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Dioscin reduces ovariectomy-induced bone loss by enhancing osteoblastogenesis and inhibiting osteoclastogenesis
Pharmacological Research 108 (2016) 90–101
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Dioscin reduces ovariectomy-induced bone loss by enhancing osteoblastogenesis and inhibiting osteoclastogenesis Xufeng Tao, Yan Qi, Lina Xu, Lianhong Yin, Xu Han, Youwei Xu, Changyuan Wang, Huijun Sun, Jinyong Peng ∗ College of Pharmacy, Dalian Medical University, Western 9 Lvshunnan Road, Dalian 116044, China
a r t i c l e
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Article history: Received 16 November 2015 Received in revised form 7 April 2016 Accepted 3 May 2016 Available online 4 May 2016 Keywords: Dioscin Osteoblasts Osteoclasts Ovariectomy Postmenopausal osteoporosis
a b s t r a c t Our previous studies showed that dioscin can promote osteoblasts proliferation and differentiation in vitro, but its anti-osteoporosis effect in vivo and the underlying mechanisms remain unclear. In the present work, the results showed that dioscin significantly increased the viability of MC3T3-E1 cells, ALP level and alizarin red S staining area, markedly decreased the numbers of RANKL-induced TRAPpositive multinucleated cells and bone resorption pits formation, enhanced the levels of some osteogenic markers including COL1A2, ALP and OC, which suggested that dioscin clearly promoted osteoblasts proliferation and suppressed osteoclasts formation. In vivo experiments demonstrated that dioscin obviously reduced OVX-induced body weight increase, and improved the biochemical indexes including ALP, StrACP, OC, DPD/Cr, HOP/Cr, BMD, biomechanics and microarchitecture. Moreover, H&E, TB, TRAP staining, and fluorescent double labeling tests indicated that dioscin enhanced osteoblastogenesis and inhibited osteoclastogenesis. Further researches demonstrated that dioscin promoted osteoblastogenesis through up-regulating OPG/RANKL ratio, and inhibited osteoclastogenesis through down-regulating the levels of RANKL induced TRAF6 and the downstream signal molecules including MAPKs, Akt, NF-B, AP-1, cathepsin K and NFATc1. In addition, dioscin also inhibited TLR4/MyD88 pathway to decrease the levels of TRAF6 and the related proteins. These findings provide new insights to elucidate the effects of dioscin against OVX-induced bone loss, which should be developed as a potential candidate for treating postmenopausal osteoporosis in the future. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Osteoporosis, a less musculoskeletal disease, is characterized by compromised bone strength inclining to an increased risk of fracture [1]. It is started by estrogen deficiency and particularly common in postmenopausal women [2]. Over 200 million people suffer from osteoporosis worldwide, which leads to 1.6 million people with hip fractures and 7.4 million people with other fractures
Abbreviations: Akt, protein kinase B; ALP, alkaline phosphatase; AP-1, transcription activator; BMD, bone mineral density; DPD, deoxypyridinoline; COL1A2, collagen, type I, alpha 2; Cr, creatinine; H&E, hematoxylin-eosin; HOP, hyroxyproline; MyD88, myeloid differentiation primary response gene (88); MAPKs, mitogen activated protein kinases; NFATc1, nuclear factor of activated T cells; NFB, nuclear factor-B; OC, osteocalcin; OPG, osteoprotegerin; OVX, ovariectomy; RANKL, receptor activator for nuclear factor-B ligand; StrACP, tartrate resistant acid phosphatase; TB, toluidine blue; TLR4, toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor; TRAP, tartrate-resistant acid phosphatase. ∗ Corresponding author. E-mail address: [email protected] (J. Peng). http://dx.doi.org/10.1016/j.phrs.2016.05.003 1043-6618/© 2016 Elsevier Ltd. All rights reserved.
annually [3,4]. Approximately half of the women and a quarter of the men over 50 years old have the lifetime risk of osteoporosisrelated fracture. In American, the direct fracture-caused costs are expected to raise from $17 billion in 2005 to over $22 billion by 2020 [5,6]. Thus, osteoporosis is one serious health and society problem. Mechanistically, postmenopausal osteoporosis (PMO) is a disorder of unbalanced bone remodeling with the increased bone resorption relative to bone formation, leading to the decreased bone mineral density (BMD) and the disruption of bone microarchitecture [7]. Bone resorption is completed by the polarization and the subsequent attachment to bone surface of osteoclasts. Meanwhile, bone formation occurs following osteoblasts phase that is to be developed into osteocytes [2]. Therefore, correcting the imbalance between bone resorption and formation is an effective therapy for PMO. It is well known that the factors including the receptor activator for nuclear factor-B (RANK) and its ligand RANKL, and osteoprotegerin (OPG, the decoy receptor for RANKL) associated with osteoporosis have been discovered [8]. Among them, RANKL is mainly produced by osteoblasts and stromal cells, and OPG is
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generated by osteoblasts. Binding RANKL to RANK provides a pivotal signal to drive osteoclasts generation from haematopoietic progenitor cells as well as to activate mature osteoclasts [9]. Meanwhile, OPG can negatively regulate the binding and then inhibit osteoclasts induced-high bone turnover [10]. Furthermore, Tolllike receptor 4 (TLR4), one germline-encoded pattern recognition receptor, can recognize multiple microbial pathogens including damage-associated molecular pattern molecules (DAMPs) and lipopolysaccharide (LPS), and promote the secretion of various inflammatory mediators to induce pre-osteoclast fusion and stimulate the survival of mature osteoclasts [11]. Therefore, the OPG/RANK/RANKL axis and TLR4 signal may be the potential antiosteoporosis targets. Presently, the methods for the treatment of PMO mainly include five kinds of agents including estrogens, bisphosphonates, calcitonin, selective estrogen receptor modulators (SERMs) and monoclonal antibody [12]. However, these agents may cause many adverse effects including invasive breast cancer, osteonecrosis, diarrhea, venous thromboembolism and serious infections of skin and urinary tract. Besides these, single drug target and high price of these anti-PMO agents also restrict their developing and applying in clinic [4,13]. Thus, it is urgent to exploit efficient agents for treating PMO. Traditional Chinese medicines (TCMs) with high efficiency and low toxicity have attracted great interest in recent years. Some active natural components including lycopene [14], resveratrol [15], and ginsenoside-Rb2 [16] have excellent activities against PMO. Therefore, it is reasonable to develop effective herbal products for the treatment of PMO. Dioscin (Supplemental Fig. 1a) is a natural steroidal saponin isolated from some medicinal plants [17,18]. Pharmacological investigations have shown that dioscin exhibits hepatoprotective [19–22], anti-tumor [23,24] and anti-ischemia reperfusion injury activities [25,26]. In addition, dioscin has obvious effects against obesity [27] and carbon tetrachloride-induced liver fibrosis [28,29], which also exerts inhibitory effects on several cytochrome P450 enzymes [30]. Our previous works have shown that dioscin can promote the proliferation and differentiation of pre-osteoblast like MC3T3-E1 cells and human osteoblast-like MG-63 cells in vitro [31]. However, its effects on PMO and the underlying mechanisms are unclear in our best knowledge. Therefore, the aim of the present paper was to further explore the effects of dioscin against PMO and then to investigate the underlying molecular mechanisms.
2. Materials and methods 2.1. Chemicals and materials Dioscin (purity >98%) was prepared from Dioscorea nipponica Makino in our laboratory [32,33]. In cell experiments, dioscin was added into the medium by dissolving with DMSO with final concentration of less than 0.1%, and it was suspended in 0.5% sodium carboxyl methyl cellulose (CMC-Na) in animals experiments. Fetal bovine serum (FBS), penicillin and streptomycin were obtained from Hyclone Laboratories, Inc. (Massachusetts, USA). Dulbecco’s minimum essential medium (DMEM) and alpha-minimum essential medium (␣-MEM) were purchased from Gibco (California, USA). Recombinant mouse macrophage colony-stimulating factor (M-CSF) and recombinant mouse RANKL were purchased from R&D Systems (Minneapolis, USA). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was provided by Roche Diagnostics (Basel, Switzerland). Alkaline phosphatase (ALP), tartrate resistant acid phosphatase (StrACP), hyroxyproline (HOP), calcium (Ca), phosphate (P) and creatinine (Cr) assay kits and
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hematoxylin-eosin (H&E), toluidine blue (TB) and TRAP staining kits were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Osteocalcin (OC) and deoxypyridinoline (DPD) ELISA kits were provided by Bogoo (Shanghai, China). alizarin red and calcein were purchased from Solarbio (Shanghai, China). 4 ,6-diamidino-2-phenylindole (DAPI) and LPS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tissue Protein Extraction Kit was purchased from KEYGEN Biotech. Co., Ltd. (Naijing, China). Bicinchoninic acid (BCA) Protein Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2.2. Cells culture The MC3T3-E1 cells were purchased from the Institute of Biochemistry Cell Biology (Shanghai, China) and cultured in DMEM medium supplemented with 10% FBS, 100 U/mL streptomycin and 100 U/mL penicillin at 37 ◦ C in a saturated humidified incubator (Thermo Fisher Scientific, Massachusetts, USA). Meanwhile, whole bone marrow cells were extracted from the femur and tibiae of 6week-old C57BL/6J mice. These cells were grown in ␣-MEM with 10% FBS, 100 U/mL streptomycin, 100 U/mL penicillin and 50 ng/mL M-CSF for 3 days to generate bone marrow-derived macrophages (BMMs). To generate osteoclasts, 50 ng/mL M-CSF and 100 ng/mL RANKL were added to ␣-MEM for 4 days of culture in a constant temperature and humidity incubator [34,35]. 2.3. Proliferation and ALP detection in MC3T3-E1 cells After adhering and growing for 24 h, the MC3T3-E1 cells were treated with dioscin (0, 0.25, 0.5 and 1.0 g/mL) for 24, 48 and 72 h. Then, the viability of MC3T3-E1 cells was detected by MTT assay. The MC3T3-E1 cells (1 × 104 cells/mL) were seeded in 24well culture plates, and then treated with dioscin (0, 0.25, 0.5 and 1.0 g/mL) for 72 h. After the cells were gently washed with iced PBS, permeated with 0.2% TritonX-100, and centrifuged, the supernatant was collected and used for detecting ALP activity and total protein concentration. 2.4. Alizarin red S staining in MC3T3-E1 cells The MC3T3-E1 cells (1 × 104 cells/mL) were seeded into culture dishes and incubated with different concentrations of dioscin (0, 0.25, 0.5 and 1.0 g/mL). At the 21 days differentiation, the cells were washed with PBS and fixed with ice-cold 70% ethanol (v/v) for 10 min, and then rinsed thoroughly with distilled water, stained with 40 mM alizarin red S solution in deionized water (pH = 4.2) for 10 min and rinsed with PBS. Eventually, the stained culture dishes were photographed using a digital camera. 2.5. Proliferation assay in BMMs The BMMs (1 × 105 cells/mL) were treated with various concentrations of dioscin (0, 0.25, 0.5, 1.0 and 2.0 g/mL) plus M-CSF (50 ng/mL) and RANKL (0 and 100 ng/mL) for 24 h. Next, the absorbance of the samples was quantified according to the MTT method. 2.6. TRAP staining in RANKL-induced osteoclasts The BMMs (1 × 105 cells/mL) were treated with M-CSF (50 ng/mL) and RANKL (100 ng/mL) for 4 days, and then various concentrations of dioscin (0, 0.25, 0.5, 1.0 g/mL) were added at the day 1, day 2 and day 3. At the fourth day, the cells were fixed and stained for TRAP activity, and the images were photographed by using an inverted microscope (Nikon, Japan).
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Fig. 1. Effects of dioscin on MC3T3-E1 and BMMs cells. (a) Effects of dioscin on the proliferation of MC3T3-E1 cells in 24, 48 and 72 h. (b) Effect of dioscin on ALP level of MC3T3-E1 cells in 72 h. (c) Effect of dioscin on alizarin red S staining color of MC3T3-E1 cells in 72 h. (d) Effects of dioscin on the expression levels of ALP, OC and COL1A2 in MC3T3-E1 cells. The MC3T3-E1 cells were treated with dioscin at different concentrations (0, 0.25, 0.5 and 1.0 g/mL) for 72 h. The cropped gels are used and full-length gels are presented in Supplemental Fig. 9. (e) Effect of dioscin on BMMs proliferation. * p < 0.05 and ** p < 0.01 versus control. Data are presented as the mean ± s.d. (n = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Effects of dioscin on RANKL-induced osteoclasts formation. (a) Effect of dioscin at different doses (0.25, 0.5, 1 g/mL) on RANKL-induced osteoclasts formation. (b) Effect of dioscin on RANKL-induced osteoclasts formation at different times. (c) Effects of dioscin on RANKL-induced osteoclasts bone resorption area (the black arrow) in vitro.
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Fig. 3. Effects of diosin on body, uterus weights, serum ALP, strACP activites in mice and rats. (a) Effects of diosin on body weights in mice. (b) Effects of diosin on body weights in rats. (c) Effects of diosin on uterus weights in mice. (d) Effects of diosin on uterus weights in rats. (e) Effects of dioscin on serum ALP activity in mice. (f) Effects of dioscin on serum ALP activity in rats. (g) Effects of dioscin on serum StrACP activity in mice. (h) Effects of dioscin on serum StrACP activity in rats. * p < 0.05 and ** p < 0.01 versus OVX. Data are presented as the mean ± s.d. (n ≥ 6).
2.7. Resorption pits formation assay The BMMs were seeded on bone slices with M-CSF (50 ng/mL) and RANKL (100 ng/mL), and then various concentrations of dioscin (0, 0.25, 0.5 and 1.0 g/mL) were supplied at the first day for 4 days of incubation. Then, the bone slices were stained with hematoxylin, and imaged using a light microscope (Leica DM4000B, Germany) with 40× magnification.
of Dalian Medical University (Dalian, China) (SCXK (Liao): 20130008). All experiments were approved by the Animal Care and Use Committee of Dalian Medical University, and the experimental procedures were performed in strict accordance with Legislation Regarding the Use and Care of Laboratory Animals of China. Before the experiments, the animals were allowed to suit the new environment for 7 days, and housed in a room under 12 h light/dark cycle, a controlled temperature at 22 ± 3 ◦ C and a relative humidity at 60 ± 10%.
2.8. Immunofluorescent examination 2.10. Pharmacological treatment The BMMs (1 × 105 cells/mL) were treated with various concentrations of dioscin (0, 0.25, 0.5 and 1.0 g/mL) plus RANKL (100 ng/mL) for 4 days. Then the cells were fixed with 4% paraformaldehyde, permeated with 0.2% Triton X-100 for 10 min, incubated with 5% goat serum albumin for 1 h at 37 ◦ C, and then incubated with the anti-TRAF6 antibody (1: 100 dilution) overnight at 4 ◦ C. At last, the cells were incubated using a TRITC-conjugated Goat Anti-Rabbit IgG (H + L) for 1 h at 37 ◦ C and then counterstained with DAPI (5 g/mL) for 10 min. Images were captured by a fluorescent microscopy (Olympus BX63, Japan) with 200× magnification. 2.9. Animals Three-month old female SD rats and six-week old female C57BL/6 mice were provided by the Experimental Animal Center
In the mice experiments, the animals were randomly divided into six groups: sham and OVX (model) groups in which the mice were treated with vehicle (0.5% CMC-Na), OVX + dioscin groups in which the mice were treated with 28, 56 and 84 mg/kg of dioscin, and the positive group in which the mice were treated with 17-Estradiol (E2 , 35 g/kg). In the rat experiments, the animals were randomly divided into six groups: sham and OVX (model) groups in which the rats were treated with vehicle (0.5% CMC-Na), OVX + dioscin groups in which the rats were treated with 20, 40 and 60 mg/kg of dioscin, and the positive group in which the rats were treated with E2 (25 g/kg). Vehicle, dioscin and E2 were all orally administrated on the week 2 after OVX for 10 weeks in the mice experiment and on the week 4 after OVX for 12 weeks in the rats experiment (Supplemental Fig. 1b). During the experiments, the
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Fig. 4. Effects of dioscin on BMD and some biochemical parameters in rats. (a) Representative images of the BMD detection of rats. (b) Effects of diosicn on total body BMD (b-BMD), proximal 1/3 tibia (t-BMD) and total femur (f-BMD) of rats. (c) Effect of dioscin on serum OC level of rats. (d) Effect of dioscin on urine DPD/Cr level of rats. (e) Effect of dioscin on urine HOP/Cr level of rats. (f) Effects of dioscin on serum Ca and P levels of rats. (g) Effects of dioscin on urine Ca and P levels of rats. * p < 0.05 and ** p < 0.01 versus OVX. Data are presented as the mean ± s.d. (n ≥ 6).
body weights of the animals were measured weekly. Urine samples were collected from the animals that were housed individually for 24 h in metabolic cages without providing food. Before euthanizing the rats, BMD of whole body, total femur and proximal 1/3 tibia in rats were measured using Dual-energy X-ray absorptiometry (Hologic, Bedford, MA, USA) and the detector attached to small animal measurement software (Version 13.3:3). After intraperitoneal (i.p.) injection of chloral hydrate (350 mg/kg), the blood of rats was collected and the serum was obtained. Uteruses of the animals were removed and immediately weighed. Urine and serum samples were then stored at −80 ◦ C. Femora and tibias of the animals were dissected, stored in physiological saline and stored at −20 ◦ C. 2.11. Biochemical parameters assays in serum and urine The serum levels of ALP and StrACP in mice were measured using commercial kits and analyzed by an U-3010 UV spectrophotometer (HITACHI, Japan). The serum levels of ALP, StrACP, Ca and P, and the urine levels of HOP, Cr, Ca and P in rats were also detected. Serumal OC and urinary DPD of rats were quantified using ELISA kits. 2.12. Three-point bending test The mechanical properties of the left femoral diaphysis were determined using a three- point bending test. Before the testing, the left femoral diaphysis was thawed at room temperature for 1 h. The femur was placed in the testing machine (SANS-10404043, Shenzhen, China) with two support points at 20 mm. The biome-
chanical quality of the left femoral diaphysis was determined using the machine at a speed of 2 mm/min. The load-deformation curve was simultaneously plotted using the specialized software, and the maximum load (ultimate strength), stiffness (slope of the linear part of the curve representing elastic deformation) was obtained. 2.13. Micro CT analysis Trabecular microarchitectures of the proximal tibias in mice and rats were evaluated using a micro-CT system for small animal (ZKKS-MCT-Sharp, Guangzhou Zhongke Kaisheng, China). The images and the levels of BS/TV, BV/TV, Tb.N, Tb.Sp, Tb.pf, BMD, DA, SMI were obtained. 2.14. Histomorphological assay The tibias of mice and rats were fixed in 10% buffered formalin for 2 days and decalcified in 14% ethylenediamine tetraacetic acid for 2 weeks. Then, the tibias were embedded in paraffin and cut into 4-m slices. The slices were stained with H&E, TB and TRAP solutions according to the manufacturer’s instructions, and the images were acquired using a light microscope (Leica DM4000B, Germany) with 200× magnification. 2.15. Calcein and alizarin red double labeling experiments The mice and rats were injected with calcein (5 mg/kg) and alizarin red (10 mg/kg) at 14 days and 4 days prior to sacrifice,
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Fig. 5. Effects of dioscin on biomechanical quality of the femurs in rats. (a) Representative images of the load-deformation curve. (b) Effect of dioscin on the ultimate load of femurs in rats. (c) Effect of dioscin on the stiffness of femurs in rats. * p < 0.05 and ** p < 0.01 versus. Data are presented as the mean ± s.d. (n ≥ 6).
respectively. After euthanizing the animals, the femurs were dissected and fixed in 10% buffered formalin. Then, the femurs were cut into 30-m slices using a hard tissue slicing machine (Leica SP1600, Germany) and the images were obtained using a laser scanning confocal microscope (Leica, TCS SP5, Germany).
2.16. Quantitative real-time PCR assay Total RNA samples from MC3T3-E1 cells and the femurs of animals were extracted using RNAiso Plus reagent (TaKaRa Biotechnology Co., Ltd., Japan) following the manufacturer’s protocol. After RNA purity determination, reverse transcripttion polymerase chain reaction (RT-PCR) was performed with PrimeScript® RT reagent Kit (TaKaRa Biotechnology Co., Ltd., Japan) following the manufacturer’s instructions using a TC-512 PCR system (TECHNE, UK). The mRNA levels were then quantified using real-time PCR with SYBR® PremixEx TaqTM II (Tli RNaseH Plus) (TaKaRa Biotechnology Co., Ltd., Japan) and ABI 7500 Real Time PCR System (Applied Biosystems, USA). The sequences of the primers are shown in Supplemental Table 1. GAPDH gene was selected as the house-keeping gene, and a no-template control was analyzed in parallel for each gene. Eventually, the unknown template was calculated by the standard curve for quantitative analysis.
2.17. Western blotting assay The protein samples from MC3T3-E1 cells and BMMs were extracted using cold lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) based on the manufacturer’s instructions. The protein samples were loaded onto the SDS-PAGE gel (10–15%), separated electrophoretically and then transferred onto a PVDF membrane (Millipore, USA). Before the membrane was individually incubated for overnight at 4 ◦ C with primary antibodies (Supplemental Table 2), it was blocked non-specific binding sites with 5% dried skim milk in TTBS at 37 ◦ C for 1 h. Next, the membrane was incubated with horseradish peroxidase- conjugated antibody (1: 5000 dilution) at room temperature for 2 h. Finally, the protein level was determined using an enhanced chemiluminescence (ECL) method and then imaged by ChemiDoc XRS (BIO-RAD, USA). The data were adjusted to correspond internal reference expression (IOD value of target protein versus IOD of correspond internal reference) to eliminate the variations of protein expression. 2.18. Statistical analysis All the data were analyzed using statistical software SPSS 18.0 and expressed as means ± s.d. Differences among groups were determined using the one way ANOVA, followed by a post
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Fig. 6. Representative images of trabecular microarchitecture of tibias in mice and rats. Trabecular microarchitecture of the proximal tibias in mice (a) and rats (b) evaluated by using a micro-CT system for small animal.
hoc LSD test. p < 0.05 and p < 0.01 were considered to be significant.
significantly increased the levels of ALP, OC and COL1A2 in MC3T3E1 cells.
3. Results
3.3. Effect of dioscin on BMMs proliferation
3.1. Effects of dioscin on MC3T3-E1 cells proliferation, differentiation and mineralization
As shown in Fig. 1e, the MTT results showed that dioscin had no significant effect on proliferation of BMMs at the concentration of lower than 2.0 g/mL, but partially suppressed cell proliferation at the concentration of more than 2.0 g/mL. Non-lethal concentrations (<2.0 g/mL) were used in subsequent experiments in order to exclude dioscin-mediated cytotoxicity.
As shown in Fig. 1a, compared with the control cells, dioscin at the concentrations of 0.25, 0.5 and 1.0 g/mL for 24, 48 and 72 h treatment significantly increased the viabilities of MC3T3-E1 cells with a dose-dependent manner. Moreover, we found that dioscin markedly increased ALP level under 72 h treatment compared with control group (Fig. 1b). In addition, as shown in Fig. 1c, the color of alizarin red S staining changed thicker with the increased concentration of dioscin in 21 days, which indicated that the mineralization of MC3T3-E1 cells was promoted by dioscin.
3.2. Effects of dioscin on the levels of osteogenic markers in MC3T3-E1 cells As shown in Fig. 1d and Supplemental Fig. 2, compared with control group, dioscin at the concentrations of 0.25, 0.5 and 1.0 g/mL
3.4. Effects of dioscin on RANKL-induced osteoclasts formation and bone resorption in vitro As shown in Fig. 2a, the cells in control group formed numerous TRAP-positive multinucleated osteoclasts, which were inhibited by dioscin at the concentrations of 0.25, 0.5 and 1.0 g/mL. Moreover, dioscin (1.0 g/mL) was added to osteoclasts differentiation cultures beginning at the days 0–3, and osteoclastogenesis was markedly inhibited by this compound during the first 2 days, while exposure of precursor cells to dioscin at days 3 was not effective in preventing osteoclastogenesis (Fig. 2b). Furthermore, dioscin sub-
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Fig. 7. Effects of dioscin on trabecular microarchitecture of tibias in mice and rats. (a) Effects of dioscin on the levels of Tb.N, DA, BMD, BS/TV, BV/TV, Tb.pf, Tb.Sp, SMI in trabecular microarchitecture in mice. (b) Effects of dioscin on the levels of Tb.N, DA, BMD, BS/TV, BV/TV, Tb.pf, Tb.Sp, SMI in trabecular microarchitecture in rats. * p < 0.05 and ** p < 0.01 versus OVX. Data are presented as the mean ± s.d. (n ≥ 6).
stantially reduced osteoclastic bone resorption pits of bone slices in vitro (Fig. 2c).
3.5. Effects of dioscin on body and uterus weights in mice and rats As shown in Fig. 3a,b, the mice or rats in different groups had a similar initial mean body weights. However, the body weights of OVX groups were significantly higher than those of in sham groups (p < 0.01) during weeks 2–12 after operation in mice experiment, and during weeks 4–16 after operation in rats experiment. While dioscin and 17-Estradiol (E2 ) markedly reduced the OVXinduced body weight gain, and no remarkable different effects produced by high-dose of dioscin and E2 were found. Furthermore, E2 significantly inhibited OVX induced- atrophy of uterine tissue compared with sham groups (p < 0.01), but dioscin did not elicit any uterotropic effects compared with OVX groups (Fig. 3c,d).
3.6. Effects of dioscin on serum ALP and strACP activities in mice and rats The serum ALP (Fig. 3e,f) and StrACP (Fig. 3g,h) activities in mice and rats were significantly increased in OVX groups compared with sham groups (p < 0.01), which were significantly suppressed
by dioscin and E2 . However, there were no remarkable different effects produced by high-dose of dioscin and E2 . 3.7. Effects of dioscin on BMD of rats As shown in Fig. 4a,b, the rats in OVX group showed lowered levels of total body BMD (b-BMD), proximal 1/3 tibia (t-BMD) and total femur (f-BMD) compared with sham group (p < 0.01). Dioscin (20, 40 and 60 mg/kg) treatment with 12 weeks significantly increased the levels of b-BMD, t-BMD and f-BMD compared with OVX group, just like E2 did. 3.8. Effects of dioscin on the biochemical parameters of serum and urine in rats The levels of serum OC (Fig. 4c), urinary DPD/Cr (Fig. 4d) and HOP/Cr (Fig. 4e) ratios in OVX group were significantly increased compared with sham group (p < 0.01), which were suppressed by dioscin and E2 . Furthermore, as shown in Fig. 4f,g, the values of serum-P and urine-P/Cr did not show significant differences among all groups (p > 0.05), while serum-Ca level was markedly increased and urine-Ca level was markedly decreased by dioscin. However, there was no significant difference of the effects produced by highdose of dioscin (60 mg/kg) and E2 (p > 0.05).
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Fig. 8. Effect of dioscin on histopathological evaluation and bone formation rate in mice and rats. (a) Effects of dioscin on HE, TB (the black arrow) and TRAP (the fuchsia area) staining of tibias in mice. (b) Effects of dioscin on HE, TB (the black arrow) and TRAP (the fuchsia area) staining of tibias in rats. (c) Effects of diosicn on the range of green line (calcein) and red line (alizarin red) in the femur of mice. (d) Effects of diosicn on the range of green line (calcein) and red line (alizarin red) in the femur of rats. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.9. Effects of dioscin on biomechanical quality of the femurs in rats As shown in Fig. 5a–c, the animals in OVX group showed a significantly decrease in ultimate load and stiffness of femur compared with sham group (p < 0.01). However, compared with OVX group, dioscin significantly increased the femur ultimate load and stiffness, and the effect produced by 60 mg/kg of dioscin was similar with the action produced by the positive drug. 3.10. Effects of dioscin on trabecular microarchitecture of mice and rats The micro CT images of proximal tibias in mice (Fig. 6a and Supplemental Fig. 3) and rats (Fig. 6b and Supplemental Fig. 4) showed the differences in trabecular microarchitecture among the groups. As shown in Fig. 7a,b, the animals in OVX groups showed the decreased levels of Tb.N, DA, BMD, BS/TV and BV/TV compared with sham groups. In contrast, compared with sham groups, the levels of Tb.pf, Tb.Sp and SMI in the tibias were significantly increased in OVX groups. Neither dioscin nor E2 restored SMI in the tibias of rats (p > 0.05). However, both dioscin and E2 reversed the above mentioned parameters compared with OVX groups. 3.11. Effects of dioscin on histopathological evaluation in mice and rats As shown in Fig. 8a,b, the H&E staining indicated that cancellous bone in metaphysis of tibia in OVX-rats form a network of bone trabeculae with a lot of bone marrow spaces compared with sham groups, which were significantly restored by dioscin and E2 . The TB (black arrow) staining indicated that the positive stained areas
were increased in the tibia of OVX mice and rats compared with sham groups. Dioscin and E2 significantly increased the TB positive stained areas compared with OVX groups. The TRAP (fuchsia area) staining demonstrated that the positive stained areas were increased in the tibias of OVX mice and rats, whereas dioscin and E2 significantly decreased the TRAP positive stained areas. 3.12. Effects of dioscin on bone formation rate in mice and rats As shown in Fig. 8c,d, the significant increased ranges of green line (calcein) and red line (alizarin red) were found in the femur of OVX groups compared with sham groups, while dioscin markedly increased the ranges compared with OVX groups, suggesting that dioscin increased the bone formation rate. 3.13. Effects of dioscin on OPG/RANKL mRNA levels in vivo and in vitro As shown in Fig. 9a, the mRNA level of OPG was significantly increased and the mRNA level of RANKL was markedly decreased in MC3T3-E1 cells compared with control cells. The ratio of OPG/RANKL was notably up-regulated by dioscin. Furthermore, in femurs from OVX mice and rats, the OPG mRNA levels and OPG/RANKL ratios were significantly decreased, and the RANKL mRNA levels were notably increased compared with sham groups, which were significantly reversed by dioscin (Fig. 9b,c). 3.14. Effects of dioscin on RANKL and TLR4 signaling pathway in BMMs As shown in Fig. 9d, obvious fluorescence intensity of tumor necrosis factor receptor- associated factor (TRAF6) (red area) in
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Fig. 9. Effects of dioscin on OPG, RANKL mRNA levels, the ratio of OPG/RANKL and TRAF6 expression. (a) Effects of diosicn on OPG, RANKL mRNA levels, and the ratio of OPG/RANKL in MC3T3-E1 cells (n = 3, * p < 0.05 versus control, ** p < 0.01 versus control). (b) Effects of dioscin on OPG, RANKL mRNA levels, and the ratio of OPG/RANKL in the femur of mice (n = 3, * p < 0.05 versus OVX, ** p < 0.01 versus OVX). (c) Effects of dioscin on OPG, RANKL mRNA levels, and the ratio of OPG/RANKL in the femur of rats (n = 3, ** p < 0.01 versus OVX). (d) Effect of dioscin on TRAF6 expression (red fluorescence) in RANKL-induced osteoclasts by immunofluorescent examination. Data are presented as the mean ± s.d. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
control cells was found, which was markedly decreased by dioscin, indicating that dioscin down-regulated TRAF6 level. As shown in Fig. 10a,b and Supplemental Figs. 5–6, after 0, 30 or 60 min treatment with RANKL (100 ng/mL) or LPS (1.0 g/mL) in BMMs, the protein levels of TLR4, myeloid differentiation primary response gene (88) (MyD88), TRAF6, phosphorylation levels of p38, c-Jun Nterminal kinase (JNK), extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) were decreased by dioscin compared with the control cells. The protein levels of nuclear factor-B (NFB) and transcription activator (AP-1) were also down-regulated by dioscin, which suggested that dioscin blocked NF-B and AP-1 nuclear translocations. As shown in Fig. 10c,d and Supplemental Fig. 7, after 0, 1, 2, 3 or 4 days treatment with RANKL or LPS plus dioscin (0 or 1.0 g/mL), we found that dioscin decreased the protein levels of cathepsin K and NFATc1, which provided further evidence that osteoclast differentiation was arrested by dioscin. 4. Discussion PMO is featured with trabecular bone loss and the increased risk of fracture. Its excessive bone remodeling is caused by the increased osteoclasts induced-bone resorption compareing with osteoblasts induced-bone formation. Previous works have shown that promoting osteoblastogenesis functions and inhibiting osteoclastogenesis represent the potential therapeutic approaches to treat PMO [36–38]. In our study, pre-osteoblast like MC3T3-E1 cells and RANKLinduced osteoclasts were used to examine the effects of dioscin on osteoblasts and osteoclasts in vitro. Dioscin (0.25, 0.5 and 1.0 g/mL) markedly increased the cell viabilities, ALP activities and alizarin red staining area, which indicated that dioscin
promoted the proliferation, differentiation and mineralization of MC3T3-E1 cells. In order to determine the effects of dioscin on osteoblastogenesis, we detected the immunoblots of the potent osteogenic markers including COL1A2, ALP and OC in MC3T3-E1 cells. The results proved that dioscin markedly enhanced the levels of them. In addition, the incubation of BMMs with dioscin (0.25, 0.5 and 1.0 g/mL) for 4 days markedly inhibited the generation of TRAP-positive multinucleated osteoclasts and the bone resorption area. However, dioscin could not reverse osteoclasts formation. OVX, an in vivo well-established experimental model of PMO [7,39,40], was confirmed by the increased body weights, increased ALP, StrACP, HOP, OC and DPD activities and urine Ca level, and decreased serum Ca level in the present paper, which were all improved by dioscin. In addition, administering E2 significantly inhibited OVX induced-atrophy of uterine tissue, but dioscin did not elicit any uterotropic effect. The results indicated dioscin showed no carcinogenesis to the uterus. Furthermore, our work also demonstrated that dioscin increased the levels of b-BMD, t-BMD, f-BMD and the femur ultimate load and stiffness, and improved the trabecular microarchitecture of tibias that were estimated by the parameters of Tb.N, DA, BMD, BS/TV, BV/TV, Tb.pf, Tb.Sp and SMI compared with OVX groups. In further experiments, a lot of bone marrow spaces were also observed in the OVX animals by H&E staining, which were effectively decreased by dioscin. In addition, the increased number of TB-positive osteoblasts and the increased number of TRAP-positive osteoclasts were observed in OVX groups. However, dioscin notably increased TB-positive areas and decreased TRAP-positive areas, which indicated that dioscin promoted osteoblastogenesis and inhibited osteoclastogenesis in vivo. Moreover, we found that dioscin markedly increased the bone formation rate by the calcein and alizarin red double label-
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Fig. 10. Effects of dioscin on RANKL and TLR4 signaling pathway in BMMs. (a) Effects of dioscin on the expression levels of TRAF6, p-p38, p-JNK, p-ERK, p-AKt, NF-B and AP-1 in RANKL-induced BMMs. (b) Effects of dioscin on the expression levels of TLR4, MyD88, TRAF6, p-p38, p-JNK, p-ERK, p-AKt, NF-B and AP-1 in LPS-induced BMMs. The BMMs were treated with dioscin at different concentrations (0, 0.25, 0.5 and 1.0 g/mL) for 4 h, followed by RANKL (100 ng/mL) or LPS (1.0 g/mL) stimulation for 0, 30 or 60 min. Effects of dioscin on the expression levels of RANKL-induced cathepsin K and NFATc1 in RANKL- (c) or LPS- (d) induced BMMs. The cells were treated with RANKL (100 ng/mL) or LPS (1.0 g/mL) with or without 1.0 g/mL dioscin for 0, 1, 2, 3 or 4 days. The cropped gels are used and full-length gels are presented in Supplemental Figs. 10–13.
ing experiment. These data indicated that dioscin exhibited potent abilities to treat PMO through enhancing osteoblastogenesis and inhibiting osteoclastogenesis. RANKL and M-CSF, two cytokines that are expressed by osteoblasts, can regulate osteoclasts differentiation, activity and survival [41]. Osteoclasts will form when RANKL activates its receptor RANK and locates on the cell surface of pre-osteoclasts or mature osteoclasts. The RANKL/RANK binding recruits TRAF6 and then activates multiple downstream signaling pathways involving inhibitor of NF-B kinase (IKK)/NF-B, JNK/AP-1, p38, ERK and Src/Akt pathways. In detailed, TRAF6 is a key adaptor to assemble signaling proteins. The activities of transcription factors including NF-kB and AP-1 are rapidly increased through signaling cascades that mediated by IKK and JNK, respectively. The stressactivated protein kinase p38 and ERK kinase are also positively regulated by the activation of TRAF6. The Src protein has been shown to bind to TRAF6 and allow RANK-mediated signaling to proceed through the phosphatidylinositol-3-kinase (PI3K) and Akt [42]. During these processes, RANKL also induces the levels of NFATc1 and cathepsin K, which are the master transcription factor in osteoclast differentiation and the osteoclast-specific marker, respectively [43]. OPG is a RANKL soluble decoy receptor produced by osteoblasts, which can inhibit the binding of RANKL and RANK [9,10,44]. The results presented here showed that dioscin significantly increased OPG/RANKL ratio and decreased the levels of TRAF6, p-p38, p-JNK, p-ERK, p-Akt, NF-B, AP-1, NFATc1 and cathepsin K in RANKL-induced osteoclasts. Thsee data suggested that dioscin inhibited osteoclastogenesis by up-regulating OPG and down-regulating RANKL-induced TRAF6 signaling pathways.
Furthermore, LPS/TLR4 can activate TRAF6 signaling pathway through triggering its adaptor protein MyD88 [45–48], and then initiate MAPKs, Akt, NF-B and AP-1 signaling pathways, which play key roles in the osteoclasts activation [11]. Our results indicated that dioscin markedly decreased the protein levels of TLR4, MyD88, TRAF6, p-p38 p-p38, p-JNK, p-ERK, p-Akt, NF-B and AP-1 in LPS/TLR4-induced osteoclasts. Moreover, dioscin inhibited the NFATc1 and cathepsin K activities. Therefore, it was obvious that dioscin inhibited osteoclastogenesis by decreasing TLR4/MyD88-induced TRAF6 signaling pathway. In summary, dioscin conferred direct actions against OVXinduced bone loss by enhancing osteoblastogenesis and inhibiting osteoclastogenesis (Supplemental Fig. 8). Accordingly, dioscin represents a novel and potent candidate for the treatment of PMO. Of course, deeply mechanisms and clinical applications of dioscin against PMO are needed to be elucidated in the further.
Conflicts of interest All authors declared no competing financial interests.
Acknowledgements This work was financially supported by the Program for Liaoning Innovative Research Team in University (LT2013019).
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Dioscin reduces ovariectomy-induced bone loss by enhancing osteoblastogenesis and inhibiting osteoclastogenesis Xufeng Tao, Yan Qi, Lina Xu, Lianhong Yin, Xu Han, Youwei Xu, Changyuan Wang, Huijun Sun, Jinyong Peng ∗ College of Pharmacy, Dalian Medical University, Western 9 Lvshunnan Road, Dalian 116044, China
a r t i c l e
i n f o
Article history: Received 16 November 2015 Received in revised form 7 April 2016 Accepted 3 May 2016 Available online 4 May 2016 Keywords: Dioscin Osteoblasts Osteoclasts Ovariectomy Postmenopausal osteoporosis
a b s t r a c t Our previous studies showed that dioscin can promote osteoblasts proliferation and differentiation in vitro, but its anti-osteoporosis effect in vivo and the underlying mechanisms remain unclear. In the present work, the results showed that dioscin significantly increased the viability of MC3T3-E1 cells, ALP level and alizarin red S staining area, markedly decreased the numbers of RANKL-induced TRAPpositive multinucleated cells and bone resorption pits formation, enhanced the levels of some osteogenic markers including COL1A2, ALP and OC, which suggested that dioscin clearly promoted osteoblasts proliferation and suppressed osteoclasts formation. In vivo experiments demonstrated that dioscin obviously reduced OVX-induced body weight increase, and improved the biochemical indexes including ALP, StrACP, OC, DPD/Cr, HOP/Cr, BMD, biomechanics and microarchitecture. Moreover, H&E, TB, TRAP staining, and fluorescent double labeling tests indicated that dioscin enhanced osteoblastogenesis and inhibited osteoclastogenesis. Further researches demonstrated that dioscin promoted osteoblastogenesis through up-regulating OPG/RANKL ratio, and inhibited osteoclastogenesis through down-regulating the levels of RANKL induced TRAF6 and the downstream signal molecules including MAPKs, Akt, NF-B, AP-1, cathepsin K and NFATc1. In addition, dioscin also inhibited TLR4/MyD88 pathway to decrease the levels of TRAF6 and the related proteins. These findings provide new insights to elucidate the effects of dioscin against OVX-induced bone loss, which should be developed as a potential candidate for treating postmenopausal osteoporosis in the future. © 2016 Elsevier Ltd. All rights reserved.
1. Introduction Osteoporosis, a less musculoskeletal disease, is characterized by compromised bone strength inclining to an increased risk of fracture [1]. It is started by estrogen deficiency and particularly common in postmenopausal women [2]. Over 200 million people suffer from osteoporosis worldwide, which leads to 1.6 million people with hip fractures and 7.4 million people with other fractures
Abbreviations: Akt, protein kinase B; ALP, alkaline phosphatase; AP-1, transcription activator; BMD, bone mineral density; DPD, deoxypyridinoline; COL1A2, collagen, type I, alpha 2; Cr, creatinine; H&E, hematoxylin-eosin; HOP, hyroxyproline; MyD88, myeloid differentiation primary response gene (88); MAPKs, mitogen activated protein kinases; NFATc1, nuclear factor of activated T cells; NFB, nuclear factor-B; OC, osteocalcin; OPG, osteoprotegerin; OVX, ovariectomy; RANKL, receptor activator for nuclear factor-B ligand; StrACP, tartrate resistant acid phosphatase; TB, toluidine blue; TLR4, toll-like receptor 4; TRAF6, tumor necrosis factor receptor-associated factor; TRAP, tartrate-resistant acid phosphatase. ∗ Corresponding author. E-mail address: [email protected] (J. Peng). http://dx.doi.org/10.1016/j.phrs.2016.05.003 1043-6618/© 2016 Elsevier Ltd. All rights reserved.
annually [3,4]. Approximately half of the women and a quarter of the men over 50 years old have the lifetime risk of osteoporosisrelated fracture. In American, the direct fracture-caused costs are expected to raise from $17 billion in 2005 to over $22 billion by 2020 [5,6]. Thus, osteoporosis is one serious health and society problem. Mechanistically, postmenopausal osteoporosis (PMO) is a disorder of unbalanced bone remodeling with the increased bone resorption relative to bone formation, leading to the decreased bone mineral density (BMD) and the disruption of bone microarchitecture [7]. Bone resorption is completed by the polarization and the subsequent attachment to bone surface of osteoclasts. Meanwhile, bone formation occurs following osteoblasts phase that is to be developed into osteocytes [2]. Therefore, correcting the imbalance between bone resorption and formation is an effective therapy for PMO. It is well known that the factors including the receptor activator for nuclear factor-B (RANK) and its ligand RANKL, and osteoprotegerin (OPG, the decoy receptor for RANKL) associated with osteoporosis have been discovered [8]. Among them, RANKL is mainly produced by osteoblasts and stromal cells, and OPG is
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generated by osteoblasts. Binding RANKL to RANK provides a pivotal signal to drive osteoclasts generation from haematopoietic progenitor cells as well as to activate mature osteoclasts [9]. Meanwhile, OPG can negatively regulate the binding and then inhibit osteoclasts induced-high bone turnover [10]. Furthermore, Tolllike receptor 4 (TLR4), one germline-encoded pattern recognition receptor, can recognize multiple microbial pathogens including damage-associated molecular pattern molecules (DAMPs) and lipopolysaccharide (LPS), and promote the secretion of various inflammatory mediators to induce pre-osteoclast fusion and stimulate the survival of mature osteoclasts [11]. Therefore, the OPG/RANK/RANKL axis and TLR4 signal may be the potential antiosteoporosis targets. Presently, the methods for the treatment of PMO mainly include five kinds of agents including estrogens, bisphosphonates, calcitonin, selective estrogen receptor modulators (SERMs) and monoclonal antibody [12]. However, these agents may cause many adverse effects including invasive breast cancer, osteonecrosis, diarrhea, venous thromboembolism and serious infections of skin and urinary tract. Besides these, single drug target and high price of these anti-PMO agents also restrict their developing and applying in clinic [4,13]. Thus, it is urgent to exploit efficient agents for treating PMO. Traditional Chinese medicines (TCMs) with high efficiency and low toxicity have attracted great interest in recent years. Some active natural components including lycopene [14], resveratrol [15], and ginsenoside-Rb2 [16] have excellent activities against PMO. Therefore, it is reasonable to develop effective herbal products for the treatment of PMO. Dioscin (Supplemental Fig. 1a) is a natural steroidal saponin isolated from some medicinal plants [17,18]. Pharmacological investigations have shown that dioscin exhibits hepatoprotective [19–22], anti-tumor [23,24] and anti-ischemia reperfusion injury activities [25,26]. In addition, dioscin has obvious effects against obesity [27] and carbon tetrachloride-induced liver fibrosis [28,29], which also exerts inhibitory effects on several cytochrome P450 enzymes [30]. Our previous works have shown that dioscin can promote the proliferation and differentiation of pre-osteoblast like MC3T3-E1 cells and human osteoblast-like MG-63 cells in vitro [31]. However, its effects on PMO and the underlying mechanisms are unclear in our best knowledge. Therefore, the aim of the present paper was to further explore the effects of dioscin against PMO and then to investigate the underlying molecular mechanisms.
2. Materials and methods 2.1. Chemicals and materials Dioscin (purity >98%) was prepared from Dioscorea nipponica Makino in our laboratory [32,33]. In cell experiments, dioscin was added into the medium by dissolving with DMSO with final concentration of less than 0.1%, and it was suspended in 0.5% sodium carboxyl methyl cellulose (CMC-Na) in animals experiments. Fetal bovine serum (FBS), penicillin and streptomycin were obtained from Hyclone Laboratories, Inc. (Massachusetts, USA). Dulbecco’s minimum essential medium (DMEM) and alpha-minimum essential medium (␣-MEM) were purchased from Gibco (California, USA). Recombinant mouse macrophage colony-stimulating factor (M-CSF) and recombinant mouse RANKL were purchased from R&D Systems (Minneapolis, USA). 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) was provided by Roche Diagnostics (Basel, Switzerland). Alkaline phosphatase (ALP), tartrate resistant acid phosphatase (StrACP), hyroxyproline (HOP), calcium (Ca), phosphate (P) and creatinine (Cr) assay kits and
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hematoxylin-eosin (H&E), toluidine blue (TB) and TRAP staining kits were obtained from Nanjing Jiancheng Institute of Biotechnology (Nanjing, China). Osteocalcin (OC) and deoxypyridinoline (DPD) ELISA kits were provided by Bogoo (Shanghai, China). alizarin red and calcein were purchased from Solarbio (Shanghai, China). 4 ,6-diamidino-2-phenylindole (DAPI) and LPS were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Tissue Protein Extraction Kit was purchased from KEYGEN Biotech. Co., Ltd. (Naijing, China). Bicinchoninic acid (BCA) Protein Assay Kit was purchased from Beyotime Institute of Biotechnology (Shanghai, China). 2.2. Cells culture The MC3T3-E1 cells were purchased from the Institute of Biochemistry Cell Biology (Shanghai, China) and cultured in DMEM medium supplemented with 10% FBS, 100 U/mL streptomycin and 100 U/mL penicillin at 37 ◦ C in a saturated humidified incubator (Thermo Fisher Scientific, Massachusetts, USA). Meanwhile, whole bone marrow cells were extracted from the femur and tibiae of 6week-old C57BL/6J mice. These cells were grown in ␣-MEM with 10% FBS, 100 U/mL streptomycin, 100 U/mL penicillin and 50 ng/mL M-CSF for 3 days to generate bone marrow-derived macrophages (BMMs). To generate osteoclasts, 50 ng/mL M-CSF and 100 ng/mL RANKL were added to ␣-MEM for 4 days of culture in a constant temperature and humidity incubator [34,35]. 2.3. Proliferation and ALP detection in MC3T3-E1 cells After adhering and growing for 24 h, the MC3T3-E1 cells were treated with dioscin (0, 0.25, 0.5 and 1.0 g/mL) for 24, 48 and 72 h. Then, the viability of MC3T3-E1 cells was detected by MTT assay. The MC3T3-E1 cells (1 × 104 cells/mL) were seeded in 24well culture plates, and then treated with dioscin (0, 0.25, 0.5 and 1.0 g/mL) for 72 h. After the cells were gently washed with iced PBS, permeated with 0.2% TritonX-100, and centrifuged, the supernatant was collected and used for detecting ALP activity and total protein concentration. 2.4. Alizarin red S staining in MC3T3-E1 cells The MC3T3-E1 cells (1 × 104 cells/mL) were seeded into culture dishes and incubated with different concentrations of dioscin (0, 0.25, 0.5 and 1.0 g/mL). At the 21 days differentiation, the cells were washed with PBS and fixed with ice-cold 70% ethanol (v/v) for 10 min, and then rinsed thoroughly with distilled water, stained with 40 mM alizarin red S solution in deionized water (pH = 4.2) for 10 min and rinsed with PBS. Eventually, the stained culture dishes were photographed using a digital camera. 2.5. Proliferation assay in BMMs The BMMs (1 × 105 cells/mL) were treated with various concentrations of dioscin (0, 0.25, 0.5, 1.0 and 2.0 g/mL) plus M-CSF (50 ng/mL) and RANKL (0 and 100 ng/mL) for 24 h. Next, the absorbance of the samples was quantified according to the MTT method. 2.6. TRAP staining in RANKL-induced osteoclasts The BMMs (1 × 105 cells/mL) were treated with M-CSF (50 ng/mL) and RANKL (100 ng/mL) for 4 days, and then various concentrations of dioscin (0, 0.25, 0.5, 1.0 g/mL) were added at the day 1, day 2 and day 3. At the fourth day, the cells were fixed and stained for TRAP activity, and the images were photographed by using an inverted microscope (Nikon, Japan).
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Fig. 1. Effects of dioscin on MC3T3-E1 and BMMs cells. (a) Effects of dioscin on the proliferation of MC3T3-E1 cells in 24, 48 and 72 h. (b) Effect of dioscin on ALP level of MC3T3-E1 cells in 72 h. (c) Effect of dioscin on alizarin red S staining color of MC3T3-E1 cells in 72 h. (d) Effects of dioscin on the expression levels of ALP, OC and COL1A2 in MC3T3-E1 cells. The MC3T3-E1 cells were treated with dioscin at different concentrations (0, 0.25, 0.5 and 1.0 g/mL) for 72 h. The cropped gels are used and full-length gels are presented in Supplemental Fig. 9. (e) Effect of dioscin on BMMs proliferation. * p < 0.05 and ** p < 0.01 versus control. Data are presented as the mean ± s.d. (n = 6). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Effects of dioscin on RANKL-induced osteoclasts formation. (a) Effect of dioscin at different doses (0.25, 0.5, 1 g/mL) on RANKL-induced osteoclasts formation. (b) Effect of dioscin on RANKL-induced osteoclasts formation at different times. (c) Effects of dioscin on RANKL-induced osteoclasts bone resorption area (the black arrow) in vitro.
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Fig. 3. Effects of diosin on body, uterus weights, serum ALP, strACP activites in mice and rats. (a) Effects of diosin on body weights in mice. (b) Effects of diosin on body weights in rats. (c) Effects of diosin on uterus weights in mice. (d) Effects of diosin on uterus weights in rats. (e) Effects of dioscin on serum ALP activity in mice. (f) Effects of dioscin on serum ALP activity in rats. (g) Effects of dioscin on serum StrACP activity in mice. (h) Effects of dioscin on serum StrACP activity in rats. * p < 0.05 and ** p < 0.01 versus OVX. Data are presented as the mean ± s.d. (n ≥ 6).
2.7. Resorption pits formation assay The BMMs were seeded on bone slices with M-CSF (50 ng/mL) and RANKL (100 ng/mL), and then various concentrations of dioscin (0, 0.25, 0.5 and 1.0 g/mL) were supplied at the first day for 4 days of incubation. Then, the bone slices were stained with hematoxylin, and imaged using a light microscope (Leica DM4000B, Germany) with 40× magnification.
of Dalian Medical University (Dalian, China) (SCXK (Liao): 20130008). All experiments were approved by the Animal Care and Use Committee of Dalian Medical University, and the experimental procedures were performed in strict accordance with Legislation Regarding the Use and Care of Laboratory Animals of China. Before the experiments, the animals were allowed to suit the new environment for 7 days, and housed in a room under 12 h light/dark cycle, a controlled temperature at 22 ± 3 ◦ C and a relative humidity at 60 ± 10%.
2.8. Immunofluorescent examination 2.10. Pharmacological treatment The BMMs (1 × 105 cells/mL) were treated with various concentrations of dioscin (0, 0.25, 0.5 and 1.0 g/mL) plus RANKL (100 ng/mL) for 4 days. Then the cells were fixed with 4% paraformaldehyde, permeated with 0.2% Triton X-100 for 10 min, incubated with 5% goat serum albumin for 1 h at 37 ◦ C, and then incubated with the anti-TRAF6 antibody (1: 100 dilution) overnight at 4 ◦ C. At last, the cells were incubated using a TRITC-conjugated Goat Anti-Rabbit IgG (H + L) for 1 h at 37 ◦ C and then counterstained with DAPI (5 g/mL) for 10 min. Images were captured by a fluorescent microscopy (Olympus BX63, Japan) with 200× magnification. 2.9. Animals Three-month old female SD rats and six-week old female C57BL/6 mice were provided by the Experimental Animal Center
In the mice experiments, the animals were randomly divided into six groups: sham and OVX (model) groups in which the mice were treated with vehicle (0.5% CMC-Na), OVX + dioscin groups in which the mice were treated with 28, 56 and 84 mg/kg of dioscin, and the positive group in which the mice were treated with 17-Estradiol (E2 , 35 g/kg). In the rat experiments, the animals were randomly divided into six groups: sham and OVX (model) groups in which the rats were treated with vehicle (0.5% CMC-Na), OVX + dioscin groups in which the rats were treated with 20, 40 and 60 mg/kg of dioscin, and the positive group in which the rats were treated with E2 (25 g/kg). Vehicle, dioscin and E2 were all orally administrated on the week 2 after OVX for 10 weeks in the mice experiment and on the week 4 after OVX for 12 weeks in the rats experiment (Supplemental Fig. 1b). During the experiments, the
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Fig. 4. Effects of dioscin on BMD and some biochemical parameters in rats. (a) Representative images of the BMD detection of rats. (b) Effects of diosicn on total body BMD (b-BMD), proximal 1/3 tibia (t-BMD) and total femur (f-BMD) of rats. (c) Effect of dioscin on serum OC level of rats. (d) Effect of dioscin on urine DPD/Cr level of rats. (e) Effect of dioscin on urine HOP/Cr level of rats. (f) Effects of dioscin on serum Ca and P levels of rats. (g) Effects of dioscin on urine Ca and P levels of rats. * p < 0.05 and ** p < 0.01 versus OVX. Data are presented as the mean ± s.d. (n ≥ 6).
body weights of the animals were measured weekly. Urine samples were collected from the animals that were housed individually for 24 h in metabolic cages without providing food. Before euthanizing the rats, BMD of whole body, total femur and proximal 1/3 tibia in rats were measured using Dual-energy X-ray absorptiometry (Hologic, Bedford, MA, USA) and the detector attached to small animal measurement software (Version 13.3:3). After intraperitoneal (i.p.) injection of chloral hydrate (350 mg/kg), the blood of rats was collected and the serum was obtained. Uteruses of the animals were removed and immediately weighed. Urine and serum samples were then stored at −80 ◦ C. Femora and tibias of the animals were dissected, stored in physiological saline and stored at −20 ◦ C. 2.11. Biochemical parameters assays in serum and urine The serum levels of ALP and StrACP in mice were measured using commercial kits and analyzed by an U-3010 UV spectrophotometer (HITACHI, Japan). The serum levels of ALP, StrACP, Ca and P, and the urine levels of HOP, Cr, Ca and P in rats were also detected. Serumal OC and urinary DPD of rats were quantified using ELISA kits. 2.12. Three-point bending test The mechanical properties of the left femoral diaphysis were determined using a three- point bending test. Before the testing, the left femoral diaphysis was thawed at room temperature for 1 h. The femur was placed in the testing machine (SANS-10404043, Shenzhen, China) with two support points at 20 mm. The biome-
chanical quality of the left femoral diaphysis was determined using the machine at a speed of 2 mm/min. The load-deformation curve was simultaneously plotted using the specialized software, and the maximum load (ultimate strength), stiffness (slope of the linear part of the curve representing elastic deformation) was obtained. 2.13. Micro CT analysis Trabecular microarchitectures of the proximal tibias in mice and rats were evaluated using a micro-CT system for small animal (ZKKS-MCT-Sharp, Guangzhou Zhongke Kaisheng, China). The images and the levels of BS/TV, BV/TV, Tb.N, Tb.Sp, Tb.pf, BMD, DA, SMI were obtained. 2.14. Histomorphological assay The tibias of mice and rats were fixed in 10% buffered formalin for 2 days and decalcified in 14% ethylenediamine tetraacetic acid for 2 weeks. Then, the tibias were embedded in paraffin and cut into 4-m slices. The slices were stained with H&E, TB and TRAP solutions according to the manufacturer’s instructions, and the images were acquired using a light microscope (Leica DM4000B, Germany) with 200× magnification. 2.15. Calcein and alizarin red double labeling experiments The mice and rats were injected with calcein (5 mg/kg) and alizarin red (10 mg/kg) at 14 days and 4 days prior to sacrifice,
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Fig. 5. Effects of dioscin on biomechanical quality of the femurs in rats. (a) Representative images of the load-deformation curve. (b) Effect of dioscin on the ultimate load of femurs in rats. (c) Effect of dioscin on the stiffness of femurs in rats. * p < 0.05 and ** p < 0.01 versus. Data are presented as the mean ± s.d. (n ≥ 6).
respectively. After euthanizing the animals, the femurs were dissected and fixed in 10% buffered formalin. Then, the femurs were cut into 30-m slices using a hard tissue slicing machine (Leica SP1600, Germany) and the images were obtained using a laser scanning confocal microscope (Leica, TCS SP5, Germany).
2.16. Quantitative real-time PCR assay Total RNA samples from MC3T3-E1 cells and the femurs of animals were extracted using RNAiso Plus reagent (TaKaRa Biotechnology Co., Ltd., Japan) following the manufacturer’s protocol. After RNA purity determination, reverse transcripttion polymerase chain reaction (RT-PCR) was performed with PrimeScript® RT reagent Kit (TaKaRa Biotechnology Co., Ltd., Japan) following the manufacturer’s instructions using a TC-512 PCR system (TECHNE, UK). The mRNA levels were then quantified using real-time PCR with SYBR® PremixEx TaqTM II (Tli RNaseH Plus) (TaKaRa Biotechnology Co., Ltd., Japan) and ABI 7500 Real Time PCR System (Applied Biosystems, USA). The sequences of the primers are shown in Supplemental Table 1. GAPDH gene was selected as the house-keeping gene, and a no-template control was analyzed in parallel for each gene. Eventually, the unknown template was calculated by the standard curve for quantitative analysis.
2.17. Western blotting assay The protein samples from MC3T3-E1 cells and BMMs were extracted using cold lysis buffer containing 1 mM phenylmethylsulfonyl fluoride (PMSF) based on the manufacturer’s instructions. The protein samples were loaded onto the SDS-PAGE gel (10–15%), separated electrophoretically and then transferred onto a PVDF membrane (Millipore, USA). Before the membrane was individually incubated for overnight at 4 ◦ C with primary antibodies (Supplemental Table 2), it was blocked non-specific binding sites with 5% dried skim milk in TTBS at 37 ◦ C for 1 h. Next, the membrane was incubated with horseradish peroxidase- conjugated antibody (1: 5000 dilution) at room temperature for 2 h. Finally, the protein level was determined using an enhanced chemiluminescence (ECL) method and then imaged by ChemiDoc XRS (BIO-RAD, USA). The data were adjusted to correspond internal reference expression (IOD value of target protein versus IOD of correspond internal reference) to eliminate the variations of protein expression. 2.18. Statistical analysis All the data were analyzed using statistical software SPSS 18.0 and expressed as means ± s.d. Differences among groups were determined using the one way ANOVA, followed by a post
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Fig. 6. Representative images of trabecular microarchitecture of tibias in mice and rats. Trabecular microarchitecture of the proximal tibias in mice (a) and rats (b) evaluated by using a micro-CT system for small animal.
hoc LSD test. p < 0.05 and p < 0.01 were considered to be significant.
significantly increased the levels of ALP, OC and COL1A2 in MC3T3E1 cells.
3. Results
3.3. Effect of dioscin on BMMs proliferation
3.1. Effects of dioscin on MC3T3-E1 cells proliferation, differentiation and mineralization
As shown in Fig. 1e, the MTT results showed that dioscin had no significant effect on proliferation of BMMs at the concentration of lower than 2.0 g/mL, but partially suppressed cell proliferation at the concentration of more than 2.0 g/mL. Non-lethal concentrations (<2.0 g/mL) were used in subsequent experiments in order to exclude dioscin-mediated cytotoxicity.
As shown in Fig. 1a, compared with the control cells, dioscin at the concentrations of 0.25, 0.5 and 1.0 g/mL for 24, 48 and 72 h treatment significantly increased the viabilities of MC3T3-E1 cells with a dose-dependent manner. Moreover, we found that dioscin markedly increased ALP level under 72 h treatment compared with control group (Fig. 1b). In addition, as shown in Fig. 1c, the color of alizarin red S staining changed thicker with the increased concentration of dioscin in 21 days, which indicated that the mineralization of MC3T3-E1 cells was promoted by dioscin.
3.2. Effects of dioscin on the levels of osteogenic markers in MC3T3-E1 cells As shown in Fig. 1d and Supplemental Fig. 2, compared with control group, dioscin at the concentrations of 0.25, 0.5 and 1.0 g/mL
3.4. Effects of dioscin on RANKL-induced osteoclasts formation and bone resorption in vitro As shown in Fig. 2a, the cells in control group formed numerous TRAP-positive multinucleated osteoclasts, which were inhibited by dioscin at the concentrations of 0.25, 0.5 and 1.0 g/mL. Moreover, dioscin (1.0 g/mL) was added to osteoclasts differentiation cultures beginning at the days 0–3, and osteoclastogenesis was markedly inhibited by this compound during the first 2 days, while exposure of precursor cells to dioscin at days 3 was not effective in preventing osteoclastogenesis (Fig. 2b). Furthermore, dioscin sub-
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Fig. 7. Effects of dioscin on trabecular microarchitecture of tibias in mice and rats. (a) Effects of dioscin on the levels of Tb.N, DA, BMD, BS/TV, BV/TV, Tb.pf, Tb.Sp, SMI in trabecular microarchitecture in mice. (b) Effects of dioscin on the levels of Tb.N, DA, BMD, BS/TV, BV/TV, Tb.pf, Tb.Sp, SMI in trabecular microarchitecture in rats. * p < 0.05 and ** p < 0.01 versus OVX. Data are presented as the mean ± s.d. (n ≥ 6).
stantially reduced osteoclastic bone resorption pits of bone slices in vitro (Fig. 2c).
3.5. Effects of dioscin on body and uterus weights in mice and rats As shown in Fig. 3a,b, the mice or rats in different groups had a similar initial mean body weights. However, the body weights of OVX groups were significantly higher than those of in sham groups (p < 0.01) during weeks 2–12 after operation in mice experiment, and during weeks 4–16 after operation in rats experiment. While dioscin and 17-Estradiol (E2 ) markedly reduced the OVXinduced body weight gain, and no remarkable different effects produced by high-dose of dioscin and E2 were found. Furthermore, E2 significantly inhibited OVX induced- atrophy of uterine tissue compared with sham groups (p < 0.01), but dioscin did not elicit any uterotropic effects compared with OVX groups (Fig. 3c,d).
3.6. Effects of dioscin on serum ALP and strACP activities in mice and rats The serum ALP (Fig. 3e,f) and StrACP (Fig. 3g,h) activities in mice and rats were significantly increased in OVX groups compared with sham groups (p < 0.01), which were significantly suppressed
by dioscin and E2 . However, there were no remarkable different effects produced by high-dose of dioscin and E2 . 3.7. Effects of dioscin on BMD of rats As shown in Fig. 4a,b, the rats in OVX group showed lowered levels of total body BMD (b-BMD), proximal 1/3 tibia (t-BMD) and total femur (f-BMD) compared with sham group (p < 0.01). Dioscin (20, 40 and 60 mg/kg) treatment with 12 weeks significantly increased the levels of b-BMD, t-BMD and f-BMD compared with OVX group, just like E2 did. 3.8. Effects of dioscin on the biochemical parameters of serum and urine in rats The levels of serum OC (Fig. 4c), urinary DPD/Cr (Fig. 4d) and HOP/Cr (Fig. 4e) ratios in OVX group were significantly increased compared with sham group (p < 0.01), which were suppressed by dioscin and E2 . Furthermore, as shown in Fig. 4f,g, the values of serum-P and urine-P/Cr did not show significant differences among all groups (p > 0.05), while serum-Ca level was markedly increased and urine-Ca level was markedly decreased by dioscin. However, there was no significant difference of the effects produced by highdose of dioscin (60 mg/kg) and E2 (p > 0.05).
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Fig. 8. Effect of dioscin on histopathological evaluation and bone formation rate in mice and rats. (a) Effects of dioscin on HE, TB (the black arrow) and TRAP (the fuchsia area) staining of tibias in mice. (b) Effects of dioscin on HE, TB (the black arrow) and TRAP (the fuchsia area) staining of tibias in rats. (c) Effects of diosicn on the range of green line (calcein) and red line (alizarin red) in the femur of mice. (d) Effects of diosicn on the range of green line (calcein) and red line (alizarin red) in the femur of rats. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
3.9. Effects of dioscin on biomechanical quality of the femurs in rats As shown in Fig. 5a–c, the animals in OVX group showed a significantly decrease in ultimate load and stiffness of femur compared with sham group (p < 0.01). However, compared with OVX group, dioscin significantly increased the femur ultimate load and stiffness, and the effect produced by 60 mg/kg of dioscin was similar with the action produced by the positive drug. 3.10. Effects of dioscin on trabecular microarchitecture of mice and rats The micro CT images of proximal tibias in mice (Fig. 6a and Supplemental Fig. 3) and rats (Fig. 6b and Supplemental Fig. 4) showed the differences in trabecular microarchitecture among the groups. As shown in Fig. 7a,b, the animals in OVX groups showed the decreased levels of Tb.N, DA, BMD, BS/TV and BV/TV compared with sham groups. In contrast, compared with sham groups, the levels of Tb.pf, Tb.Sp and SMI in the tibias were significantly increased in OVX groups. Neither dioscin nor E2 restored SMI in the tibias of rats (p > 0.05). However, both dioscin and E2 reversed the above mentioned parameters compared with OVX groups. 3.11. Effects of dioscin on histopathological evaluation in mice and rats As shown in Fig. 8a,b, the H&E staining indicated that cancellous bone in metaphysis of tibia in OVX-rats form a network of bone trabeculae with a lot of bone marrow spaces compared with sham groups, which were significantly restored by dioscin and E2 . The TB (black arrow) staining indicated that the positive stained areas
were increased in the tibia of OVX mice and rats compared with sham groups. Dioscin and E2 significantly increased the TB positive stained areas compared with OVX groups. The TRAP (fuchsia area) staining demonstrated that the positive stained areas were increased in the tibias of OVX mice and rats, whereas dioscin and E2 significantly decreased the TRAP positive stained areas. 3.12. Effects of dioscin on bone formation rate in mice and rats As shown in Fig. 8c,d, the significant increased ranges of green line (calcein) and red line (alizarin red) were found in the femur of OVX groups compared with sham groups, while dioscin markedly increased the ranges compared with OVX groups, suggesting that dioscin increased the bone formation rate. 3.13. Effects of dioscin on OPG/RANKL mRNA levels in vivo and in vitro As shown in Fig. 9a, the mRNA level of OPG was significantly increased and the mRNA level of RANKL was markedly decreased in MC3T3-E1 cells compared with control cells. The ratio of OPG/RANKL was notably up-regulated by dioscin. Furthermore, in femurs from OVX mice and rats, the OPG mRNA levels and OPG/RANKL ratios were significantly decreased, and the RANKL mRNA levels were notably increased compared with sham groups, which were significantly reversed by dioscin (Fig. 9b,c). 3.14. Effects of dioscin on RANKL and TLR4 signaling pathway in BMMs As shown in Fig. 9d, obvious fluorescence intensity of tumor necrosis factor receptor- associated factor (TRAF6) (red area) in
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Fig. 9. Effects of dioscin on OPG, RANKL mRNA levels, the ratio of OPG/RANKL and TRAF6 expression. (a) Effects of diosicn on OPG, RANKL mRNA levels, and the ratio of OPG/RANKL in MC3T3-E1 cells (n = 3, * p < 0.05 versus control, ** p < 0.01 versus control). (b) Effects of dioscin on OPG, RANKL mRNA levels, and the ratio of OPG/RANKL in the femur of mice (n = 3, * p < 0.05 versus OVX, ** p < 0.01 versus OVX). (c) Effects of dioscin on OPG, RANKL mRNA levels, and the ratio of OPG/RANKL in the femur of rats (n = 3, ** p < 0.01 versus OVX). (d) Effect of dioscin on TRAF6 expression (red fluorescence) in RANKL-induced osteoclasts by immunofluorescent examination. Data are presented as the mean ± s.d. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
control cells was found, which was markedly decreased by dioscin, indicating that dioscin down-regulated TRAF6 level. As shown in Fig. 10a,b and Supplemental Figs. 5–6, after 0, 30 or 60 min treatment with RANKL (100 ng/mL) or LPS (1.0 g/mL) in BMMs, the protein levels of TLR4, myeloid differentiation primary response gene (88) (MyD88), TRAF6, phosphorylation levels of p38, c-Jun Nterminal kinase (JNK), extracellular signal-regulated kinase (ERK) and protein kinase B (Akt) were decreased by dioscin compared with the control cells. The protein levels of nuclear factor-B (NFB) and transcription activator (AP-1) were also down-regulated by dioscin, which suggested that dioscin blocked NF-B and AP-1 nuclear translocations. As shown in Fig. 10c,d and Supplemental Fig. 7, after 0, 1, 2, 3 or 4 days treatment with RANKL or LPS plus dioscin (0 or 1.0 g/mL), we found that dioscin decreased the protein levels of cathepsin K and NFATc1, which provided further evidence that osteoclast differentiation was arrested by dioscin. 4. Discussion PMO is featured with trabecular bone loss and the increased risk of fracture. Its excessive bone remodeling is caused by the increased osteoclasts induced-bone resorption compareing with osteoblasts induced-bone formation. Previous works have shown that promoting osteoblastogenesis functions and inhibiting osteoclastogenesis represent the potential therapeutic approaches to treat PMO [36–38]. In our study, pre-osteoblast like MC3T3-E1 cells and RANKLinduced osteoclasts were used to examine the effects of dioscin on osteoblasts and osteoclasts in vitro. Dioscin (0.25, 0.5 and 1.0 g/mL) markedly increased the cell viabilities, ALP activities and alizarin red staining area, which indicated that dioscin
promoted the proliferation, differentiation and mineralization of MC3T3-E1 cells. In order to determine the effects of dioscin on osteoblastogenesis, we detected the immunoblots of the potent osteogenic markers including COL1A2, ALP and OC in MC3T3-E1 cells. The results proved that dioscin markedly enhanced the levels of them. In addition, the incubation of BMMs with dioscin (0.25, 0.5 and 1.0 g/mL) for 4 days markedly inhibited the generation of TRAP-positive multinucleated osteoclasts and the bone resorption area. However, dioscin could not reverse osteoclasts formation. OVX, an in vivo well-established experimental model of PMO [7,39,40], was confirmed by the increased body weights, increased ALP, StrACP, HOP, OC and DPD activities and urine Ca level, and decreased serum Ca level in the present paper, which were all improved by dioscin. In addition, administering E2 significantly inhibited OVX induced-atrophy of uterine tissue, but dioscin did not elicit any uterotropic effect. The results indicated dioscin showed no carcinogenesis to the uterus. Furthermore, our work also demonstrated that dioscin increased the levels of b-BMD, t-BMD, f-BMD and the femur ultimate load and stiffness, and improved the trabecular microarchitecture of tibias that were estimated by the parameters of Tb.N, DA, BMD, BS/TV, BV/TV, Tb.pf, Tb.Sp and SMI compared with OVX groups. In further experiments, a lot of bone marrow spaces were also observed in the OVX animals by H&E staining, which were effectively decreased by dioscin. In addition, the increased number of TB-positive osteoblasts and the increased number of TRAP-positive osteoclasts were observed in OVX groups. However, dioscin notably increased TB-positive areas and decreased TRAP-positive areas, which indicated that dioscin promoted osteoblastogenesis and inhibited osteoclastogenesis in vivo. Moreover, we found that dioscin markedly increased the bone formation rate by the calcein and alizarin red double label-
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Fig. 10. Effects of dioscin on RANKL and TLR4 signaling pathway in BMMs. (a) Effects of dioscin on the expression levels of TRAF6, p-p38, p-JNK, p-ERK, p-AKt, NF-B and AP-1 in RANKL-induced BMMs. (b) Effects of dioscin on the expression levels of TLR4, MyD88, TRAF6, p-p38, p-JNK, p-ERK, p-AKt, NF-B and AP-1 in LPS-induced BMMs. The BMMs were treated with dioscin at different concentrations (0, 0.25, 0.5 and 1.0 g/mL) for 4 h, followed by RANKL (100 ng/mL) or LPS (1.0 g/mL) stimulation for 0, 30 or 60 min. Effects of dioscin on the expression levels of RANKL-induced cathepsin K and NFATc1 in RANKL- (c) or LPS- (d) induced BMMs. The cells were treated with RANKL (100 ng/mL) or LPS (1.0 g/mL) with or without 1.0 g/mL dioscin for 0, 1, 2, 3 or 4 days. The cropped gels are used and full-length gels are presented in Supplemental Figs. 10–13.
ing experiment. These data indicated that dioscin exhibited potent abilities to treat PMO through enhancing osteoblastogenesis and inhibiting osteoclastogenesis. RANKL and M-CSF, two cytokines that are expressed by osteoblasts, can regulate osteoclasts differentiation, activity and survival [41]. Osteoclasts will form when RANKL activates its receptor RANK and locates on the cell surface of pre-osteoclasts or mature osteoclasts. The RANKL/RANK binding recruits TRAF6 and then activates multiple downstream signaling pathways involving inhibitor of NF-B kinase (IKK)/NF-B, JNK/AP-1, p38, ERK and Src/Akt pathways. In detailed, TRAF6 is a key adaptor to assemble signaling proteins. The activities of transcription factors including NF-kB and AP-1 are rapidly increased through signaling cascades that mediated by IKK and JNK, respectively. The stressactivated protein kinase p38 and ERK kinase are also positively regulated by the activation of TRAF6. The Src protein has been shown to bind to TRAF6 and allow RANK-mediated signaling to proceed through the phosphatidylinositol-3-kinase (PI3K) and Akt [42]. During these processes, RANKL also induces the levels of NFATc1 and cathepsin K, which are the master transcription factor in osteoclast differentiation and the osteoclast-specific marker, respectively [43]. OPG is a RANKL soluble decoy receptor produced by osteoblasts, which can inhibit the binding of RANKL and RANK [9,10,44]. The results presented here showed that dioscin significantly increased OPG/RANKL ratio and decreased the levels of TRAF6, p-p38, p-JNK, p-ERK, p-Akt, NF-B, AP-1, NFATc1 and cathepsin K in RANKL-induced osteoclasts. Thsee data suggested that dioscin inhibited osteoclastogenesis by up-regulating OPG and down-regulating RANKL-induced TRAF6 signaling pathways.
Furthermore, LPS/TLR4 can activate TRAF6 signaling pathway through triggering its adaptor protein MyD88 [45–48], and then initiate MAPKs, Akt, NF-B and AP-1 signaling pathways, which play key roles in the osteoclasts activation [11]. Our results indicated that dioscin markedly decreased the protein levels of TLR4, MyD88, TRAF6, p-p38 p-p38, p-JNK, p-ERK, p-Akt, NF-B and AP-1 in LPS/TLR4-induced osteoclasts. Moreover, dioscin inhibited the NFATc1 and cathepsin K activities. Therefore, it was obvious that dioscin inhibited osteoclastogenesis by decreasing TLR4/MyD88-induced TRAF6 signaling pathway. In summary, dioscin conferred direct actions against OVXinduced bone loss by enhancing osteoblastogenesis and inhibiting osteoclastogenesis (Supplemental Fig. 8). Accordingly, dioscin represents a novel and potent candidate for the treatment of PMO. Of course, deeply mechanisms and clinical applications of dioscin against PMO are needed to be elucidated in the further.
Conflicts of interest All authors declared no competing financial interests.
Acknowledgements This work was financially supported by the Program for Liaoning Innovative Research Team in University (LT2013019).
X. Tao et al. / Pharmacological Research 108 (2016) 90–101
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