevalence of bone resorptive disease in men with non-metastatic prostate cancer who were not treated with ADT [2].

Only a few therapies are available to treat or prevent osteoporosis, such as hormone replacement therapy or treatment with bisphosphonates, calcitonin, vitamin D analogues, and anabolic steroids, and all have severe adverse effects. Bisphosphonates, a class of widely used drugs for osteoporosis, are associated with adverse effects that include nausea, vomiting, diarrhea, peptic ulceration, abdominal pain, constipation, allergic skin disease, and the possibility of osteomalacia and osteonecrosis [11,12]. Therefore, research on natural substances that can serve as alternatives to chemical agents has increased. Rubus coreanus Miquel (RCM) is a species of raspberry that has been used in traditional Asian herbal medicine. In recent years, RCM has been reported to have various beneficial properties, including anti-inflammatory, anti-obesity, anti-bacterial, anti-protozoan, and anti-osteoporotic functions [13–17]. RCM has also been shown to have anti-osteoporotic effects in patients with diabetic osteoporosis at the post-menopausal stage [14] through the simultaneous regulation of osteoblasts and osteoclasts by the partial upregulation of the endocannabinoid system [18].

In the present study, we investigated the effects of RCM extracts on osteoporosis associated with N-methyl-N-nitrosourea (MNU)-induced prostatic dysplasia. In addition, we investigated RCM-induced changes in the endocannabinoid system, which is known to play a critical role in bone homeostasis.

Materials and methods

Animals

Forty-two 7-week-old male Wistar rats were obtained from Orient Chemical Co. Ltd. (Gyeonggi-do, Republic of Korea). The animals were used after 7 d of quarantine and acclimation to the facility conditions. The animals were housed in polycarbonate cages (2–3 per cage), wherein they were placed on a bed and exposed to a 12-h light/dark cycle (room temperature, 22 ± 2 °C; relative humidity, 50 ± 10%). They were fed a basal diet (LabDiet®, MO) and had access to tap water ad libitum. An individual identification card that showed the group and animal numbers was attached to each cage. Each animal was identified by a tail-marking method with an indelible marker. All experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee of Konkuk University (Seoul, Republic of Korea).

MNU-induced prostatic hyperplasia

The rats were randomly divided into three groups: healthy controls (Control, n = 10), MNU treated (MNU, n = 16), and MNU and RCM treated (RCM, n = 16). Time sequential treatment of hormone and carcinogen followed protocols previously published [19,20]. The rats then received intraperitoneal injections of cyproterone acetate (50 mg/kg; Sigma Chemical Co., St. Louis, MO) in olive oil for 21 d. One day after the last injection of cyproterone acetate, the rats received intraperitoneal injections of testosterone propionate (100 mg/kg; Tokyo Chemical Industry, Tokyo, Japan) in propylene glycol for 3 d. One day after the last injection of testosterone propionate, the rats received a single intravenous injection of MNU (30 mg/kg; Sigma Chemical Co.) in sterile saline. One week after the injection of MNU, 2 Silastic tubes (Korea Ace Scientific, Seoul, Republic of Korea) containing 40 mg of crystalline testosterone were implanted into each rat subcutaneously. Oral administration of RCM (400 mg/kg) was started 1 week before administration of MNU (5 d/week for 10 weeks). The detailed composition of the RCM extract was the same as that used in our previous study [14]. All animals were euthanized by Zoletil® (Virbac, Carros, France) and Rompun® (Bayer, Leverkusen, Germany) at 10 weeks after starting treatment with RCM. Before necropsy, prostatic enlargement was determined using ultrasonography (Sonoace 9900, Medison, Seoul, Republic of Korea).

Serum biochemistry

After non-invasive evaluation of the prostate gland, blood samples were collected from the abdominal vein through a laparotomy incision. The serum samples were stored at −70 °C until further analyses. Serum alkaline phosphatase (ALP) levels were estimated by using an automated serum biochemistry analyzer (Cobas C111; Roche Co., Seoul, Republic of Korea) to evaluate osteoblast activity. Serum levels of pyridinoline (PYD), a bone turnover marker, were measured by using an enzyme-linked immunosorbent assay with the Metra® PYD enzyme immunoassay kit (Quidel Co., San Diego, CA) following the manufacturer’s instructions. In brief, filtered serum samples (25 µL) were incubated overnight in the dark with 75 µL of the PYD antibodies and reagent at 4 °C. After three washings, the samples were incubated with an enzyme conjugate for 60 min at room temperature. Then, 150 µL of the substrate solution was added to the samples and incubated for 40 min at room temperature. A Sunrise™ absorbance reader (Tecan Group Ltd., Maennedorf, Switzerland) (wavelength, 405 nm) was used for the assay quantification, and the optical density values were converted to PYD concentrations by using a standard curve.

Histopathology and immunohistochemistry

The right femur was fixed in 10% neutral buffered formalin and soaked with a decalcifying solution, RapidCal Immuno™ (BBC Biochemical, WA), then subjected to routine tissue processing procedures. Sections were stained with hematoxylin and eosin for general microscopic evaluation and with Azan for differentiation of osteoid from mineralized bone in the femur. On histopathological examinations of the femur, the distal metaphyseal trabeculae were traced by using the Image J program (version 1.43 u) for calculation of the changes in trabecular dimension and trabecular thickness.

Immunohistochemistry was performed to identify the expression of bone ALP, an indicator of osteoblast activation in the bone. The sections were deparaffinized, rehydrated, and incubated in a methanol solution containing 3% hydrogen peroxidase for 30 min. The tissue slides were then treated with monoclonal anti-ALP primary antibody at a dilution of 1:100 (Santa Cruz Biotechnology Inc., Heidelberg, Germany) and visualized by using an avidin–biotin–peroxidase complex kit (Vector Laboratories, Burlingame, CA). The control group was treated with normal rabbit serum instead of the monoclonal anti-ALP primary antibody. We used 3,3-diaminobenzidine (Zymed Laboratories, San Francisco, CA) as the chromogen and methyl green for counterstaining. The intensity of immunoreactivity was measured by using the Leica Application Suite (Version 2.8.1; Leica Microsystems, Heerbrugg, Switzerland), and the relative ratio of bone ALP expression was examined.

On histopathological examination, the prostatic acini were evaluated considering a cumulative histoscore system for degree of crowding and intraluminal villosity, loss of basal nuclear polarity, and number of hyperplastic nodules [21]. This scoring system, expressed in arbitrary units, allowed us to make a better comparative assessment of the prostatic hyperplastic conditions in rats (Table 1).

Table 1. Cumulative chart score of histopathological findings in the rat prostate.

Low-power magnification (objective 10×)
Lumen shape	regular (1), villamentous (3), papillary (4), cribriform (5)
Acinar shape	tubular (1), branched (3), irregular (5)
Interacinar space	large (1), moderate (1), back to back (5)
Stroma	fine (1), moderate (3), fibrosis (5)
High-power magnification (objective 25–40×)
Epithelial shape	flat (1), cuboidal (1), cylindrical (3), hexagonal (5)
Nuclear shape	round (1), small/large (2/2), irregular (5)
Mitoses per field	absent (0), isolated [1–2] (2), abundant [3–5] (5), excessive [>6] (10)
Numbers of layers	mono (1), oligo [2–3] (3), pluri [>5] (5)
Incidence	focal (3), diffuse (5)
Cell alignment	polar (1), apolar (3), pilling up (3), budding out (5), periacinar (3), isolated clusters (5)
Lesion distribution	unilobar: isolated (2), multiple (6); bilobar: isolated (4), multiple (8)
Basal membrane	intact (1), thin (1), thick (5), interrupted (5)

Semi-quantitative reverse-transcription polymerase chain reaction

Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) was performed to identify the expression of receptor activators of nuclear factor kappa B ligand (RANKL), osteoprotegerin (OPG), osteocalcin (OCN), cannabinoid receptor (CB)1, CB2, Runt-related transcription factor 2 (RUNX2), activating transcription factor (ATF) 4, and embryonic stem cell phosphatase (Esp). The purity of the extracted RNA was confirmed by visualizing two ribosomal RNA bands on electrophoresis gels and calculating the OD₂₆₀/OD₂₈₀ ratio from the measured absorbance values. The extracted RNA was reverse transcribed into single-stranded complementary DNA by using 1 µg of extracted RNA, reverse transcriptase, random primers, deoxynucleotide triphosphates, and ribonuclease inhibitor (Takara Bio Inc., Shiga, Japan). The AccuPower® RT PreMix (Bioneer, Daejeon, Republic of Korea) was used to amplify 25 ng of complementary DNA in a Mastercycler (Eppendorf, Hamburg, Germany). The primer sets used for RT-PCR are listed in Table 2. The PCR products were separated on agarose gels by using electrophoresis, and the intensity of ethidium bromide fluorescence was evaluated by using the ProXima image system (Isogen Life Science, De Meern, Netherlands) to detect ethidium bromide staining. Serial changes in expression of the target genes were evaluated by using semi-quantitative RT-PCR with the GAPDH gene as the control.

Table 2. Primer sequences for semi-quantitative RT-PCR.

Gene	Source	Sequence (5′–3′)	Predicted length (bp)
RANKL	NM_057149.1	F: TCA GGA GTT CCA GCT ATG AT R: CCA TCA GCT GAA GAT AGT CC	298
OPG	NM_012870.2	F: GAC GAG ATT GAG AGA ACG AG R: GGT GCT TGA CTT TCT AGG TG	502
OCN	NM_013414.1	F: TGA GGA CCC TCT CTC TGC TC R: GAG CTC ACA CAC CTC CCT GT	221
CB1	NM_012784.4	F: TGC ACT CCC GCA GTC TCC GA R: GTT GGC GTG CTT GTG CAG GC	887
CB2	NM_001164143.1	F: GGA GTA CAT GAT CTT GAG TGA T R: AGA ACA GGG ACT AGG ACA AC	472
RUNX2	NM_001278484.1	F: GAC AGC CCC AAC TTC CTG T R: ATG CGC CCT AAA TCA CTG AG	407
ATF4	NM_024403.1	F: AAC AGC GAA GTG TTG GCG GGG R: CGC CTT GTC GCT GGA GAA CCC	141
Esp	NM_033099.1	F: GGC GGA CCC ATC GGC CTA TCC CCA R: GTC TCT TTA TTC CTG GAC TCT GCC	1866
GAPDH	AF106860.2	F: TAC ATT TTG CTG ATG ACT GG R: TGA ATG GTA GGA GCT TGA CT	202

Statistical analysis

Statistical analysis was performed by using the SPSS software package (version 19.0). All data are expressed as mean and standard deviation. Inter-group analysis was performed by using the two-tailed unpaired t-test and one-way analysis of variance followed by the Tukey post-hoc test. p-values of 0.05, 0.01 or 0.001 were considered statistically significant.

Results

Effects of RCM on body weight

Changes in body weight during the experimental period are shown in Figure 1(A). The control group showed continuous body weight gain, while suppression of body weight gain was observed in the MNU and RCM groups. Treatment with RCM had little effect on body weight gain. The body weights of the control, MNU, and RCM groups at 10 weeks were 600 ± 43.4 g, 454.2 ± 22.2 g, and 438.5 ± 30.2 g, respectively.

Figure 1. (A) Changes in body weight during the experimental period. At 10 weeks of treatment, the control, MNU, and RCM groups had a 58.31%, 26.37%, and 30.56% increase in body weight, respectively. Results are expressed as mean ± SD. (B) Serum biochemical analysis of ALP and PYD. Compared with the MNU group, the RCM group showed a significant increase in serum ALP levels and a significant decrease in serum PYD levels, indicating reduced osteoclastic resorption. Results are expressed as mean ± SD. #p < 0.05, significantly different from the MNU group. (C) Ultrasonographic image showing the prostate gland close to the urinary bladder and heterogeneous echogenicity. The width (dotted arrow) and length (arrow) of the prostate gland were higher in the MNU and RCM groups. (D) The size and the weight of the prostate gland, as observed on ultrasonographic images, in both the MNU and RCM groups were 1.6–1.9 times the corresponding values of the control group. Results are expressed as mean ± SD. *p < 0.05 and **p < 0.01, significantly different from the control group, #p < 0.001 significantly different from the MNU group. SD, standard deviation; ALP, alkaline phosphatase; MNU, N-methyl-N-nitrosourea; PYD, pyridinoline; RCM, Rubus coreanus Miquel.

Effects of RCM on serum ALP and PYD levels

The serum ALP levels were significantly higher in the RCM group than in the MNU group (p < 0.05). The serum PYD levels were the highest in the MNU group, and the levels in the RCM group were similar to those in the control group (Figure 1B).

Effects of RCM on prostatic hyperplasia on non-invasive examination

The prostate gland was attached to the bladder neck, and it showed heterogeneous echogenicity. The MNU and RCM groups had a larger prostate area than the control group, but there were no observable differences between the prostate area in the MNU and RCM groups (Figure 1C). The width and length of the prostate gland were measured by using ultrasonography, and their product was used to obtain an estimate of its size. The estimated size of the prostate gland was normalized according to the body weight (width on ultrasonography/body weight). On necropsy, the wet weight of the prostate gland was recorded. Subsequently, each wet weight value was normalized according to the individual body weight (prostate gland weight/body weight). The width on ultrasonography/body weight and prostate gland weight/body weight values in the MNU and RCM groups were significantly higher than those in the control group (Figure 1D).

Effects of RCM on hyperplastic changes in the prostate gland

Histopathological evaluation revealed marked hyperplastic changes in the prostate gland in both the MNU and RCM groups. The MNU group had the most severe hyperplastic changes in the prostate gland. The control group showed regular-shaped acini within fine interstitial stroma. The acini of monolayered cuboidal cells were surrounded by a thin, intact basement membrane. The monomorphic epithelial cells consisted of abundant eosinophilic cytoplasm and rounded nuclei without mitotic figures. In contrast, the high-scoring prostate gland in the MNU group showed intraluminal villous projections developing in the prostate gland with pluri-layered cylindrical cells and a partially interrupted basement membrane. The hyperplastic epithelial cells contained round to ovoid nuclei and mitosis with relatively high-collagenous stroma. In some cases, epithelial hyperplastic nodules, either piling or budding, were observed (Figure 2A, arrowheads). These results were quantified with a histoscore system, and although the difference was not statistically significant, the RCM group had a lower histoscore than the MNU group (Figure 2B).

Figure 2. Histopathological evaluation of the prostate gland. (A) On low-power magnification (40×; left images), the control group showed regular-shaped acini with fine collagenous stroma. The MNU and RCM groups showed tubular or branch-shaped acini with variable stroma components, although the RCM group showed slightly smaller lesions. On high-power magnification (200×; right images), the acini of the MNU and RCM groups showed cuboidal to cylindrical epithelial cells with round to ovoid nuclei and an interrupted basement membrane. Hyperplastic and dysplastic nodules were frequently observed in the MNU group and were shown to be budding out, pilling up, or forming isolated clusters (arrowheads); meanwhile, the RCM group showed few hyperplastic nodules. (B) The MNU group had the highest histoscore, indicating marked dysplastic changes in the prostate gland. The RCM group had a lower histoscore than the MNU group. Results are expressed as mean ± SD. **p < 0.01, significantly different from the control group. SD, standard deviation; MNU, N-methyl-N-nitrosourea; RCM, Rubus coreanus Miquel.

Effects of RCM on the histopathology of trabecular bone

The RCM group had better restoration of trabecular dimension and thickness than the MNU group, and histopathological analysis of the femur showed similar results (Figure 3A and B). Immunohistochemical analysis of bone ALP levels showed that the trabecular bone of the control and RCM groups was lined with ALP-positive osteoblasts, whereas ALP positivity was barely observed in the MNU group (Figure 3C). The intensity of immunoreactivity for bone ALP was scored from 1 (weak) to 5 (strong), and the RCM group had the strongest level of intensity among the three groups. The number of osteoclasts was significantly higher in the MNU group than in the control group and was lower in the RCM group (Figure 3D).

Figure 3. Histopathological evaluation and immunohistochemical analysis of the markers in the femur. (A) The control group showed thick trabecular bone with abundant bone marrow cells, whereas the femur of the MNU and RCM groups had smaller trabecular dimensions (magnification 40×). (B) The RCM group showed slightly higher bone volume, which was related to increased trabecular thickness and lower trabecular separation, than the MNU group. Results are expressed as mean ± SD. (C) The femur of the RCM group showed the strongest reactivity for bone ALP (magnification 200×). (D) The relative expression of bone ALP was significantly higher in the RCM group than in the MNU group. The number of osteoclasts in the MNU group was moderately higher than that in the control group, whereas the RCM group had similar values as the control group. Results are expressed as mean ± SD. **p < 0.05, ***p < 0.001; significantly different from the Control group, ##p < 0.05, ###p < 0.001 significantly different from the MNU group. SD, standard deviation; ALP, alkaline phosphatase; MNU, N-methyl-N-nitrosourea; RCM, Rubus coreanus Miquel.

Effects of RCM on levels of RANKL, OPG, OCN, CB1, and CB2 in bone

Expression levels of RANKL, OPG, OCN, CB1, and CB2 in bone (Figure 4A) and the prostate gland (Figure 4B) were measured by using semi-quantitative RT-PCR. Compared with the control group, the MNU group had higher RANKL expression and lower OCN expression, indicating excessive formation of osteoclasts and reduction of osteoblastic activation. However, the RCM group had higher expression levels of OPG, CB1, and CB2 and lower expression levels of RANKL than the MNU group (Figure 4A). There were no differences in OCN expression among the three experimental groups. As OPG inhibits osteoclast activation, an increased RANKL/OPG ratio indicates excessive bone loss; the RANKL/OPG ratio in the MNU group (3.00) was significantly higher than that in the control group (1.96) and was lower than that in the RCM group (0.49). This result showed that RCM prevents the progression of osteoclastic bone loss.

Figure 4. Semi-quantitative RT-PCR for RANKL, OPG, OCN, CB1, and CB2. (A) The MNU group, compared with the control group, showed higher RANKL expression in the femur. However, the expression levels of other genes were similar to those in the control group. Compared with the MNU group, the RCM group showed higher expression of OPG, CB1, and CB2 as well as lower expression of RANKL. (B) For the prostate gland, OPG levels were significantly higher (p < 0.05) in the RCM group than in the control group. RANKL expression showed OPG-related changes. Compared with the MNU group, the RCM group showed an approximately 42% increase in OPG levels. However, the mRNA levels of CB1 and CB2 significantly lower in the RCM. Results are expressed as mean ± SD. *p < 0.05, significantly different from the control group. SD, standard deviation; CB, cannabinoid receptor; MNU, N-methyl-N-nitrosourea; OPG, osteoprotegerin; OCN, osteocalcin; RANKL, receptor activators of nuclear factor kappa beta ligand; RCM, Rubus coreanus Miquel.

Effects of RCM on prostatic hyperplasia in relation to maintaining bone mass

OPG levels in the RCM group were significantly higher (p < 0.05) than those in the control group. Compared with the MNU group, the RCM group had an approximately 42% increase in OPG levels (Figure 4B). The RANKL messenger RNA (mRNA) level showed OPG-related changes. The mRNA expression levels of ATF4 and RUNX2 were similar in the MNU group (Figure 5), and the levels of these two transcription factors were lower in the RCM group. In addition, the Esp levels were significantly higher whereas the OCN levels decreased in the RCM group compared to the other groups (Figure 4A and B). However, the mRNA levels of CB1 and CB2 showed a significant decrease in the RCM group.

Figure 5. Expression of RUNX2, ATF4, and Esp in the prostate gland was determined by using semi-quantitative RT-PCR. The level of two transcription factors was higher in the MNU group. Expression of Esp significantly increased in the MNU and RCM groups. Results are expressed as mean ± SD. *p < 0.05, significantly different from the control group. SD, standard deviation; ATF, activating transcription factor; Esp, embryonic stem cell phosphatase; MNU, N-methyl-N-nitrosourea; RCM, Rubus coreanus Miquel; RT-PCR, reverse-transcription polymerase chain reaction; RUNX, Runt-related transcription factor 2.

Discussion

ADT for prostate cancer is an important risk factor for osteoporosis, which is also highly induced before hormonal treatment [22,23]. In some cases of prostatic disease, the acini of the prostate gland can secrete soluble factors such as interleukin-1, interleukin-6, and parathyroid hormone-related proteins to stimulate RANKL production and can also directly produce RANKL and receptor activators of nuclear factor kappa beta (RANK) [7,24]. RANKL, a tumor necrosis factor ligand superfamily member, is a membrane-bound protein on the surface of osteoblasts that binds to RANK on the surface of osteoclast precursors to induce osteoclastogenesis and activation of mature osteoclasts [7,24]. For these reasons, increased levels of RANKL and RANK expression in prostatic disease can induce excessive osteoclastic osteolysis. In addition, ATF4 accumulates in osteoblasts and influences the bone mass as well as regulatory role in endocrine disorder-related cellular metabolism [25]. Upregulation of ATF4 and RUNX2 can influence osteoclastic differentiation. A high proportion of patients with prostate cancer have a high risk of osteoporosis because of advanced age, poor nutrition, and vitamin D deficiency, indicating that prostatic disease is typically associated with an increase in bone loss [26]. RCM is a traditionally used remedy; however, scientific studies highlighting the efficacy and mechanism of RCM are limited, and only a few studies have been reported to date [14,17,18]. Therefore, the present study was performed to investigate the anti-osteoporotic effects of RCM on MNU-induced prostatic hyperplasia. MNU-induced prostatic hyperplasia was selected to serve as a model of osteoporosis in aging men. Administration of MNU with testosterone has been reported to be an effective method to induce prostatic adenocarcinoma in rats but takes more than 40 weeks [27]. After short-term treatment with MNU and testosterone combination, the animal develops prostatic hyperplasia, which leads to dysplasia and intraepithelial neoplasia; therefore, we selected this animal model for our experiments [19,20,27,28].

The MNU-induced prostatic hyperplasia rat model showed marked dysplastic changes in the prostate gland as well as severe trabecular bone loss in the femur. However, the results of serum biochemical analysis for ALP and PYD showed that treatment with RCM reduced bone resorption and maintained bone volume compared with controls. Furthermore, rats treated with RCM had increased trabecular bone dimension and activation of osteoblasts as well as a reduction in the number of osteoclasts in the femur. The histopathological improvements observed in the femur after RCM treatment were correlated to the increased expression of OPG and decreased expression of RANKL. In addition, histopathological evaluation revealed that treatment with RCM reduced MNU-induced hyperplastic changes in the prostate gland, although the difference was not significant.

Furthermore, RCM-treated rats had higher CB1 and CB2 expression in the bone tissue than the MNU-treated rats. Cannabinoids are heterogeneous molecules that bind to cannabinoid receptors, and there are two distinct subtypes of cannabinoids, CB1 and CB2, depending on the specific tissue or organ [29]. The presence and functions of the endocannabinoid system have been identified in various tissues. In particular, in the skeletal system, activated CB1 regulates bone formation by influencing osteoblast differentiation of bone marrow stromal cells [16,30,31]. In contrast, CB2 is expressed in both osteoblastic and osteoclastic roles in the differentiation and activation of bone cells. Activation of CB2 triggers expansion of the preosteoblastic pool and stimulates the functioning of differentiated osteoblasts in bone remodeling. It also restrains mitogenesis at the monocytic stage and suppresses osteoclast formation by repressing RANKL expression in osteoblasts [16,24,29,30]. Therefore, we suggest that the anti-osteoporotic effect of RCM in prostatic dysplasia is at least partly influenced by the endocannabinoid system-dependent upregulation of osteoblastogenesis, and this leads to inhibition of osteoclastic osteolysis as well as downregulation of tumorigenesis in the prostate gland. CB1 is normally expressed in the epithelial cells of the prostatic duct, and it plays a role in the maintenance of prostatic homeostasis, including the architecture and functioning of the prostate gland [32,33]. Considering our results for CB1 and CB2 expression in the prostate gland, we suggest that treatment with RCM mediates both inhibition of prostatic dysplasia and maintenance of bone mass by cannabinoid signaling.

It is assumed that some useful compounds interact in combination for the effectiveness of RCM, but in the present study, it was not enough to identify a fractional component from the RCM extracts. Although the present study has some limitations, including examination of CB1 and CB2 changes at the mRNA level, the observation that RCM prevents MNU-induced osteoporosis by the simultaneous regulation of osteoblasts and osteoclasts supports further research on the use of RCM as a useful functional food supplement for the prevention and treatment of osteoporosis induced by prostatic disease.

Declaration of interest

This work was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD, Basic Research Promotion Fund) (NRF-2008-331-E00383). The authors report no declarations of interest.