Cistanche Deserticola Polysaccharide Inhibits OVX-induced Bone Loss in Mice And RANKL-induced Osteoclastogenesis

Please contact oscar.xiao@wecistanche.com for more information

ABSTRACT

Osteoporosis is a serious bone disease affecting the aging population. Cistanche deserticola (CD), a tonic and medicinal food widely used in China, has been proved to provide effective treatment for osteoporosis. Cistanche deserticola polysaccharide (CDP), extracted from CD, possesses various pharmacological properties, but its role in osteoclastic formation and function, as well as osteoporosis, remains unknown. The purpose of this study was to extract and purify CDP to further explore its potential mechanism of action on osteoporosis. Results showed that the CDP treatment prevented OVX-induced osteoporosis and ameliorated bone loss by repressing osteoclast activity and function. Furthermore, CDP treatment significantly inhibited RANKL-induced osteoclastogenesis, bone resorption, and osteoclast-specific gene expression. Mechanistically, CDP inhibited RANKL-induced NF-κB and MAPKs signaling pathways activation and consequently affected the downstream NFATc1 activation. The findings in this study suggest that CDP is a potentially safe drug for treating osteoporosis.(10 benefits the Cistanche can give )

Please click here to know more

Introduction

Osteoporosis—a disease characterized by decreased bone mass, abnormal bone tissue microstructure, increased bone fragility, and fracture (De Martinis, Di Benedetto, Mengoli, & Ginaldi, 2006)— currently affects more than 200 million people worldwide and presents a substantial medical and socioeconomic burden on modern society (Strom et al., 2011). The clinical treatment of osteoporosis mainly focuses on hormone replacement, bisphosphonate, or denosumab therapy. These methods are effective but pose long-term side effects such as the potential risk of breast cancer and atypical femur fractures (Black, Bauer, Schwartz, Cummings, & Rosen, 2012; Rachner, Khosla, & HofBauer, 2011). Hence, there is an urgent need to explore drugs that can not only inhibit osteoporosis but also possess fewer undesirable side effects. Cistanche deserticola (CD), a tonic and medicinal food widely used in China, known as “desert ginseng”, is the dried succulent stem with scale leaf of C. deserticola YC Ma (Gu, Yang, & Huang, 2016) and has diverse and effective pharmacological activities, such as immune regulation, anti-oxidation, and anti-osteoporosis properties (Hu et al., 2020; Li et al., 2012; Zhang et al., 2014). Previous studies on the active sub-stances of CD on osteoporosis treatment mainly focused on phenylethanoid glycosides (Li, Jiang, & Gu, 2018; Xu, Zhang, Wang, Yao, & Ma, 2017). However, the CD also contains other effective components such as iridoids, lignans, polysaccharides (Wang, Zhang, & Xie, 2012). The clinical application of traditional Chinese medicine (TCM) is mainly water decocting. Polysaccharides are water-soluble components and are most likely to be the main pharmacodynamic components of TCM. Polysaccharides extracted from TCM were wildly reported to have an anti-osteoporosis effect and a few undesirable side effects, which have been generally used in clinics (e.g., polysaccharides extracted from Epimedium brevicornum (Zheng, He, Wu, Cai, & Wei, 2020), Achyranthes bidentata (Zhang, Zhang, Zhang, Wang, & Yan, 2018), and Morinda officinalis (Yan et al., 2019)). Therefore, we hypothesized that Cistanche deserticola polysaccharide (CDP) extracted from CD may be a potentially safe drug for the treatment of osteoporosis. Typically, bone resorption and bone formation maintain a dynamic balance during bone remodeling in coordination with several types of cells, including osteoclasts, osteoblasts, bone lining cells, and osteocytes (Kular, Tickner, Chim, & Xu, 2012). A break in this balance can result in a variety of bone metabolic diseases, such as osteoporosis (Zhu et al., 2018) and osteosclerosis (Ihde et al., 2011). Osteoclasts, as enddifferentiated cells derived from the mononuclear/macrophage lineage, are the only cells with bone resorption function (Teitelbaum, 2000). There are two key cytokines that participate in the osteoclast differentiation and maturation (Koga et al., 2004): macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor κB (NF-κB) ligand (RANKL). M-CSF mediates the differentiation of hematopoietic stem cells into osteoclast progenitors and promotes the differentiation of osteoclasts by upregulating the expression of the RANK receptor (Takayanagi, 2007). The binding of RANKL and RANK on the osteoclast cell surface results in the following: (1) recruitment of signaling adapter molecules such as TNF receptor-associated factor 6 (TRAF6); (2) activation of multiple downstream targets, including NF- κB and mitogen-activated protein kinases (MAPKs); and (3) upregulation of the expression levels of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1) (Liu et al., 2019; Yamashita et al., 2007). The activation of these signaling pathways directly regulates the expression of osteoclast genes, including acid phosphatase 5 (Acp5) [encoding tartrate-resistant acid phosphatase (TRAcP)], matrix metalloproteinase 9 (MMP9), and cathepsin K (CTSK) (Boyle, Simonet, & Lacey, 2003). Therefore, the inhibition of RANKL-induced osteoclast differentiation-related signaling pathways is a potential therapeutic method for osteoporosis. In this study, we aimed to determine the effects of CDP treatment on the ovariectomized (OVX)-induced osteoporosis mouse model in vivo and on RANKL-induced osteoclast activity in vitro, with a focus on the expression of osteoclast-specific genes and the activation of NFATc1 and the NF-κB and MAPKs signaling pathways. Our findings may provide new insights into the potential of CDP as a safe and effective drug for treating osteoporosis. 2. Materials and methods 2.1. Chemicals and sample collection CD (no. 180801) was purchased from Anhui Jishun Traditional Chinese Medicine Co., Ltd. (Anhui, China) and identified by Professor Haibo Huang from the School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China. Estradiol valerate tablets were purchased from Bayer (Leverkusen, North-RhineWestphalia, Germany). Alpha-modified minimal essential medium (α-MEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin/streptomycin and the TRAcP staining kit were acquired from Solarbio (Beijing, China). Recombinant mouse M-CSF and recombinant mouse RANKL were procured from R&D Systems (Minneapolis, MN, USA). Hydroxyapatite-coated plates were purchased from Corning Life Sciences (St. Lowell, MA, USA). The primary antibodies for NFATc1 and CTSK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); IκB-α, p65, P-p65, p38, P-p38, ERK1/2, P-ERK1/2, JNK, and P-JNK (Cell Signaling Technology, Danvers, MA, USA); and β-actin (CWBIO, Beijing, China) was also obtained. The other reagents used in this study were of analytical grade, and the water was purified by the Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. CDP extraction The CD was ground to a fine powder and mixed with petroleum ether thrice to defeat it. The solid residue was collected by filtration and then dried at room temperature. The polysaccharides were extracted from the pre-treated samples thrice with water for 2 h, concentrated, precipitated by the addition of anhydrous ethanol to a final concentration of 80% (v/ v), and stored at 4 ◦C for 24 h. The precipitates were collected by centrifugation at 3000 rpm for 20 min, dissolved in distilled water, and purified (removal of free proteins) using the Sevag method (Zhao et al., 2019). The recovered polysaccharides were dialyzed, dried, and stored until further use. Furthermore, the chemical composition results showed that the total sugar, uronic acid, sulfate, and protein contents of CDP were 67.63 ± 0.98%, 21.05 ± 0.49%, 1.90 ± 0.24%, and 8.81 ± 0.36%. The monosaccharide composition and Fourier transform infrared analysis of CDP were shown in Supplementary Fig. 1 and 2. 2.3. OVX-induced osteoporosis mouse model All animal experiments were approved by the Animal Ethics Committee of the Guangzhou University of Chinese Medicine (approved SYXK 2019-0202). Thirty 6-week-old female C57BL/6 mice were supplied by the Experiment Animal Center of the Guangzhou University of Chinese Medicine. After acclimatization for 1 week, one group of mice was given a sham operation (Sham group), while the remaining mice were subjected to an ovariectomy operation (OVX group). After surgery, the mice were allowed to recover for 1-week. Then, mice successfully modeled were randomly divided into 5 groups, with 6 mice per group: (1) Sham group: treated with distilled water, (2) OVX group: treated with distilled water, (3) OVX + E2 group (E2): treated with 0.13 mg/kg estradiol valerate tablets, (4) OVX + CDP low dose group (CDP-L): treated with 300 mg/kg of CDP, (5) OVX + CDP high dose group (CDPH): treated with 600 mg/kg of CDP. Distilled water, E2, or CDP was administered by gavage once daily. All mice were sacrificed after the 12- week treatment period (Fig. 1A). The uteruses were collected and weighed, and the left tibias were collected for subsequent examinations. Whole blood samples were collected and centrifuged at 5000 rpm for 15 min at 4 ◦C. The serum supernatants were aspirated and stored at − 80 ◦C until further analysis. 2.4. Micro-computed tomography (micro-CT) analysis and bone histomorphometry The left tibias were fixed with 4% paraformaldehyde for 48 h and subsequently placed in centrifuge tubes containing normal saline. The fixed samples were scanned with Skyscan 1172 micro-CT instrument (Bruker micro-CT, Skyscan, Kontich, Belgium) using the following settings: voltage, 80 kV; source current, 100 μA; Al, 0.5 mm filter; pixel size, 9.76 μm; and rotation step, 0.6◦. Several parameters of the trabecular bone, including bone mineral density (BMD), bone volume fraction (BV/TV), bone surface per total volume (BS/TV), trabecular number (Tb. N), and trabecular spacing (Tb.Sp) were measured using CTAnalyser® software (Bruker micro-CT, Skyscan). Three-dimensional images were generated using CTVol software (Bruker micro-CT. Skyscan). Following micro-CT analysis, the left tibias were decalcified in 14% EDTA solution and embedded into paraffin for sectioning. The samples were sliced into 5 µm sections using a microtome prior to the hematoxylin and eosin (H&E) and TRAcP staining experiments. The stained sections were examined and documented using Olympus CX 31 microscope (Olympus Optical Co., Ltd, Tokyo, Japan). 2.5. Serum analysis The calcium (Ca) and phosphorus (P) concentrations were determined according to the kit instructions designed by the Nanjing Jiancheng Bioengineering Institute (Nanjing, China). TRAcP-5b and RANKL levels in the serum were analyzed using TRAcP-5b ELISA kit (CUSABIO, Wuhan, China) and RANKL ELISA kit (Cloud-CloneCorp., Wuhan, China), respectively.

Chemicals and sample collection CD (no. 180801) was purchased from Anhui Jishun Traditional Chinese Medicine Co., Ltd. (Anhui, China) and identified by Professor Haibo Huang from the School of Pharmaceutical Sciences, Guangzhou University of Chinese Medicine, Guangzhou, China. Estradiol valerate tablets were purchased from Bayer (Leverkusen, North-RhineWestphalia, Germany). Alpha-modified minimal essential medium (α-MEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Waltham, MA, USA). Penicillin/streptomycin and the TRAcP staining kit were acquired from Solarbio (Beijing, China). Recombinant mouse M-CSF and recombinant mouse RANKL were procured from R&D Systems (Minneapolis, MN, USA). Hydroxyapatite-coated plates were purchased from Corning Life Sciences (St. Lowell, MA, USA). The primary antibodies for NFATc1 and CTSK (Santa Cruz Biotechnology, Santa Cruz, CA, USA); IκB-α, p65, P-p65, p38, P-p38, ERK1/2, P-ERK1/2, JNK, and P-JNK (Cell Signaling Technology, Danvers, MA, USA); and β-actin (CWBIO, Beijing, China) was also obtained. The other reagents used in this study were of analytical grade, and the water was purified by the Milli-Q water purification system (Millipore, Bedford, MA, USA). 2.2. CDP extraction The CD was ground to a fine powder and mixed with petroleum ether thrice to defeat it. The solid residue was collected by filtration and then dried at room temperature. The polysaccharides were extracted from the pre-treated samples thrice with water for 2 h, concentrated, precipitated by the addition of anhydrous ethanol to a final concentration of 80% (v/ v), and stored at 4 ◦C for 24 h. The precipitates were collected by centrifugation at 3000 rpm for 20 min, dissolved in distilled water, and purified (removal of free proteins) using the Sevag method (Zhao et al., 2019). The recovered polysaccharides were dialyzed, dried, and stored until further use. Furthermore, the chemical composition results showed that the total sugar, uronic acid, sulfate, and protein contents of CDP were 67.63 ± 0.98%, 21.05 ± 0.49%, 1.90 ± 0.24%, and 8.81 ± 0.36%. The monosaccharide composition and Fourier transform infrared analysis of CDP were shown in Supplementary Fig. 1 and 2. 2.3. OVX-induced osteoporosis mouse model All animal experiments were approved by the Animal Ethics Committee of the Guangzhou University of Chinese Medicine (approved SYXK 2019-0202). Thirty 6-week-old female C57BL/6 mice were supplied by the Experiment Animal Center of the Guangzhou University of Chinese Medicine. After acclimatization for 1 week, one group of mice was given a sham operation (Sham group), while the remaining mice were subjected to an ovariectomy operation (OVX group). After surgery, the mice were allowed to recover for 1-week. Then, mice successfully modeled were randomly divided into 5 groups, with 6 mice per group: (1) Sham group: treated with distilled water, (2) OVX group: treated with distilled water, (3) OVX + E2 group (E2): treated with 0.13 mg/kg estradiol valerate tablets, (4) OVX + CDP low dose group (CDP-L): treated with 300 mg/kg of CDP, (5) OVX + CDP high dose group (CDPH): treated with 600 mg/kg of CDP. Distilled water, E2, or CDP was administered by gavage once daily. All mice were sacrificed after the 12- week treatment period (Fig. 1A). The uteruses were collected and weighed, and the left tibias were collected for subsequent examinations. Whole blood samples were collected and centrifuged at 5000 rpm for 15 min at 4 ◦C. The serum supernatants were aspirated and stored at − 80 ◦C until further analysis. 2.4. Micro-computed tomography (micro-CT) analysis and bone histomorphometry The left tibias were fixed with 4% paraformaldehyde for 48 h and subsequently placed in centrifuge tubes containing normal saline. The fixed samples were scanned with Skyscan 1172 micro-CT instrument (Bruker micro-CT, Skyscan, Kontich, Belgium) using the following settings: voltage, 80 kV; source current, 100 μA; Al, 0.5 mm filter; pixel size, 9.76 μm; and rotation step, 0.6◦. Several parameters of the trabecular bone, including bone mineral density (BMD), bone volume fraction (BV/TV), bone surface per total volume (BS/TV), trabecular number (Tb. N), and trabecular spacing (Tb. Sp) were measured using CTAnalyser® software (Bruker micro-CT, Skyscan). Three-dimensional images were generated using CTVol software (Bruker micro-CT. Skyscan). Following micro-CT analysis, the left tibias were decalcified in 14% EDTA solution and embedded into paraffin for sectioning. The samples were sliced into 5 µm sections using a microtome prior to the hematoxylin and eosin (H&E) and TRAcP staining experiments. The stained sections were examined and documented using Olympus CX 31 microscope (Olympus Optical Co., Ltd, Tokyo, Japan). 2.5. Serum analysis The calcium (Ca) and phosphorus (P) concentrations were determined according to the kit instructions designed by the Nanjing

Jiancheng Bioengineering Institute (Nanjing, China). TRAcP-5b and RANKL levels in the serum were analyzed using TRAcP-5b ELISA kit (CUSABIO, Wuhan, China) and RANKL ELISA kit (Cloud-CloneCorp., Wuhan, China), respectively. 2.6. In vitro osteoclastogenesis assay BMMs were isolated from the tibia and femur of 6-week-old female C57BL/6 mice. The isolated cells were cultured in α-MEM medium containing 25 ng/mL M-CSF, 10% FBS and 1% penicillin/streptomycin. Upon reaching confluence, the BMMs (1 × 104 cells/well) were seeded in 96-well plates and treated with CDP in the presence of 50 ng/mL RANKL. The medium and CDP were replaced every 2 days. After 7 days, the cells were fixed with 4% paraformaldehyde and stained for the presence of trace. The number of osteoclasts, defined as TRAcPpositive cells with three or more nuclei, was counted and documented using a light microscope. 2.7. Cell proliferation assay The BMMs (1 × 104 cells/well) were seeded in a 96-well plate and incubated overnight. Following this, the cells were treated with different concentrations of CDP (0, 0.625, 1.25, 2.5, 5, 10, 20, and 40 µg/mL) for 48 h. The cell confluence was detected and analyzed using IncuCyte ZOOM® (Essen BioScience, Ann Arbor, MI, USA), a long-term, real-time dynamic live cell imaging system. 2.8. Hydroxyapatite resorption assay The BMMs (1 × 104 cells/well) were seeded in a hydroxyapatite-coated plate, treated with CDP at the indicated concentrations (0, 5 and 10 µg/mL), and cultured in complete α-MEM containing 25 ng/mL M-CSF and 50 ng/mL RANKL. After 7 days, the cells were washed with a 10% bleach solution to remove the cell components. The images of hydroxyapatite resorption areas were captured using IncuCyte ZOOM® and analyzed using ImageJ software (NIH, Bethesda, MD, USA) (Abramoff, Magelhaes, & Ram, 2003). 2.9. RNA isolation and real-time reverse transcription-quantitative PCR (RT-qPCR) analysis The BMMs (1 × 105 cells/well) were seeded in a 6-well plate and stimulated with RANKL and M-CSF in the presence of CDP at different concentrations (0, 5 and 10 µg/mL) for 5 days. Total RNA was isolated from the cells or bone tissue of mice using TRIzol reagent (SigmaAldrich). Complementary DNA was synthesized from 2 µg total RNA using a reverse transcriptase kit (TransGen Biotech, Beijing, China). The RT-qPCR reactions were prepared using PerfectStart™ Green qPCR SuperMix (TransGen Biotech) and detected by ABI 7500 system (Applied Biosystems, Thermo Fisher Scientific, Inc., Waltham, MA, USA). The cycling parameters for PCR were set as follows: 95 ◦C for 5 min, followed by 40 cycles of 95 ◦C for 15 s, and 60 ◦C for 30 s. The specific primers used are shown in Table 1, and the quantity of each target gene was normalized to GAPDH (internal control).

2.10. Western blot analysis The BMMs (5 × 105 cells/well) were seeded in a 6-well plate. For short time-course experiments, the cells were pre-incubated with the different concentrations of CDP (0, 5, and 10 µg/mL) for 1 h, and then treated with 50 ng/mL RANKL for 30 min. For a long time course, the cells were stimulated with 50 ng/mL RANKL on days 3, 5, and 7 in the presence of CDP (0, 5, and 10 µg/mL). Total proteins were extracted using the RIPA lysis buffer (CWBIO). Proteins were separated by 10% SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% skim milk at room temperature for 2 h, incubated with primary antibodies overnight at 4 ◦C, and then with the corresponding secondary antibodies for 1.5 h. The membranes were developed using ECL reagents (Millipore Corp., Billerica, MA, USA), and images were taken using Tanon 5200 Chemiluminescence Imaging System (Tanon Science and Technology, Shanghai, China). 2.11. Detection of NFATc1 translocation using immunofluorescence test The BMMs was seeded onto 12-well coverslips, stimulated with RANKL and M-CSF and treated with 10 µg/mL CDP for 5 days. The cells were fixed with 4% paraformaldehyde for 15 min, washed thrice with PBS, and permeabilized with 0.1% Triton X-100 for 10 min. The cells were mixed with 10% goat serum (CWBIO) and incubated for 2 h. Subsequently, the cells were incubated with the primary antibody overnight at 4 ◦C and then with Alexa Fluor 488 goat anti-mouse secondary antibody (Abcam, Cambridge, MA, USA) in the dark for 1 h. The coverslips were washed with PBS, mounted in Prolong Gold Antifade Reagent with 4′, 6-diamidino-2-phenyl in-dole (DAPI) (Solar), and inspected using Zeiss LSM 800 with Airyscan confocal microscope (Carl Zeiss, Oberkochen, Germany). 2.12. Statistical analysis All experimental data were analyzed using SPSS 25.0 statistical software (IBM Corporation, Armonk, NY, USA). The data for animal and cell studies are expressed as means ± SEM and means ± SD, respectively. Results are using one-way analysis of variance or Student’s t-test. Values of p < 0.05 were considered statistically significant. 3. Results 3.1. CDP prevents OVX-induced bone loss in vivo We used an OVX-induced osteoporosis mouse model to investigate the effects of CDP treatment on osteoporosis in vivo. Compared with the Sham group, the bodyweight of OVX mice significantly increased whereas the uterine index decreased, indicating that the osteoporosis model was constructed successfully (Fig. 1B and D). micro-CT analysis of the tibias in the OVX group showed an extensive bone loss. Compared with the OVX group, the number of bone trabeculae increased in the CDP-L group, while the increase in the CDP-H and E2 groups was more significant, and the trabeculae in the latter two groups became coarser and the gap became smaller (Fig. 1C). Quantitative analysis confirmed that several bone histomorphometric parameters, including BMD, BV/TV, BS/TV, and Tb. N had significantly increased values, whereas Tb. Sp showed decreased values in the CDP-H group (Fig. 1E and F). Hispathological examination using H&E staining revealed that the number of bone trabeculae in the CDP-H and E2 groups was significantly higher than that in the OVX group while there was no significant improvement in the CDP-L group (Fig. 1G). Additionally, TRAcP staining showed that the number of osteoclasts markedly increased in the OVX group but decreased in the CDP-H and E2 groups (Fig. 1H). CDP-L, CDP-H, and E2 groups showed inhibited the OVX-induced increase of P content but increased the Ca content in the blood serum. The bone absorption markers, RANKL and TRAcP-5b showed a decreasing trend in the CDP-L.

the group without a statistical difference, while they were significantly decreased in the CDP-H group (Fig. 1I). Furthermore, we examined the effect of CDP on the expression of osteoclast marker genes in the bone tissues by RT-qPCR. The mRNA levels of NFATc1, Acp5, Mmp9, and CTSK in the OVX group were significantly higher than those in the Sham group. CDP-H groups showed decreased the mRNA level of NFATc1, Acp5, and CTSK by 1.74-, 1.70- and 2.00-fold, compared with the OVX group. (Fig. 1J). 3.2. CDP suppresses RANKL-induced osteoclastogenesis in vitro We monitored the cell proliferation of CDP-treated BMMs using a long-term real-time dynamic live cell imaging analyzer. Compared to the control group, cell confluence in the CDP-treated groups did not change at a dose <10 µg/mL, but was significantly reduced at the 20 and 40 µg/mL dosages (Fig. 2A). To investigate the effect of CDP on RANKLinduced osteoclastogenesis, BMMs were stimulated with RANKL and MCSF in the presence of CDP for 7 days. TRAcP, a characteristic enzyme of osteoclasts, is considered as an indicator of osteoclast function (Minkin, 1982). TRAcP staining showed that the osteoclasts treated with >1.25 µg/mL CDP showed significantly reduced size and number (Fig. 2B and C). These results showed that CDP effectively inhibited the expected RANKL-induced osteoclastogenesis without affecting the cell viability. 3.3. CDP inhibits RANKL-induced osteoclastic resorption activity and osteoclast-specific gene expression We further determined the effects of CDP treatment on the bone resorption activity of osteoclasts using hydroxyapatite-coated plates. Bone resorption is the main function and standard for measuring the activity and ability of osteoclasts (Novack & Faccio, 2011). The results showed that CDP can significantly reduce the bone-resorptive area of osteoclasts in a dose-dependent manner, indicating that CDP inhibited the bone resorption activity induced by RANKL (Fig. 3A and B). In particular, the osteoclastic bone-resorptive area decreased to approximately 6.3% after treatment with 10 µg/mL CDP. To better figure out the hindrance of CDP on osteoclastogenesis, we measured the transcription levels of osteoclast-related genes. Without treatment with CDP, induction with RANKL promoted the expression of NFATc1, CTSK, Acp5, Atp6v0d2, and Mmp9 genes. On the contrary, the concomitant application of CDP and RANKL significantly downregulated these five genes (Fig. 3C and D).

3.4. CDP inhibits RANKL-induced NFATc1 activation TRAcP staining revealed that CDP treatment significantly reduced the expected increase in the number of mature osteoclasts after RANKL stimulation for 3–7 days (Fig. 4A and B). Furthermore, we investigated whether CDP treatment can effectively impair osteoclast differentiation by inhibiting NFATc1 expression. Results showed that NFATc1 was highly expressed after RANKL stimulation for 5 days, which was significantly inhibited after CDP treatment (Fig. 4C and D). In addition, CDP treatment suppressed the expression levels of CTSK, a protein required for normal osteoclast formation and function (Bossard et al., 1996) (Fig. 4E and F). We selected a point-in-time representation of RANKL stimulation for 5 days to further investigate the effect of 10 µg/ mL CDP treatment on NFATc1 translocation. As expected, CDP treatment significantly inhibited the RANKL-induced NFATc1 nuclear translocation (Fig. 4G and H).

Fig. 4. CDP suppresses RANKL-induced NFATc1 activation. (A) The BMMs were seeded in a 6-well plate and stimulated with RANKL and M-CSF in the presence of CDP (0, 5, and 10 µg/mL) for the indicated days. Representative images showing TRAcP-positive multinucleated cells. Magnification, ×100. Scale bars = 100 μm. (B) Quantification of TRAcP-positive multinucleated cells (nuclei ≥ 3). (C-F) Western blot analysis of NFATc1 and CTSK protein expressions, in BMMs, stimulated with MCSF and RANKL for 3–7 days in the presence of CDP. (G) The BMMs were treated with 10 μg/mL CDP and stimulated by M-CSF and RANKL for 5 days, then immunostained with NFATc1 (green) and DAPI (blue) and observed using a confocal microscope. Magnification, ×200. Scale bars = 20 μm. (H) Fluorescence intensity was analyzed using Image-Pro Plus. Values are expressed as means ± SD (n = 3). *p < 0.05, **p < 0.01, ***p < 0.001 vs. Control group; #p < 0.05, ##p < 0.01, ###p < 0.001 vs. RANKL group. 3.5. CDP inhibits RANKL-induced activation of NF-κB and MAPKs signaling pathways To elucidate the mechanisms involved in the CDP-induced inhibition of osteoclast differentiation, we examined the impact of CDP treatment on the NF-κB and MAPKs pathways. Results revealed that CDP treatment had suppressive effects on IκB-α degradation and p65 phosphorylation in a dose-dependent manner (Fig. 5A and B). Phosphorylation of three MAPKs family members, specifically extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were upregulated.

Discussion

Osteoporosis is a common and degenerative bone disease. At present, there are many chemical synthesis drugs used in clinical treatments for osteoporosis, such as estrogen and bisphosphonate. However, the long-term use of these drugs will gradually weaken clinical efficacy and even cause many serious side-effects, including increased risk of ovarian, breast, and endometrial cancers and osteonecrosis of the jaw (Beral, 2003; Khan et al., 2009; Lacey et al., 2002). While in this study, we selected CDP, an effective component extracted from CD, as the therapeutic agent and demonstrated that CDP had a protective effect against bone loss in OVX mice and inhibited RANKL-induced osteoclast formation and function. Meanwhile, according to the “kidney governing bone” traditional Chinese medicine theory, bone loss is attributed to kidney deficiencies. As a kidney-tonifying medical food, the CD has been proved to provide effective treatment for osteoporosis (Fan et al., 2019; Jiang, Wang, Li, & Zhang, 2016). Thus, CDP extracted from this natural herb is a better and safer drug option. Currently, the OVX mouse model is a classic model of post-menopausal bone loss in women, investigating the causes of bone loss and conducting intervention (Wehrle et al., 2015). Accordingly, we performed bilateral oophorectomy on C57BL/6J female mice to construct an osteoporosis model. In previous studies, the CDP doses given to animals were 50–200 mg/kg (low dose) and 1800 mg/kg (high dose) (Guo et al., 2016; Zhang et al., 2018). Here, the high dose of CDP in mice was 600 mg/kg, which was based on the high clinical dosage of 30 g CD in decoction; thus, it is similar to current clinical practice. In this study, treatment with CDP significantly inhibited OVX-induced body weight gain, which is a common phenomenon observed in OVX mice (Davis et al., 2019). By using micro-CT scanning and 3D reconstruction, we found that OVX led to a noticeable reduction in BMD, BV/TV, BS/TV, and Tb.N, and an increase in Tb.Sp, while CDP treatment reversed all these alterations. In particular, compared with the positive drug, CDP could significantly improve the BMD of OVX mice, which is an important marker of bone quality and used to reflect the degree of osteoporosis (Fuggle et al., 2019), indicating that CDP has an advantage over the positive drug in improving BMD. Furthermore, the histological results indicated that CDP treatment could reduce the increased amount of TRAcP-positive osteoclasts induced by OVX. These results suggest that CDP administration effectively mitigated bone loss in OVX-induced osteoporosis, possibly by inhibiting osteoclast formation. Consistent with the above results, we found CDP dose-dependently inhibited RANKL-induced osteoclastic formation and function, while CDP had little effect on M-CSF-induced osteoclast precursor cell proliferation in our in vitro experiments. The binding of RANKL and RANK can lead to the recruitment of TRAF6 and the subsequent activation of several downstream signaling pathways, such as NF-κB and MAPKs (Boyle et al., 2003; Huang et al., 2006). The NF-κB signaling pathway plays an important role in the regulation of osteoclast differentiation, and the silencing of genes coding for key proteins in this pathway can lead to abnormal bone development (Leibbrandt & Penninger, 2008). It is well established that NF-κB is present in the cytoplasm of unstimulated cells in a complex with IκB but rapidly enters into the nucleus after RANKL stimulation (Boyle et al., 2003). The released IκB is then rapidly degraded, whereas NF-κB activates specific gene transcription, consequently promoting osteoclast differentiation, maturation, and apoptosis (Abdelmagid et al., 2015). In this study, results showed that CDP treatment suppressed RANKLinduced activation of the NF-κB signaling pathway, as demonstrated by inhibition of the degradation of IκBα and phosphorylation of p65 in a dose-dependent manner, which may involve in its anti-osteoclastogenic effect. In addition, the MAPKs signaling pathway, including ERK, JNK, and p38, are closely involved in RANKL-induced osteoclastogenesis (Li et al., 2002). Furthermore, the dominant inhibitors of p38 and JNK can prevent RANKL-induced osteoclastogenesis (Chang et al., 2008; Kim et al., 2019), whereas ERK plays a significant role in osteoclast survival (Miyazaki et al., 2000). It is well established that RANKL activates the MAPKs signaling pathway by increasing the phosphorylation of ERK, JNK, and p38 (Mizukami et al., 2002). In our study, we demonstrated that CDP suppressed RANKL-induced phosphorylation of key proteins in the MAPKs signaling pathway. Taken together, these results indicate that inhibiting the activation of the NF-κB and MAPKs signaling pathways is contributed to the inhibitory effect of CDP against osteoclastogenesis. Activation of the NF-κB and MAPKs signaling pathways promotes the expression of several key transcription factors, such as cellular oncogene fos, activator protein 1, and NFATc1 (Huang et al., 2006; Wagner & Matsuo, 2003). NFATc1, an important member of the NFAT family, is involved in terminal osteoclast differentiation as a master transcriptional regulator (Asagiri et al., 2005). It has been reported that NFATc1- deficient embryonic stem cells can not differentiate into osteoclasts under the stimulation of RANKL and the ectopic expression of NFATc1 causes precursor cells to undergo differentiation bypassing RANKL signaling, which suggests that NFATc1 has a critical role in osteoclast differentiation (Takayanagi et al., 2002). The results of our study showed the suppressive effect of CDP on NFATc1 expression and its following protein CTSK. Interestingly, we observed that the protein expression level of NFATc1 first increased and then decreased, reaching the highest level on the 5th day after RANKL stimulation. This was consistent with the results of Baek et al. (2014), who observed M-CSFinduced NFATc1 degradation during late-stage osteoclastogenesis through the Cbl-induced ubiquitination of NFATc1 in an Src kinase-dependent manner. Immunofluorescence assay results consistently supported the inhibitory effect of CDP on NFATc1 transcriptional activation. Furthermore, osteoclast-specific genes, including CTSK, Acp5, Mmp9, and Atp6v0d2, are all regulated by NFATc1 directly (Y. Kim et al., 2005), were suppressed by CDP. These findings illustrate that the NFATc1 is a target of the inhibitory effect of CDP on the NF-κB and MAPKs signaling pathways.

Conclusion

In summary, we extracted and purified the active polysaccharide from CD and demonstrated that CDP exhibited a protective effect on OVX-induced osteoporosis and ameliorated bone loss by repressing osteoclast activity and function. Furthermore, CDP can inhibit RANKLinduced osteoclast differentiation and function by interfering with the NF-κB and MAPKs signaling pathways and consequently affecting the downstream NFATc1 activation. In conclusion, our findings provide the foundation for the expansion and application of CDP in the treatment of osteoporosis.

This article comes from Contents lists available at ScienceDirect Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff