Abstract
Background/Aim: Receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG), regulate the cognate receptor RANK on osteoclast precursor cells. Herein we examined the inhibitory effects of palmatine on bone metabolism using ovariectomized (OVX) mice. Materials and Methods: The first experimentaI set was designed to histologically and biochemically examine mice randomly divided into four groups: sham-operated, OVX, and OVX-palmatine intake groups (1 mg/kg and 10 mg/kg). The second experimental set examined the influence of palmatine on osteoblast-like cells in vitro. Results: Palmatine caused significant suppression of osteoclast numbers in tissues. In palmatine-treated mice, RANKL and OPG expression decreased. In the culture supernatant of MC3T3-E1 cells, RANKL and OPG levels were significantly reduced by palmatine addition. Conclusion: Palmatine may attenuate osteoclast differentiation through inhibition of RANKL and OPG expression by osteoblasts. Therefore, palmatine might be a candidate anti-resorptive agent for osteoporosis therapy.
- Receptor activator of nuclear factor kappa-B ligand
- osteoprotegerin
- ovariectomized mice
- palmatine
- postmenopausal osteoporosis
Osteoporosis is a major global health problem, resulting in more than 200 million osteoporotic fractures each year worldwide, including 1.6 million hip fractures (1). Osteoporosis affects both men and women of all races. White and Asian women, particularly older post-menopausal women, are at the highest risk. Bone mass peaks in the twenties and gradually decreases after that due to bone metabolism. In women, bone resorption accelerates with menopause due to a decline in the estrogen level. During menopause, a high turnover rate of bone loss characterized by increased osteoclast activity is observed. Later, the system swings back to normalized osteoclast activity with a slight deficit in osteoblast function and a low turnover of bone loss (1).
Bisphosphonates are commonly used for the treatment of osteoporosis and to improve the quality of life in a vast majority of patients (2). However, a number of reports have described necrotic maxillofacial bone lesions in patients who have received high dosage of bisphosphonates, particularly when the patient undergoes subsequent dental procedures. Despite numerous investigations, the problem of the treatment and prevention of osteoporosis remains unresolved.
Palmatine is a quaternary protoberberine alkaloid. It is typically yellow in color and is an active constituent of a number of plants, such as Coptidis rhizoma (3). The alkaloid has been used in the treatment of jaundice, dysentery, hypertension, inflammation, and liver-related diseases (4). It has been reported that berberine, an isoquinoline alkaloid which is a close structural analog of palmatine, inhibited bone loss in an osteoporosis model (5). However, there are few reports on the effect of palmatine on bone loss.
Ovariectomy has been extensively used to establish an experimental animal model for post-menopausal osteoporosis. Ovariectomized animals exhibited a significant decrease in bone mineral density (BMD) and bone formation (6).
Therefore, in order to evaluate the osteoimmunological effects of a component derived from the crude drug on post-menopausal osteoporosis, we examined whether palmatine could prevent bone loss in OVX mice in vivo and control cytokine secretion of osteoblasts in vitro.
Materials and Methods
Reagents. Palmatine chloride (Wako Pure Chemical Industries, Ltd., Tokyo, Japan) was used in the present study. It was dissolved in distilled water to be used for in vivo experiments or with culture medium for in vitro experiments. Culture medium was Dulbecco's modified Eagle's medium (DMEM; Sigma–Aldrich Corporation, St. Louis, MO, USA) supplemented with 10% heat-inactivated fetal calf serum (FCS; Nihon Bio-Supply Center, Tokyo, Japan), sterilized by passing through 0.2-μm pore filters, and stored at 4°C until use.
Animals. Specific 7-week-old pathogen-free ICR female mice were purchased from Japan CLEA Co., Ltd. (Tokyo, Japan). Mice were maintained on a 12-h light/dark schedule (lights on at 8 a.m.) at 25±2°C, humidity 50±2%, and provided with food and water ad libitum. After an acclimation period of one week, mice were subjected to bilateral ovariectomy (OVX) or sham operation (Sham) via dorsolateral incision under anesthesia with sodium pentobarbital (50 mg/kg i.p. (7, 8). The ovaries were excised, oviducts were replaced, and incisions were closed. The effect of ovariectomy was confirmed by measuring body and uterine weights at the time of the experiment (9, 10). Mice were fed with a normal diet (CE-2, CLEA Japan, Tokyo, Japan) for 13 weeks from the day after surgery. Mice were randomly divided into four groups of five mice/group: Sham group; OVX group (OVX-P0); group of OVX plus 1 mg/kg palmatine (OVX-P1); and a group of OVX plus 10 mg/kg palmatine (OVX-P10). One week after the ovariectomy, OVX-P1 and OVX-P10 groups were given 200 μl palmatine solution via gavage once a day. The sham and OVX groups were left untreated to exhibit constitutive levels of enzymes. The animals were sacrificed 24 h after the last treatment, and tibias were immediately excised for histological analysis. Blood was obtained by cardiac puncture and sera was obtained and stored at −80°C until use. This study was approved by the Ethics Committee of Showa University for animal experiments (No. 05092).
Histochemical demonstration of tartrate-resistant acid phosphatase (TRAP) activity. After sacrificing mice, tibias were dissected free of surrounding tissues. Samples were then fixed in 4% neutral buffered formalin, de-calcified in 10% EDTA–TBS, dehydrated, and embedded in paraffin. Serial 1-μm-thick sections were then stained with TRAP (TRAP/ALP stain kit; Wako Pure Chemical Industries, Ltd., Tokyo, Japan). Briefly, deparaffinized and rehydrated tissue sections were incubated at room temperature in acetate-tartaric acid buffer (pH 5.0) for 20 min. Sections were then incubated at 37°C for 15-20 min in freshly prepared acetate-tartaric acid buffer, pH 5.0, containing 0.5 mg/ml naphthol ASMX phosphate and 1.1 mg/ml Fast Red TR (TRAP substrate). After washing with distilled water, tissues were counterstained with aqueous hematoxylin and mounted with Vectamount (Vector Laboratories, Inc., Burlingame, CA, USA) (11).
Quantification of TRAP-positive cells. The number of osteoclasts appearing in digital images were captured with a System Microscope BX53 and a DP21 digital camera (Olympus Co., Tokyo, Japan) and counted within the primary spongiosa of the proximal tibia in a region of interest starting 1.0 mm proximal to the growth plate and extending 1.0 mm in length and 3.0 mm medially from the periosteum. The number of cells was determined within five tissue sections. Data are expressed as the mean number of cells per 1.0 mm of trabecular bone (11).
Osteoblast-like cellular cytokine secretion. MC3T3-E1 cell line was purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan) and resuspended at a density of 5×105 cells/ml in DMEM-FCS and cultured with different concentrations of palmatine in 24-well plates in triplicate. After 24 h, culture supernatants were obtained and stored at −80°C until use for protein measurements of receptor activator of nuclear factor-κB (NFκB) ligand (RANKL) and osteoprotegerin (OPG) by enzyme-linked immunosorbent assay (ELISA; see below). Additionally, MC3T3-E1 cells were collected and used for the measurement of cytokine mRNA expression by real-time reverse transcription polymerase chain reaction (PCR; see below). Furthermore, we evaluated the effects of palmatine for TNF-α family, RANKL and OPG, secretion on osteoblast under lipopolysaccharide (LPS; Sigma–Aldrich Corporation, St. Louis, MO, USA), which activates NF-κB. LPS (1 μg/ml) was added to the culture medium in the examination of cytokine secretion and mRNA expression.
Assay for cytokine production. Cytokine levels in serum samples and culture supernatants were examined using commercially available mouse RANKL or OPG ELISA kits (MTR00, MOP00; R&D Systems, Inc., Minneapolis, MN, USA) according to the manufacturer's recommendations. The sensitivity of the RANKL assay kit was 5.0 pg/ml and OPG assay kit was 6.9 pg/ml. The absorbance at 450 nm was measured by a Multiskan™ GO instrument (Thermo Fisher Scientific Inc. Waltham, MA, USA).
Cell viability in cytotoxicity CCK-8 assays. The cell viability was measured using the Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) (12). Cells were seeded in a 96-well flat-bottomed microplate (5×103 cells/well) and cultured in 100 μl of growth medium at 37°C and 5% CO2 for 24 h. The cell culture medium in each well was then replaced with 100 μl of cell growth medium containing palmatine at concentrations of 1, 5, 10, 40, 100, and 200 μM (13, 14). After incubation for 3 days at 37°C, palmatine was washed out with PBS three times then 10 μl of CCK-8 dye and 100 μl of α-minimum essential medium were added to each well, and cells were incubated for another 1 h at 37°C. The cell viability was calculated using the following formula: where OD (experimental sample) refers to the absorbance of a well with treated cells and CCK-8, while OD (blank) was the absorbance of a well with medium and CCK-8 but without cells, and OD (control) was the absorbance of a well with untreated cells and CCK-8 (12). The absorbance at 450 nm was measured by a Multiskan™ GO instrument, and the results are presented as the mean±SD from triplicate wells.
PCR primers and reagent kits. The reagents used for mRNA isolation (TaqMan Gene Expression Cells-to-Ct™) and real-time RT-PCR (TaqMan Gene Expression Assays) were purchased from Applied Biosystems (Foster City, CA, USA). These assays were performed according to the manufacturer's instructions (15). For real-time reverse transcription (RT)-PCR comparison of gene expression, we selected RANKL (Tnfsfll: TaqMan Gene Expression Assays; Assay ID: Mm00441906_m1) and OPG (Tnfrsfl: TaqMan Gene Expression Assays; Assay ID: Mm0205928_m1 related to vascularization). Rn18s, 18S ribosomal RNA, was used as a housekeeping gene to normalize for RNA loading.
mRNA isolation, quantitative RT-PCR. Total RNA was isolated from MC3T3-E1 cells using 50 μl Lysis Solution (P/N4383583). Each sample of total RNA was subjected to RT using 20× RT Enzyme Mix (P/N 4383585) and 2× RT Buffer (P/N43833586) with Applied Biosystems 2720 Thermal Cycler (Applied Biosystems). After the RT reaction, the cDNA templates were amplified by PCR using TaqMan Gene Expression Assays, PCR primers, and RT Master Mix (P/N 4369016). Pre-designed and validated gene-specific TaqMan Gene Expression Assays (15, 16) from Applied Biosystems were used in duplicate for quantitative RT-PCR according to the manufacturer's protocol. PCR assays were performed as follows: 10 min denaturation at 95°C, 40 cycles of 15 s denaturation at 95°C, and 1 min annealing and extension at 60°C. Samples were analyzed using an ABI Prism 7900HT Fast Real-Time PCR System (Applied Biosystems) (16, 17). Relative quantification (RQ) studies (18) were prepared from collected data (threshold cycle numbers, referred to as Ct) with ABI Prism 7900HT Sequence-Detection System (SDS) software 2.3 (Applied Biosystems).
Statistical analysis. Data are expressed as means±standard deviations. All assays were repeated three times to ensure reproducibility. Statistical significance of effuses between control and experimental groups was analyzed by one-way analysis of variance followed by the Scheffe test. A probability (p) value of less than 0.05 was considered statistically significant.
Results
Histochemistry for bone tissue (tibias). Shinbones were preserved in formalin and embedded in paraffin. To identify the dose-dependent effects of orally administered palmatine on trabecular bone architecture, proximal tibias were observed with an optical microscope (Figure 1). This specimen slice was stained with TRAP and osteoclasts were counted (dark purple cell indicated by the arrow). The blue area in the Figure is the epiphyseal cartilage, and the pink area is the trabecular bone. The trabecular bone quantity decreased in the OVX-P0 group compared to the sham group but seemed to remain steady in the OVX-P10 group. The number of osteoclasts within the spongiosa in the proximal tibia was counted. Data are expressed as mean cell number per mm of the trabecular bone. Compared to animals of the shown group, the number of TRAP-positive osteoclasts increased in animals of the OVX-P0 group (non-palmatine addition). In addition, compared to OVX-P0 animals, the number of osteoclasts decreased in animals of the OVX-P1 and OVX-P10 groups (Figure 2).
Serum cytokine levels. The experiments were conducted to examine the influence of palmatine on cytokine secretion into serum in vivo. The serum cytokine levels were examined by ELISA. As shown in Figure 3, serum RANKL levels of the OVX-P0 group increased in comparison to the sham group; both serum RANKL levels and OPG levels decreased depending on the concentration of palmatine administered. Furthermore, the RANKL/OPG ratio also decreased depending on the concentration of palmatine.
Cell viability. The proliferation of MC3T3-E1 cells cultured with different doses of palmatine was evaluated using a CCK-8 assay. There was no significant difference in cell proliferation between palmatine-treated groups, even at high doses of 100 μM, compared to that in the control group (Figure 4). This result suggests that MC3T3-E1 cell proliferation for different dose groups followed a normal growth trend.
Cytokine levels in cell culture supernatant. Experiments were conducted to examine the influence of palmatine on cytokine production from MC3T3-E1 cells under LPS in vitro. The cytokine content in culture supernatants were examined by ELISA. As shown in Figure 5, 10 μM palmatine significantly reduced RANKL and OPG levels in the culture supernatant of MC3T3-E1 cells.
Effect of palmatine on the RANKL and OPG mRNA expression. The final experiments were conducted to examine whether addition of palmatine would change RANKL and OPG mRNA expression in MC3T3-E1 cells. As shown in Figure 6, 10 μM palmatine significantly reduced RANKL and OPG mRNA expressions in the culture supernatant of MC3T3-E1 cells under LPS.
Discussion
Bone is a living tissue that is constantly being degraded and replaced. Osteoporosis occurs when creation of new bone does not keep up with the removal of old bone. Older postmenopausal women are at the highest risk for developing osteoporosis. In the current study, we examined the effects of daily palmatine administration on bone metabolism in normal and ovariectomized mice. Ovariectomy increased the trabecular bone quantity in the shinbone metaphysis by suppressing bone resorption. We also examined the appearance of osteoclasts, which affect bone resorption in the epiphyseal area. When compared to animals of the OVX groups, the number of TRAP-positive osteoclasts decreased in animals receiving 10 mg/kg palmatine.
The RANK/RANKL/OPG system is actively involved in the differentiation and function of osteoclasts and seems to play a central role in most pathophysiological mechanisms (19, 20). Therefore, the aim of the second study was to examine the influence of palmatine on RANKL and OPG in OVX mice serum. Serum RANKL and OPG levels obtained from normal and ovariectomized mice were measured. In the palmatine-administered group, serum RANKL and OPG levels decreased in comparison to those in other groups. Furthermore, the RANKL/OPG ratio also decreased depending on the concentration of palmatine. Administration of 10 mg/kg palmatine inhibited the decrease of trabecular bone by osteoclasts. RANKL is now considered a member of the tumor necrosis factor (TNF) family and is an osteoclast differentiation factor is produced by osteoblasts or stromal cells (21, 22). Activation of the receptor for RANKL (RANK) on the surface of osteoclast precursors delivers an essential signal leading to osteoclast differentiation and bone resorption (23). In contrast, OPG is a secreted member of the TNF receptor family that is inhibitory of osteoclastogenesis due to its ability to bind to RANKL as a decoy receptor, and inhibits secretion of RANKL in osteoblasts (24). The decrease in RANKL expression and in RANKL/OPG ratio caused by palmatine in our study may be related to the reaction induced for the suppression of bone resorption. Proteins of the TNF family regulate various physiological functions, including immune responses, organ development, and hematopoiesis (25). Interactions between these proteins and their respective cognate receptors activate intracellular signaling pathways that regulate the transcriptional activation of the NF-κB and activator protein-1, as well as the activation of the caspase cascade (25, 26). Thus, proteins of the TNF family control the proliferation, survival, differentiation, and apoptosis of their target cells (25).
In our case, there are two possible explanations for the suppression of bone resorption by palmatine. The first is that palmatine directly reduces the number of osteoclasts, and the second is that the metabolism of palmatine alters the microenvironment that regulates osteoblasts. In the second part of this study, we also examined the influence of palmatine-induced cytokine production from MC3T3-E1 cells (similar to osteoblasts used in the earlier experiments) in vitro. Palmatine (10 μM) significantly reduced RANKL and OPG levels in the culture supernatant of MC3T3-E1 cells. These results show the possibility that palmatine controls cytokine, RANKL, and OPG secretion of osteoblasts and regulates crosstalk with osteoclasts. The results of PCR reveal the possibility that palmatine regulates the downstream gene expression of RANKL and OPG. RANKL and OPG are present in lysosomes and Golgi apparatus in osteoblasts. Additionally, these cytokines regulate each other's secretion by forming complexes. Palmatine may regulate the secretion or the transportation of cytokine in at least osteoblasts (27). Treatment of osteoporosis with antibodies to RANKL is currently in clinical trials and is showing efficacy in vertebral, hip, and other bone fractures in both men and women (28, 29).
Our findings provide evidence that palmatine may be beneficial for the treatment of osteoporosis similar to an antibody to RANKL. We show that palmatine regulates osteoclasts via cytokine secretion by osteoblasts. However, it is possible that palmatine contributes to osteoclasts directly. Therefore, we believe that it is necessary to further investigate the influence of palmatine on osteoclast differentiation and function.
Footnotes
This article is freely accessible online.
Conflicts of Interest
The Authors declare that there are no conflicts of interest regarding the publication of this article.
- Received September 4, 2015.
- Revision received October 12, 2015.
- Accepted October 15, 2015.
- Copyright © 2015 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved