Magnolol prevents ovariectomy‑induced bone loss by suppressing osteoclastogenesis via inhibition of the nuclear factor‑κB and mitogen‑activated protein kinase pathways
- Authors:
- Published online on: February 18, 2019 https://doi.org/10.3892/ijmm.2019.4099
- Pages: 1669-1678
-
Copyright: © Fei et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
Abstract
Introduction
Bone homeostasis is dependent on the balance between bone formation and bone resorption, which are mediated by osteoblasts and osteoclasts (1). In patients with osteoporosis, principally postmenopausal women, the rate of osteoclastogenesis surpasses that of osteogenesis, resulting in decreased bone density and strength (2-4). Osteoporosis is associated with an increased incidence of bone fractures, which are particularly dangerous in the elderly, and with a reduced quality of life (5,6). The prevalence of osteoporosis is increasing worldwide (7). In the United States, ~10 million individuals aged >50 years suffer from osteoporosis and 34 million suffer from osteopenia. In China, the prevalence of osteoporosis was 14.94% prior to 2008 and 27.96% between 2012 and 2015 (8).
Osteoclasts differentiate from the mononuclear phagocyte system and are involved in bone re sorption. Inflammation leads to overactivation of osteoclasts, and thus suppression of inflammation inhibits osteoclastogenesis (3). Osteoclastogenesis has been reported to involve several signaling pathways (6,9,10). Receptor activator for nuclear factor-κB ligand (RANKL) is vital for the formation and function of osteoclasts (11,12), and binding of RANKL to its receptor RANK results in activation of the nuclear factor (NF)-κB, mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) signaling pathways. The activation of these pathways induces the expression of several osteoclast-specific genes, including cathepsin K, calcitonin receptor (CTR), matrix metalloproteinase 9 (MMP9), TNF receptor-associated factor 6 (TRAF6), and tartrate-resistant acid phosphatase (TRAP) (13-15).
Magnolol is the active component of the Chinese medicine Magnolia officinalis and is used clinically for its anti-inflammatory and antioxidant effects (16-19). Magnolol reportedly inhibits RANKL-induced osteoclastic differentiation of RAW264.7 macrophages (20). However, further research is required to assess the effect of magnolol on osteoporosis and the underlying mechanism(s). Therefore, the present study aimed to investigate the effect of magnolol on osteoclastogenesis in vivo and in vitro, as well as to examine the underlying mechanism at the molecular level.
Materials and methods
Reagents and cells
Magnolol was provided by Shanghai Nodel Biotech Co., Ltd. (Shanghai, China) and reconstituted in phosphate-buffered saline (PBS), serving as the vehicle. RAW264.7 cells were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China). Fetal bovine serum (FBS), penicillin, streptomycin and antibodies were purchased from BioTNT (Shanghai, China). The primary antibodies (all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) used included anti-cathepsin K (1:500; cat. no. sc-6506), anti-CTR (1:200; cat. no. sc-8858), anti-MMP9 (1:400; cat. no. sc-21733), anti-TRAF6 (1:250; cat. no. sc-8409), anti-TRAP (1:350; cat. no. sc-100314), anti-p65 (1:350; cat. no. sc-398442), anti-phosphorylated (p)-p65 (1:500; cat. no. sc-136548), anti-p50 (1:250; cat. no. sc-166580), anti-p-p50 (1:400; cat. no. sc-271908), anti-inhibitor of κB (IκB; 1:350; cat. no. sc-74451), anti-p-IκB (1:500; cat. no. sc-8404), anti-extracellular signal-regulated kinase (ERK; 1:200; cat. no. sc-135900), anti-p-ERK (1:400; cat. no. sc-81492), anti-c-Jun N-terminal kinase (JNK; 1:500; cat. no. sc-7345), anti-p-JNK (1:400; cat. no. sc-135642), anti-p38 (1:300; cat. no. sc-136210), anti-p-p38 (1:300; cat. no. sc-7973) and anti-β actin (1:1,000; cat. no. sc-8432). Horseradish peroxidase-conjugated anti-mouse (cat. no. ZB2301) and anti-rabbit (cat. no. ZB2305) antibodies (1:5,000; OriGene Technologies, Inc., Beijing, China) were also used. Dulbecco’s modified Eagle’s medium was purchased from Corning Inc. (Corning, NY, USA).
Animals and experimental design
Experiments involving mice were approved by the Ethics Committee on Animal Experiments of the Central Hospital of Zibo (Shandong University, Zibo, China). A total of 36 C57BL/6 female mice (8-week-old; weight, 20-25 g) were obtained from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China). The mice were housed under standard laboratory conditions (20-25°C; humidity, 40-70%; 16/8-h light/dark cycle), and provided with food and water ad libitum. The mice were randomly divided into the sham-operated, saline-treated ovariectomy (OVX), and magnolol-treated OVX groups (n=6 per group). OVX was performed using a standard protocol (21) Sham operation was performed following the same procedure as OVX without the resection of ovaries. At 24 h postoperatively, the OVX mice were treated with an intraperitoneal injection of 10 ml/kg magnolol or saline daily for 6 weeks. The mice were then euthanized by cervical dislocation, and femur and blood samples were obtained.
Bone histomorphometric analysis
Femur samples were decalcified for 2 weeks in an aqueous solution of 10% tetrasodium-EDTA at 4°C. Next, bone sections (4 µm) were stained with hematoxylin and eosin (H&E) at room temperature for 5-10 min, followed by TRAP staining (cat. no. BB-4421-1; BestBio, Shanghai, China) to identify osteoclasts according to the manufacturer’s protocol. Bone trabecula was observed in H&E-stained sections, and the osteoclasts were observed in TRAP-stained sections. Histomorphometric measurements were then performed under a microscope, and the trabecular bone area was calculated using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).
Microcomputed tomography analysis
Bone samples were analyzed by microcomputed tomography (Skyscan; Bruker Corporation, Billerica, MA, USA) using the following parameters: 80 kV, 124 µA and 8-µm resolution. The built-in software was used to analyze various indices, including bone mineral density (BMD), bone surface area/bone volume (BS/BV), BS/total volume (BS/TV), BV/TV and trabecular number (Tb.N).
Serum biochemistry
Following anesthesia, blood samples were collected from the fundus venous plexus. Subsequent to centrifugation (1,811 × g; 15 min; 4°C), the serum levels of C-terminal telopeptide of type 1 collagen (CTX-1; cat. no. MBS726456; MyBiosource, Inc., San Diego, USA), interleukin (IL)-6 (cat. no. MEC1008; Anogen-Yes Biotech Laboratories, Ltd., Mississauga, Canada), tumor necrosis factor α (TNF-α; cat. no. MEC1003; Anogen-Yes Biotech Laboratories, Ltd.) and TRAP 5b (TRAcp5B; cat. no. MBS776993; MyBiosource, Inc.) were determined using enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturer’s protocol.
In vitro osteoclastogenesis assay
Bone marrow monocytes (BMMCs) were isolated from the bone marrow of healthy C57BL/6 mice by flushing femurs and tibias with α-MEM and culturing in α-MEM with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin and macrophage colony-stimulating factor (M-CSF; 30 ng/ml) overnight. Non-adherent cells were collected and further cultured in the presence of M-CSF (30 ng/ml) for 3 days. Floating cells were discarded and adherent cells were used as bone marrow-derived macrophages. BMMCs and RAW264.7 cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with glucose, 10% heat-inactivated FBS, penicillin (100 U/ml) and streptomycin (100 mg/ml) at 37°C in an atmosphere containing 5% CO2. The culture medium was changed once per day during the first 3 days. After removing nonadherent cells, colonies were cultured for 14 days, then passaged and subcultured. BMMCs treated with 0, 5, 10, 20, 40, 80 or 160 µg/ml magnolol were subjected to an MTT assay.
Based on the MTT assay results, third-generation BMMCs (5×103/well) and RAW264.7 cells (3×103/well) were cultivated in 96-well plates and divided into a control group and four magnolol-treated (0, 5, 10 and 20 µg/ml) groups. To induce osteoclast formation, 20 ng/ml M-CSF and 50 ng/ml RANKL were applied. After 5 days, staining for TRAP was performed using the TRAP Staining kit (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). TRAP-positive cells with three or more nuclei were regarded as osteoclasts. In addition, in order to visualize F-actin rings by fluorescent microscopy, cells treated with M-CSF/RANKL were cultured for 48 h with 4% paraformaldehyde in PBS, permeabilized with 0.1% Triton X-100 and incubated with rhodamine-conjugated phalloidin (cat. no. ab235138; Abcam, Cambridge, MA, USA) for 60 min at room temperature. Fluorescein isothiocyanate-conjugated F-actin antibodies (1:600; cat. no. ab205; Abcam) were used to detect F-actin. Assays were performed in at least triplicate, and mean values were calculated.
Bone-pit formation assay
RAW264.7 cells were divided into five groups, including the untreated control and four groups treated with magnolol of various concentration. Next, cells (3×103/well) were seeded in 48-well plates containing a collagen gel matrix, and then treated with 20 ng/ml M-CSF and 50 ng/ml RANKL for 6 days. The resulting mature osteoclasts were digested with collagenase and seeded onto a biosynthetic bone surface (Osteo Assay Surface 24-well Multiple-Well Plates; Corning, Inc., Corning, NY, USA) and incubated for 7 days at room temperature. The medium was changed on day 3, and the plates were washed with PBS and air-dried for 3 h on day 7. The bone resorption area was assessed by light microscopy (BX53; Olympus Corporation, Tokyo, Japan) and Image-Pro Plus software.
Western blotting
In order to assess the effects of magnolol on the NF-κB and MAPK signaling pathways, RAW264.7 cells (3×103/well) were seeded into 6-well plates and treated with RANKL without or with magnolol (0 or 20 µg/ml). Following incubation for 0, 15, 30, 45 or 60 min, the phosphorylation levels of p38, p50, p65, IκB, ERK and JNK were evaluated by western blotting. In addition, in order to assess the protein expression levels of osteoclastogenesis markers, RAW264.7 cells (3×103/well) were cultured for 7 days in the presence of RANKL without or with 0, 5, 10, or 20 µg/ml magnolol. Subsequently, the expression levels of cathepsin K, CTR, MMP9, TRAF6, and TRAP were assessed by western blotting using antibodies obtained from Abcam (Cambridge, MA, USA).
The total protein was extracted from the cells using M-PER mammalian protein extraction reagent (Pierce; Thermo Fisher Scientific, Inc., Waltham, MA, USA). Equal amounts of protein (10 µg/lane), estimated by a bicinchoninic acid protein assay kit (Pierce; Thermo Fisher Scientific, Inc.) were loaded onto 11% SDS-PAGE gels and transferred onto nitrocellulose membranes. Blocking was performed with blocking solution (cat. no. P0023B; Beyotime Institute of Biotechnology, Haimen, China) at room temperature for 1 h. Membranes were then incubated with the primary (4°C; overnight) and secondary antibodies (room temperature; 1 h) detailed above (p38, p50, p65, IκB, ERK, JNK, cathepsin K, CTR, MMP9, TRAF6 and TRAP). Bands were detected by chemiluminescence (cat. no. E003-1007; Sea Biotech, Shanghai, China) and imaged with X-ray films. β-actin was used as an endogenous reference for normalization. ImageJ software (v1.46; National Institutes of Health, Bethesda, MD, USA) was used to analyze densitometry.
Immunofluorescence staining
The effect of magnolol on the nuclear translocation of p65 was evaluated by immunofluorescence staining. Briefly, BMMCs treated with RANKL in the absence or presence of 20 µg/ml magnolol were fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 in PBS for 5 min. The cells were washed with 0.1% BSA-PBS and incubated with an anti-p65 monoclonal antibody overnight at 4°C, followed by a biotinylated goat anti-mouse IgG and fluorescein-conjugated streptavidin (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature and Hoechst (Vector Laboratories) was used for counterstaining for 2 days. The localization of p65 was visual-ized by immunofluorescence analysis (×400 magnification) and the percentage of nuclei fluorescence intensity of p65 in 3 random fields were measured in each group with ImageJ software.
Statistical analysis
Data are presented as the mean ± standard deviation. Student’s t-test was used for comparisons of two groups, while one-way analysis of variance was performed to compare three or more groups. A value of P<0.05 was considered to denote a difference that was statistically significant.
Results
Magnolol prevents OVX-induced bone loss
According to H&E staining, magnolol prevented the loss of trabecular bone in OVX mice (Fig. 1A). This was further confirmed by microcomputed tomography, and the three-dimensional structure of the bone is shown in Fig. 1B. When comparing Sham and OVX groups, it was observed that the BMD (P<0.001), BS/BV (P<0.05), BS/TV (P<0.001), BV/TV (P<0.001) and Tb.N (P<0.001) values were decreased in OVX mice. When comparing OVX and OVX+Magnolol groups, the BMD (P<0.05), BS/BV (P<0.01), BS/TV (P<0.001), BV/TV (P<0.01) and Tb.N (P<0.001) values were increased in OVX+Magnolol mice, indicating that magnolol treatment reversed OVX-induced bone loss (Fig. 1C).
Magnolol inhibits osteoclastogenesis in vivo
To determine whether magnolol suppressed OVX-induced bone loss by inhibiting the formation of osteoclasts, TRAP staining was performed, and the serum levels of markers of osteoclastogenesis and inflammation were assessed. It was observed that there were more TRAP-positive cells in OVX mice when compared with the sham group. However, magnolol reduced the number TRAP-positive cells in OVX+Magnolol mice compared with the OVX group (Fig. 2A). As determined by ELISA, the serum CTX-1 (P<0.001), IL-6 (P<0.001), TNF-α (P<0.01) and TRAcp5B (P<0.001) levels were significantly increased in OVX mice compared with those in the sham group. However, magnolol treatment markedly decreased the serum levels of CTX-1 (P<0.001), IL-6 (P<0.001), TNF-α (P<0.01) and TRAcp5B (P<0.001) in the OVX+Magnolol group compared with the OVX group (Fig. 2B), suggesting inhibition of osteoclastogenesis and inflammation.
Magnolol inhibits osteoclastogenesis in vitro
Based on the results of the MTT assays, magnolol concentrations of 0, 5, 10 and 20 µg/ml were selected for use in subsequent experiments, since the cell proliferation was not markedly reduced following treatment at these doses (Fig. 3A). Compared with the positive control (RANKL alone), magnolol decreased the number of TRAP-positive BMMCs (Fig. 3B) and RAW264.7 cells (Fig. 3C) in a concentration-dependent manner. Therefore, magnolol appeared to suppress RANKL-induced osteoclastogenesis in BMMCs and RAW264.7 cells.
Magnolol inhibits osteoclast function in vitro
To evaluate the effect of magnolol on osteoclast function in vitro, F-actin ring formation and bone-pit formation were assayed. Magnolol inhibited F-actin ring formation in M-CSF-stimulated and RANKL-stimulated RAW264.7 cells in a concentration-dependent manner (Fig. 4). In addition, magnolol significantly decreased the area of bone resorption pits (Fig. 5). Taken together, these results suggested that magnolol inhibited RANKL-induced osteoclast activity.
Magnolol suppresses the expression of osteoclastogenesis markers
To evaluate the mechanism underlying the effect of magnolol, the protein expression levels of cathepsin K, CTR, MMP9, TRAF6 and TRAP were assessed. It was observed that magnolol significantly suppressed the RANKL-induced expression of cathepsin K, CTR, MMP9, TRAF6 and TRAP (Fig. 6). Therefore, magnolol suppressed the expression levels of osteoclastogenesis markers.
Magnolol suppresses the NF-κB and MAPK pathways
To assess the effect of magnolol on the NF-κB pathway, immunofluorescence staining of p65 was performed. The results revealed that the RANKL-induced translocation of p65 to the nucleus was inhibited by magnolol (Fig. 7A). Furthermore, the phosphorylation of the key proteins of the NF-κB pathway, namely IκB, p65 and p50, were assessed by western blotting. Magnolol inhibited the phosphorylation of IκB, p65 and p50 (Fig. 7B) at different time points. These results indicate that magnolol suppressed the NF-κB signaling pathway.
The MAPK pathway is also important in osteoclastogenesis (13,15); thus, the phosphorylation of the key proteins, namely ERK, p38 and JNK were further examined by western blotting. It was observed that magnolol suppressed the RANKL-induced phosphorylation of ERK, p38 and JNK at different time points (Fig. 8). Therefore, magnolol may also suppress the activation of the MAPK signaling pathway.
Discussion
A large number of middle-aged and elderly individuals worldwide suffer from osteoporosis (2,22). The pathogenesis of osteoporosis involves an imbalance between bone formation and resorption (23), which reflects an imbalance between osteoblasts and osteoclasts (24). Patients with osteoporosis are more vulnerable to fractures, which may lead to hypostatic pneumonia, bedsores, deep-vein thrombosis, pulmonary embolism, and urinary tract infection due to long-term immobilization (2,6,23). Calcium supplements and vitamin D are used to reverse bone loss (25); however, the clinical outcomes are unsatisfactory (6,23,25). For this reason, effort has been focused on developing drugs that are effective against osteoporosis (13,15,26).
Estrogen suppresses inflammation and inhibits osteoclast formation and function in bone tissue; therefore, postmenopausal women are more likely to develop osteoporosis (27,28). In the present study, the femurs of OVX mice were subjected to H&E staining and microcomputed tomography, as bone loss typically begins in regions with abundant cancellous bone (24). The data indicated that OVX-induced bone loss was significantly attenuated by magnolol treatment. To identify the underlying mechanism, TRAP-positive cells were counted and the concentration of TRAcp5B was determined, which reflects the degree of osteoclastogenesis (15); the results suggested that magnolol inhibits osteoclastogenesis. Therefore, magnolol assists the restoration of the osteoclast-osteoblast balance by suppressing the formation of osteoclasts. Magnolol alleviated OVX-induced osteoporosis by restraining osteoclastogenesis in a concentration-dependent manner. However, magnolol at greater than threshold concentrations suppressed cell proliferation. Furthermore, inflammation is an important factor in the pathogenesis of osteoporosis (3). In the current study, the elevated serum IL-6 and TNF-α levels in OVX mice were reversed by magnolol treatment. Thus, inflammation serves an important role in osteoclastogenesis and bone resorption, and consequently anti-inflammatory agents may be useful against osteoporosis.
Traditional Chinese medicine provides an abundant source for drug development. A number of active components of Chinese herbs, such as matrine and epoxyeicosatrienoic acids, can prevent bone loss (26,29,30). Magnolol is extracted from Magnolia officinalis, and has been reported to exhibit antioxidant, anti-inflammatory and anticancer effects (16-21). In addition, magnolol promotes the growth of osteoblasts and inhibits the differentiation of osteoclasts in vitro (27). Previous research has demonstrated that magnolol was able to ameliorate ligature-induced periodontitis in rats, which is an osteoclast-associated disease (31).
In the present study, magnolol suppressed osteoclastogenesis in vivo and in vitro by blocking the NF-κB and MAPK signaling pathways. In vitro, TRAP staining revealed that magnolol inhibited RANKL-induced osteoclastogenesis by BMMCs and RAW264.7 cells. Osteoclast function, in terms of F-actin ring formation and bone-pit formation, was also inhibited by magnolol. Furthermore, the expression levels of the osteoclastogenesis markers cathepsin K, CTR, MMP9, TRAF6 and TRAP were suppressed by magnolol.
RANKL serves a vital role in osteoclast maturation (32). RANKL binds to RANK on immature osteoclasts, resulting in the activation of the NF-κB and MAPK pathways (11,12,20). The NF-κB pathway is closely associated with osteoclastogen-esis and inflammation, and agents targeting this pathway are used to treat osteoporosis and inflammatory conditions (13,28). Following the induction of RANKL, p65 translocates to the nucleus to activate gene transcription, and this translocation was suppressed by magnolol in the present study. Furthermore, magnolol decreased the phosphorylation of IκB, p65 and p50 in a time-dependent manner, which subsequently suppressed gene transcription. It was also observed that magnolol suppressed the MAPK pathway, as indicated by inhibition of the phosphorylation of ERK, p38 and JNK.
As mentioned earlier, magnolol was demonstrated to inhibit osteoclastogenesis in vivo and in vitro; however, it remains unclear whether magnolol affects osteoblast formation. A previous study reported that magnolol promoted the function of MC3T3-E1 osteoblasts (27), but further research is required on this subject. Furthermore, the present study demonstrated that magnolol inhibited osteoclast maturation by suppressing the NF-κB and MAPK pathways. However, whether magnolol affects the AKT pathway or other signaling transduction pathways remains unclear. Identification of the genes targeted by magnolol that mediate its inhibition of osteoclast formation would provide further insight on the pathways involved. Finally, in the current study, magnolol inhibited bone loss in a model involving estrogen; however, the effects of magnolol on other osteoclastogenesis-associated diseases, including arthritis and bone tumors, warrant further investigation.
In conclusion, the current data indicated that magnolol suppressed osteoclastogenesis in vivo and in vitro by blocking the NF-κB and MAPK pathways, suggesting its potential use in the treatment of osteoporosis and associated disorders.
Funding
The present study was supported by funds from the Young Medical Key Talent Project of Jiangsu province (grant no. QNRC2016458), Jiangsu Provincial Medical Innovation Team (grant no. CXTDB2017004) and Clinical Study of Jiangsu University (grant no. JLY20160005).
Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors’ contributions
The study was conceived and designed by WYF and TL. WYF and LJQ conducted most of the experiments with assistance from QH and PQZ. The paper was written by WYF, with contributions from TL. All authors read and approved the final manuscript.
Ethics approval and consent to participate
The experiments on mice were approved by the Ethics Committee on Animal Experiments of the Central Hospital of Zibo (Zibo, China).
Patient consent for publication
No patients were involved in this study.
Competing interests
The authors declare that they have no competing interests and no financial associations to disclose.
Acknowledgments
The authors would like to thank the Orthopedic Basic and Translational Research Center in Wuxi, China, and Shanghai Geekbiotech Company for technical support.
References
Bateman ME, Strong AL, McLachlan JA, Burow ME and Bunnell BA: The effects of endocrine disruptors on adipogenesis and osteogenesis in mesenchymal stem cells: A review. Front Endocrinol (Lausanne). 7:1712016. | |
Noel SE, Mangano KM, Griffith JL, Wright NC, Dawson-Hughes B and Tucker KL: Prevalence of osteoporosis and low bone mass among Puerto Rican older adults. J Bone Miner Res. 33:396–403. 2018. View Article : Google Scholar : | |
Aldaghri NM, Aziz I, Yakout S, Aljohani NJ, Al-Saleh Y, Amer OE, Sheshah E, Younis GZ and Al-Badr FB: Inflammation as a contributing factor among postmenopausal Saudi women with osteoporosis. Medicine (Baltimore). 96:e57802017. View Article : Google Scholar | |
Paschalis EP, Gamsjaeger S, Hassler N, Fahrleitner-Pammer A, Dobnig H, Stepan JJ, Pavo I, Eriksen EF and Klaushofer K: Vitamin D and calcium supplementation for three years in postmenopausal osteoporosis significantly alters bone mineral and organic matrix quality. Bone. 95:41–46. 2017. View Article : Google Scholar | |
Gielen E, Bergmann P, Bruyère O, Cavalier E, Delanaye P, Goemaere S, Kaufman JM, Locquet M, Reginster JY, Rozenberg S, et al: Osteoporosis in frail patients: A consensus paper of the belgian bone club. Calcif Tissue Int. 101:111–131. 2017. View Article : Google Scholar : PubMed/NCBI | |
Ensrud KE and Crandall CJ: Osteoporosis Ann Intern Med. 167:ITC17–ITC32. 2017. View Article : Google Scholar | |
Office of the Surgeon General (US): Bone Health and Osteoporosis: A Report of the Surgeon General. Office of the Surgeon General; Rockville, MD: 2004 | |
Peng C, Li Z and Hu Y: Prevalence of osteoporosis in China: A meta-analysis and systematic review. Bmc Public Health. 16:10392016. View Article : Google Scholar | |
Zhou Y, Guan X, Chen X, Yu M, Wang C, Chen X, Shi J, Liu T and Wang H: Angiotensin II/Angiotensin II receptor blockade affects osteoporosis via the AT1/AT2-mediated cAMP-dependent PKA pathway. Cells Tissues Organs. 204:25–37. 2017. View Article : Google Scholar : PubMed/NCBI | |
Xu L, Zhang L, Zhang H, Yang Z, Qi L, Wang Y and Ren S: The participation of fibroblast growth factor 23 (FGF23) in the progression of osteoporosis via JAK/STAT pathway. J Cell Biochem. 119:3819–3828. 2018. View Article : Google Scholar | |
Wang Y, van Assen AHG, Reis CR, Setroikromo R, van Merkerk R, Boersma YL, Cool RH and Quax WJ: Novel RANKL DE-loop mutants antagonize RANK-mediated osteoclastogenesis. FEBS J. 284:2501–2512. 2017. View Article : Google Scholar : PubMed/NCBI | |
Okabe I, Kikuchi T, Mogi M, Takeda H, Aino M, Kamiya Y, Fujimura T, Goto H, Okada K, Hasegawa Y, et al: IL-15 and RANKL play a synergistically important role in osteoclastogenesis. J Cell Biochem. 118:739–747. 2017. View Article : Google Scholar | |
Yang X, Gao W, Wang B, Wang X, Guo H, Xiao Y, Kong L and Hao D: Picroside II Inhibits RANKL-mediated Osteoclastogenesis by attenuating the NF-κB and MAPKs signaling pathway in vitro and prevents bone loss in lipopolysaccharide treatment mice. J Cell Biochem. 118:4479–4486. 2017. View Article : Google Scholar : PubMed/NCBI | |
Ueno M, Cho K, Isaka S, Nishiguchi T, Yamaguchi K, Kim D and Oda T: Inhibitory effect of sulphated polysaccharide porphyran (isolated from Porphyra yezoensis) on RANKL-induced differentiation of RAW264.7 cells into osteoclasts. Phytother Res. 32:452–458. 2018. View Article : Google Scholar | |
Chen X, Zhi X, Pan P, Cui J, Cao L, Weng W, Zhou Q, Wang L, Zhai X, Zhao Q, et al: Matrine prevents bone loss in ovariectomized mice by inhibiting RANKL-induced osteoclastogenesis. FASEB J. 31:4855–4865. 2017. View Article : Google Scholar : PubMed/NCBI | |
Dong J, Ding H, Liu Y, Yang Q, Xu N, Yang Y and Ai X: Magnolol protects channel catfish from Aeromonas hydrophila infection via inhibiting the expression of aerolysin. Vet Microbiol. 211:119–123. 2017. View Article : Google Scholar : PubMed/NCBI | |
Zhang Li M, Wang F, Wu X, Zhang X, Zhang B, Wu N, Wang W, Weng Z, Liu HS, et al: Magnolol inhibits growth of gallbladder cancer cells through the p53 pathway. Cancer Sci. 106:1341–1350. 2015. View Article : Google Scholar : PubMed/NCBI | |
Wei W, Dejie L, Xiaojing S, Tiancheng W, Yongguo C, Zhengtao Y and Naisheng Z: Magnolol inhibits the inflammatory response in mouse mammary epithelial cells and a mouse mastitis model. FEBS J. 38:16–26. 2015. | |
Yang B, Xu Y, Yu S, Huang Y, Lin L and Liang X: Anti-angiogenic and anti-inflammatory effect of Magnolol in the oxygen-induced retinopathy model. Inflamm Res. 65:81–93. 2016. View Article : Google Scholar | |
Park KR, Kim JY, Kim EC, Yun HM and Hong JT: RANKL-induced osteoclastogenesis is suppressed by 4-O-methylhonokiol in bone marrow-derived macrophages. Arch Pharm Res. 40:933–942. 2017. View Article : Google Scholar : PubMed/NCBI | |
Jeong JH, Jin ES, Kim JY, Lee B, Min J, Jeon SR, Lee M and Choi KH: The effect of biocomposite screws on bone regeneration in a rat osteoporosis model. World Neurosurg. 106:964–972. 2017. View Article : Google Scholar : PubMed/NCBI | |
Chen P, Li Z and Hu Y: Prevalence of osteoporosis in China: A meta-analysis and systematic review. Bmc Public Health. 16:10392016. View Article : Google Scholar : PubMed/NCBI | |
McClung M, Baron R and Bouxsein M: An update on osteoporosis pathogenesis, diagnosis, and treatment. Bone. 98:372017. View Article : Google Scholar : PubMed/NCBI | |
Chen X, Wang Z, Duan N, Zhu G, Schwarz EM and Xie C: Osteoblast-osteoclast interactions. Connect Tissue Res. 59:99–107. 2018. View Article : Google Scholar | |
Stoecker WV, Carson A, Nguyen VH, Willis AB, Cole JG and Rader RK: Addressing the crisis in the treatment of osteoporosis: Better paths forward. J Bone Miner Res. 32:1386–1387. 2017. View Article : Google Scholar : PubMed/NCBI | |
Liu Q, Wang T, Zhou L, Song F, Qin A, Feng HT, Lin XX, Lin Z, Yuan JB, Tickner J, et al: Nitidine chloride prevents OVX-induced bone loss via suppressing NFATc1-mediated osteoclast differentiation. Sci Rep. 6:366622016. View Article : Google Scholar : PubMed/NCBI | |
Kwak EJ, Lee YS and Choi EM: Effect of magnolol on the function of osteoblastic MC3T3-E1 cells. Mediators Inflamm. 2012.829650:2012. | |
Park JW, Yoon HJ, Kang WY, Cho S, Seong SJ, Lee HW, Yoon YR and Kim HJ: G protein-coupled receptor 84 controls osteoclastogenesis through inhibition of NF-κB and MAPK signaling pathways. J Cell Physiol. 233:1481–1489. 2018. View Article : Google Scholar | |
Chen X, Zhi X, Cao L, Weng W, Pan P, Hu H, Liu C, Zhao Q, Zhou Q, Cui J and Su J: Matrine derivate MASM uncovers a novel function for ribosomal protein S5 in osteoclastogenesis and postmenopausal osteoporosis. Cell Death Dis. 8:e30372017. View Article : Google Scholar : PubMed/NCBI | |
Guan H, Zhao L, Cao H, Chen A and Xiao J: Epoxyeicosanoids suppress osteoclastogenesis and prevent ovariectomy-induced bone loss. FASEB J. 29:1092–1101. 2015. View Article : Google Scholar | |
Lu SH, Huang RY and Chou TC: Magnolol ameliorates ligature-induced periodontitis in rats and osteoclastogenesis: In vivo and in vitro study. Evid Based Complement Alternat Med. 2013.634095:2013. | |
Kanzaki H, Shinohara F, Itohiya K, Yamaguchi Y, Katsumata Y, Matsuzawa M, Fukaya S, Miyamoto Y, Wada S and Nakamura Y: RANKL induces Bach1 nuclear import and attenuates Nrf2-mediated antioxidant enzymes, thereby augmenting intracellular reactive oxygen species signaling and osteoclastogenesis in mice. FASEB J. 31:781–792. 2017. View Article : Google Scholar |