Postmenopausal osteoporosis (PMOP) is characterized by increased bone loss due to enhanced osteoclastogenesis and bone resorption. A Chinese herbal formula, jiangugranule (JG), exhibited great efficacy in the clinical treatment of PMOP. However, the molecular mechanisms underlying the therapeutic effects remain unclear. The present study aimed to examine the effects of JG-containing serum on receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis. Osteoclast precursor RAW264.7 cells were cultured and treated with JG-containing serum in the presence of RANKL. Following 6 days of culture, the cells were stained with tartrate-resistant acid phosphatase and the rate of differentiation was calculated. In addition, cells were treated with JG-containing serum for 24, 48 and 96 h and total RNA and proteins were extracted for reverse transcription-quantitative polymerase chain reaction and western blot analysis to detect mRNA and protein expression, respectively, of key molecules in the RANK/RANKL signaling pathway, including RANK, tumor necrosis factor receptor-associated factor 6, NF-κB (p50 and p52 subunits), c-Fos and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1). The results revealed that JG-containing serum inhibited RANKL-induced osteoclastogenesis and reduced mRNA and protein expression of RANK, c-Fos and NFATc1. The results suggested that JG may regulate osteoclast differentiation through the RANK/RANKL signaling pathway, which may be a possible mechanism for the therapeutic effects of JG on PMOP.
Primary osteoporosis is a systemic skeletal disease that is characterized by low bone mass, microstructural damage of bone tissue and weakened bone strength (
In Traditional Chinese Medicine (TCM) it is believed that kidney deficiency is the main pathogenesis of PMOP, and spleen deficiency is also involved (
JG is a TCM prescription that comprises calcined
An Agilent 1260 Liquid Chromatography system (Agilent Technologies, Inc., Santa Clara, CA, USA), equipped with a G1311C quaternary solvent delivery system, a G7617B autosampler and a G1315D diode array detector was used to detect icariin from
A total of 20 male specific-pathogen-free Sprague-Dawley rats (age, 8–10 weeks; weight, 300±30 g) were obtained from Shanghai Laboratory Animal Center (Shanghai, China) and provided with food and water
Osteoclast precursor RAW264.7 cells were obtained from The Cell Bank of Type Culture Collection of Chinese Academy of Science (Shanghai, China) and cultured in a differentiation medium comprising: α-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum (FBS) (both from Gibco; Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 50 ng/ml RANKL (R&D Systems, Inc., Minneapolis, MN, USA). The culture medium was replaced every 48 h. Cells were incubated at 37°C in a humid atmosphere containing 5% CO2 for 6 days to obtain osteoclasts.
For differentiation assays, cells were seeded in 24-well plates at a density 1×104 cells/well in differentiation medium (without FBS) with various concentrations (2, 5, 10, 15 and 20%) of either JG-containing serum or Blank serum during the entire culture period of 6 days. The differentiation rate of each well was calculated and compared to determine the most effective concentration of JG-containing serum.
For RANK/RANKL pathway tests, cells were divided into 2 groups: i) The Blank group, which was treated with 50 ng/ml RANKL and 10% Blank serum; and ii) the JG group, which was treated with 50 ng/ml RANKL and 10% JG-containing serum. Following 24, 48 and 96 h incubation, the cells were harvested for reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis. Cells cultured in common medium (α-MEM supplemented with 10% Blank serum, without RANKL) as negative control (0 h of Blank group). All experiments were repeated at least 3 times.
Osteoclast differentiation rate was measured by counting the number of tartrate-resistant acid phosphatase (TRAP)-positive stained cells, using the TRAP Staining kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China), according to the manufacturer's protocol. RAW264.7 cells cultured in differentiation medium at a density of 1×104 cells/well for 6 days were collected and fixed with the fixative solution in the kit at room temperature for 10 min, rinsed thoroughly with deionized water and stained with naphthol AS-BI phosphate for 1 h at 37°C, followed by hematoxylin counterstaining at room temperature for 2 min. Osteoclasts were determined to be TRAP-positive stained multinuclear (containing ≥3 nuclei) cells under light microscopy. A total of 6 fields/well (magnification, 100x) were examined.
To detect the F-actin containing podosome belt of osteoclasts, rhodaminephalloidin staining (1:200 dilution with PBS; Cytoskeleton Inc., Denver, CO, USA) was performed, according to the manufacturer's protocol. RAW264.7 cells cultured in a differentiation medium at a density of 1×104 cells/well for 6 days were collected, and fixed at room temperature with 4% paraformaldehyde for 10 min, permeabilized with 1% Triton X-100 for 10 min, washed with PBS for 3 min and incubated with rhodaminephalloidin at room temperature in the dark for 30 min, followed by 3 washes with PBS for 5 min each. Nuclei were counterstained with 100 nM DAPI in PBS at room temperature for 5 min. Osteoclasts were observed under a fluorescence microscope (Leica DMI4000B; Leica Microsystems GmbH, Wetzlar, Germany).
Cells from the Blank control group, JG group and negative control group were collected. RNA was extracted by TRIzol (Invitrogen; Thermo Fisher Scientific, Inc.) from cells at a density of 1×107 cells/ml and reverse transcribed to cDNA using Prime Script First-Strand cDNA Synthesis kit (Takara Biotechnology Co., Ltd., Dalian, China) for use as qPCR template. The primers were as follows: RANK forward, 5′-GGCTGGCTACCACTGGAACT-3′ and reverse, 5′-TCCTGTAGTAAACGCCGAAGA−3′; TRAF6 forward, 5′-TCATTATGATCTGGACTGCCCAAC-3′ and reverse, 5′-TTATGAACAGCCTGGGCCAAC-3′; NF-κB forward, 5′-ACCACTGCTCAGGTCCACTGTC-3′ and reverse, 5′-GCTGTCACTATCCCGGAGTTCA3-'; NFATc1, 5′-CAAGTCTCACCACAGGGCTCACTA-3′ and reverse, 5′-TCAGCCGTCCCAATGAACAG-3′;c-Fos forward, 5′-ACGTGGAGCTGAAGGCAGAAC-3′ and reverse, 5′-AGCCACTGGGCCTAGATGATG−3′; and β-actin forward, 5′-AGGCTGTGTTGTCCCTGTA-3′ and reverse, 5′-ATGTCACGCACGATTTCC−3′. PCR was performed using SYBR Green qPCR Mix (Takara Biotechnology Co., Ltd.) and a Real-Time PCR system (ABI7500; Thermo Fisher Scientific, Inc.) with the following program: 1 cycle at 95°C for 30 sec, followed by 40 cycles of 95°C for 5 sec, 60°C for 34 sec. qPCR was carried out on three replicates per sample. β-actin was used as a reference gene for RNA correction of all samples; the relative standard curve method (2−ΔΔCq method) was used for the calculation of fold changes in gene expression (
Cells from the Blank control, JG and negative control groups were collected. Total protein was extracted in protein lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) from cells at a density of 5×107 cells/ml. Protein concentrations were determined by BCA Protein assay kit (Beyotime Institute of Biotechnology). Equal amounts of proteins (30 µg) were resolved by SDS-PAGE on a 12% gel and transferred to polyvinylidene difluoride membranes (EMD Millipore, Billerica, MA, USA). Non-specific interactions were blocked with 5% skim milk at 4°C for 2 h and the membranes were incubated with 1:500 diluted primary antibodies [rabbit anti-RANK polyclonal antibody (cat no. ab200369), rabbit anti-TRAF6 monoclonal antibody (cat no. ab33915), rabbit anti-NF-κB p105/p50 monoclonal antibody (cat no. ab32360), rabbit anti-NFATc1 polyclonal antibody (cat no. ab25916), rabbit anti-c-Fos polyclonal antibody (cat no. ab190289), Rabbit anti-β-Actin monoclonal antibody (cat. no. ab8227) all Abcam, Cambridge, MA, USA]. Rabbit anti-NF-κB p100/p52 monoclonal antibody (cat no. 52583; Cell Signaling Technology, Inc., MA, USA) overnight at 4°C, followed by incubation with horseradish peroxidase (HRP)-conjugated secondary antibodies (goat anti-rabbit immunoglobulin G H&L HRP; 1:5,000; cat no. ab6721; Abcam, Cambridge, MA, USA). Protein bands were visualized with the enhanced chemiluminescence reagent (Invitrogen; Thermo Fisher Scientific, Inc.). Densitometric values were quantified for each band with the Image Pro-Plus program (version 5.0; Media Cybernetics, Inc., Rockville, MD, USA). Relative expression data are expressed as a ratio of the optical intensity of the band of the target protein over that of the internal control protein (β-actin).
All calculations were performed using SPSS version 17.0 for Windows software (SPSS Inc., Chicago, IL, USA). Results are presented as the mean ± standard deviation. All data were analyzed using one-way analysis of variance and Fisher's least significant difference test. P<0.05 were considered to indicate a statistically significant difference.
For HPLC analysis, four standard compounds (
The results of TRAP staining demonstrated a notable increase in TRAP-positive multinuclear cells in the RAW264.7 cells incubated with RANKL for 6 days (
The differentiated osteoclasts were treated with various concentrations of either JG-containing serum or Blank serum for 6 days, and subsequently stained with TRAP (
The results of RT-qPCR and western blot analysis were consistent compared with the negative control (0 h of Blank group), the mRNA and protein expression levels of NF-κB, NFATc1 and c-Fos significantly increased in JG-containing serum group and the Blank serum group following 24, 48 and 96 h of RANKL stimulation (P<0.0l). Compared with Blank group, the mRNA and protein expression of RANK, NFATc1 and c-Fos of JG group significantly decreased following treatment for 24 and 48 h (P<0.0l). The differences of TRAF6 and NF-κB expression between the Blank group and JG group were not significant (
In TCM it is believed that the kidney is the origin of congenital constitution. Bones are governed and nourished by the kidney, which means that the development and the quality of bones depend on the functions of the kidneys (
Bone is a dynamic organ that undergoes continuous remodeling; that is, the osteoclasts resorb old and damaged bone, which is replaced with new bone by osteoblasts (
Osteoclasts are large, multinucleated cells that are derived from the monocyte/macrophage lineage. The RANK/RANKL pathway serves a crucial role in osteoclast differentiation and bone resorption (
In the present study, key molecules of the RANK signaling pathway, including RANK, TRAF6, NF-κB (p50 and p52), NFATc1 and c-Fos were examined to investigate the effects of JG treatment on RANKL-induced osteoclastogenesis. The results demonstrated that RANKL stimulation increased mRNA and protein expression of NF-κB, c-Fos and NFATc1, and consequently led to osteoclast differentiation, which was consistent with the results of previous studies (
In conclusion, the present study demonstrated the effects of JG treatment on inhibiting osteoclast differentiation, which may be achieved through the RANK/RANKL signaling pathway. This study provides an experimental rationale for the application of JG in clinical therapy of PMOP.
This work was supported by the Natural Science Foundation of China (grant nos. 81574003 and 81473706), the Guiding Project Foundation of Fujian Science and Technology Department (grant no. 2015Y0069) and the Science Foundation of Fujian Province (grant nos. 2015J01690 and 2014J01355).
High-performance liquid chromatographychromatogram of JG. (A) Mixed standard solution of four components of JG. (B) JG sample analysis. 1, morroniside; 2, loganin; 3, aurantiamarin; 4, icariin; JG, jiangu granule; mAU, milli-absorbance units.
Osteoclast differentiation in the presence of RANKL. (A) Untreated RAW264.7 cells. (B) Tartrate-resistant acid phosphatase staining of RAW264.7 cells treated with RANKL (50 ng/ml) for 6 days. (C) F-actin staining by rhodaminephalloidin (red) of RAW264.7 cultures treated with RANKL (50 ng/ml) for 6 days; nuclei were counterstained with DAPI (blue). Scale bar, 50 µm. RANKL, receptor activator of nuclear factor-κB ligand.
Osteoclast formation of RAW264.7 treated with various concentrations of JG-containing serum in the presence of RANKL. (A) RAW264.7 cells were treated with RANKL (50 ng/ml) for 6 days, and examined using TRAP staining. (B) Total number of TRAP-positive stained cells, which indicates differentiated osteoclasts. Data are presented as the mean ± standard deviation of 3 cultures. *P<0.05 vs. Blank-serum group. TRAP, tartrate-resistant acid phosphatase.
Effects of JG-containing serum on mRNA expression of RANK/RANKL pathway components in RAW264.7 cells. Results of reverse transcription-quantitative polymerase chain reaction indicating the relative mRNA expression levels of (A) RANK, (B) TRAF6, (C) NF-κB, (D) NFATc1 and (E) c-Fos. Data are presented as the mean ± standard deviation of 3 cultures. *P<0.05 vs. Blank group, **P<0.01 vs. Blank group; #P<0.05 vs. negative control (0 h of Blank group), ##P<0.01 vs. negative control (0 h of Blank group). JG, jiangu granule; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; NF-κB, nuclear factor-κB; RANK, receptor activator of nuclear factor-κB ligand; RANKL, RANK ligand; TRAF6, tumor necrosis factor receptor-associated factor 6.
Effects of JG-containing serum on protein expression levels of RANK/RANKL pathway components. (A) Images of the protein expression as analyzed by western blot assay. Relative protein expression levels of (B) RANK, (C) TRAF6, (D) NF-κBp52, (E) NF-κBp50, (F) NFATc1 and (G) c-Fos. Data are presented as the mean ± standard deviation of 3 cultures. *P<0.05 vs. Blank group; **P<0.01 vs. Blank group; #P<0.05 vs. negative control (0 h of Blank group); ##P<0.01 vs. negative control (0 h of Blank group). JG, jiangu granule; NFATc1, nuclear factor of activated T cells, cytoplasmic 1; NF-κB, nuclear factor-κB; RANK, receptor activator of nuclear factor-κB ligand; RANKL, RANK ligand; TRAF6, tumor necrosis factor receptor-associated factor 6.