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Rifampin suppresses osteoclastogenesis and titanium particle-induced osteolysis via modulating RANKL signaling pathways
Accepted Manuscript Rifampin suppresses osteoclastogenesis and titanium particle-induced osteolysis via modulating RANKL signaling pathways Liang Zhu, Hui Kang, Chang-an Guo, Wen-shuai Fan, Yi-ming Wang, Lian-fu Deng, Zuo-qin Yan PII:
S0006-291X(17)30105-5
DOI:
10.1016/j.bbrc.2017.01.071
Reference:
YBBRC 37133
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 3 January 2017 Accepted Date: 15 January 2017
Please cite this article as: L. Zhu, H. Kang, C.-a. Guo, W.-s. Fan, Y.-m. Wang, L.-f. Deng, Z.-q. Yan, Rifampin suppresses osteoclastogenesis and titanium particle-induced osteolysis via modulating RANKL signaling pathways, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.01.071. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title page
Rifampin suppresses osteoclastogenesis and titanium
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particle-induced osteolysis via modulating RANKL
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signaling pathways
Liang zhu1,2*; Hui Kang2*; Chang-an Guo1; Wen-shuai Fan1; Yi-ming Wang1; Lian-fu Deng2; Zuo-qin Yan1#
1 Zhongshan Hospital of Fudan University, 180 Fenglin Road; Shanghai 200032, China
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2 Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Orthopaedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiaotong University
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School of Medicine, Shanghai, China
# Corresponding author: Prof. Zuoqin Yan
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E-mail: [email protected], Tel: +86-13818009668, Fax: 86-021-64037269 Address: Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China * These authors contributed equally to this work.
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Abstract Background: Wear particles liberated from the surface of prostheses are considered to be
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main reason for osteoclast bone resorption and that extensive osteoclastogenesis leads to peri-implant osteolysis and subsequent prosthetic loosening. The aim of this study was to assess the effect of rifampin on osteoclastogenesis and titanium (Ti) particle-induced
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osteolysis. Methods: The Ti particle-induced osteolysis mouse calvarial model and bone marrow-derived macrophages (BMMs) were used. Rifampin, at dose of 10 or 50
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mg/kg/day, was respectively given intraperitoneally for 14 days in vivo. The calvariae were removed and processed for Further histological analysis. In vitro, osteoclasts were generated from mouse BMMs with receptor activator of nuclear factor-κB ligand (RANKL) and the macrophage colony stimulating factor. Rifampin at different concentrations was added to the medium. The cell viability, tartrate-resistant acid
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phosphatase (TRAP) staining, TRAP activity and resorption on bone slices were analysis. Osteoclast-specific genes and RANKL-induced MAPKs signaling were tested for further study of the mechanism. Results: Rifampin inhibited Ti-induced osteolysis and
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osteoclastogenesis in vivo. In vitro data indicated that rifampin suppressed osteoclast
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differentiation and bone resorption in a dose-dependent manner. Moreover, rifampin significantly reduced the expression of osteoclast-specific markers, including TRAP, cathepsin K, V-ATPase d2, V-ATPase a3, c-Fos, and nuclear factor of activated T cells (NFAT) c1. Further investigation revealed that rifampin inhibited osteoclast formation by specifically abrogating RANKL-induced p38 and NF-κB signaling. Conclusion: Rifampin had significant potential for the treatment of particle-induced peri-implant osteolysis and other diseases caused by excessive osteoclast formation and function.
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Keywords: Osteolysis; Rifampin; NF-κB; P38; Wear particles Background
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Total hip arthroplasty (THA) is thought to be the highly successful treatment for severe trauma, osteoporotic fracture, and other arthritic joint diseases. However, particle-induced periprosthetic osteolysis and subsequent aseptic loosening remain the
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major causes of arthroplasty failure, especially in long-term complication[1]. Particulate wear particles from the interface between implant component and surrounding bone are
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thought to play a central role in osteolysis and aseptic loosening[2]. The osteoclast is the specialized cell responsible for the removal of bone in normal metabolism and various pathological processes. It is likely that a significant amount of the bone loss occurring in the osteolysis associated with the aseptic loosening of an implant is mediated by the
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classical bone resorptive activity of osteoclasts[3]. Although the pathophysiology mechanisms of particle-induced osteolysis remains unclear, it was generally accepted that the wear particles stimulate macrophages, phagocytes, fibroblasts, and T lymphocytes to
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secrete high concentrations of cytokines and chemokines[4-6]. This can accelerate osteoclastic bone resorption[7].
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Rifampin, an important antibiotic agent, is widely used as a major drug for the
treatment of mycobacterial infections. Unexpected properties of rifampin were reported that a subset of pregnane X receptor (PXR) activators, such as rifampin, vitamin K2, might function as effective therapeutic agents for the management of osteoporosis[8]. In PXR knockout
(PXRKO) mice,
the bone turnover of
the trabecular
bones, bone formation is reduced, whereas bone resorption is enhanced compare with the WT mice[9]. However, the beneficial effects of rifampin on bone osteoylsis have not
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previously been evaluated. Three major subfamilies of the MAPK signaling pathways[10] (p38, JNK 1/2, ERK1/2) and NF-κB pathway were crucial for osteoclast formation and development[11, 12]. Due to the importance of osteoclasts in abnormal osteolytic disease,
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inhibitors that can suppress osteoclast formation and/or function are candidates for the prevention and treatment of wear particle-induced osteolysis and pathological bone
rifampin treatment on osteoclastogenesis.
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Methods
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loss[13]. We hypothesized that NF-κB and MAPK may be invovled behind the effects of
Media and reagents Commercial pure titanium (Ti) particles, with average diameter of 5.3 mm, were purchased from Johnson Matthey (Catalog #00681;Ward Hill, MA, USA) (Figure. 1A/C). The cell counting kit (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Recombinant soluble human M-CSF and mouse RANKL were obtained from
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R&D (R&D Systems, Minne-apolis, MN, USA). Rifampin was purchased from Sigma Aldrich (St Louis, MO, USA). The TRAP staining kit and all other reagents were purchased from Sigma Aldrich, unless stated otherwise.
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Titanium particle-induced calvarial osteolysis model The mouse calvarial osteolysis
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model was established according to Jin[14] to determine the preventative effects of rifampin on osteolysis in vivo. All experiment procedures were performed in accordance with the Animal Care Committee of Fundan University. Briefly, 24 healthy 8-week-old C57BL/J6 mice were assigned randomly into four groups: sham PBS control (sham), Ti particles with PBS (vehicle), and Ti particles with low (10 mg/kg/day) and high (50 mg/kg/ day) concentrations of rifampin (low and high rifampin, respectively). The way to remove endotoxins adherent on Ti particles was according to the Liu et al ’s article[15].
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The mice were anesthetized, and the cranial periosteum was separated from the calvarium by sharp dissection. Then, 30 mg of Ti particles were embedded under the periosteum at the middle suture of the calvaria. Mice in the low and high rifampin groups were injected
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intraperitoneally with rifampin at 10 or 50 mg/kg/day, respectively, for 14 days. Mice in the sham and vehicle groups received PBS daily. At the end of the experiment, the mice were sacrificed, and the calvaria were excised and fixed in 4% paraformaldehyde for
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micro-CT analysis. During the whole experiment the animals were housed in stainless steel cages at conventional controlled conditions (temperature 25 ± 2°C, relative humidity
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of 50 ± 10%, 12 h light–dark cycle) and had free access to the standard laboratory food and tap water.
Micro CT scanning The fixed calvaria samples were analyzed using a high-resolution micro-CT (Skyscan 1072; Skyscan, Aartselaar, Belgium). The scanning protocol was set
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at an isometric resolution at 8.3 µm, and X-ray energy settings of 80 kV and 80 mA. After reconstruction, a ROI around the midline suture was selected for further qualitative and quantitative analysis. BV/TV, Bone mineral density (BMD) and percentage of total
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porosity of each sample measured.
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Histological and histomorphometric analysis After micro-CT scanning, the calvaria samples were decalcified in 10% EDTA for 3 weeks, and then embedded in paraffin. Histological sections were prepared for TRAP and H&E staining. The specimens were then examined and photographed under a high quality microscope. The percentage of TRAP-positive multinucleated osteoclasts area pre bone area was assessed in each sample.
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Cell culture, cell viability and osteoclast differentiation Bone marrow-derived macrophages (BMMs) were isolated from the femur and tibiae bone marrow as previously described[16] and cultured in a-MEM supplemented with 10% FBS, 1%
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penicillin/streptomycin, and 10 ng/mL M-CSF for 24 h. Non-adherent cells were then removed, and the adherent cells were cultured in a 37°C, 5% CO2 incubator for a further 3-4 days until cells were confluent. For cell viability assay, the BMMs were then seeded
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into a 96-well plate at a density of 8×103 cells/well with 30 ng/mL M-CSF for 24 h. Cells were then treated with different concentrations of rifampin (0, 1.25, 2.5, 5, 10, 20, 40, 80,
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160 or 320 µg/ml). 10 µl of CCK-8 reagent were added to each well 1 h before the end of incubation. The optical density (OD) value of each sample was measured at a wavelength of 450 nm on a microplate reader. The result of cell viability measurement is expressed as the absorbance at OD450. For osteoclast differentiation, cells were cultured in complete
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a-MEM supplemented with 30 ng/mL M-CSF, 50 ng/mL RANKL, and different concentrations of rifampin (0, 0.5, 5 or 50 µg/ml). Culture media were replaced every 2 days until mature osteoclasts had formed. Cells were then washed twice with PBS, fixed
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with 4% paraformaldehyde for 20 min, and stained for TRAP according to the manufacturer’s instruction. TRAP-positive cells with more than three nuclei were
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counted under a microscope and the area of TRAP-positive cells was quantified using Image Pro-Plus 5.0 (Media Cybernetics, Bethesda, MD, USA). The supernatant of the cell lysates was tested for the TRAP activity by using Tartrate Resistant Acid Phosphatase Assay Kit (Beyotime Institute of Biotechnology, Hai men, China). RNA extraction and quantitative PCR assay BMMs were seeded in 6-well plates a density of 1×105 cells/well with 30 ng/mL M-CSF for 24 h. Cells were then cultured in
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complete a-MEM supplemented with 30 ng/mL M-CSF, 50 ng/mL RANKL, and different concentrations of rifampin (0, 5 or 50 µg/ml) for 3 days. Total RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies; Carlsbad, CA, USA)
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following the manufacturer’s instructions. Total RNA (2 µg) was used to generate single-stranded cDNA, which was then used as template in real-time PCR. PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems 7500 System,
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Foster City, CA, USA) using SYBR Green PCR Master Mix (Applied Bio-systems). GAPDH was used as the housekeeping gene, and all reactions were run in triplicate. The
NFATc1
and
cathepsin
K
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mouse primer sequences of GAPDH, TRAP, V-ATPase a3, V-ATPase d2, c-fos, were
as
follows:
GAPDH
forward
5'-ACCCAGAAGACTGTGGATGG-3'
and
reverse
5'-CACATTGGGGGTAGGAACAC-3';
TRAP
forward
V-ATPase
a3
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5'-TCCCCAATGCCCCATTC-3' and reverse 5'-CGGTTCTGGCGATCTCTTTG-3'; forward
5'-GCCTCAGGGGAAGGCCAGATCG-3'
and
reverse
5'-GGCCACCTCTTCACTCCGGAA-3'; V-ATPase d2 forward 5'-AAGCCTTTGTTT-
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GACGCTGT-3' and reverse 5'-TTCGATGCCTCTGTGAGATG-3'; c-Fos forward 5'-AGGCCCAGTGGCTCAGAGA-3'
and
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5'-GCTCCCAGTCTGCTGCATAGA-3'; 5'-ACCACCTTTCCGCAACCA-3' cathepsin
K
forward
and
reverse
NFATc1 reverse
forward
5'-TTCCGTTTCCCGTTGCA-3';
5'-GGCTGTGGAGGCGGCTAT-3'
and
reverse
5'-AGAGTCAATGCCTCCGTTCTG-3'. Western blotting BMMs were seeded in 6-well plates a density of 1×105 cells/well with 30 ng/mL M-CSF for 24 h. Cells were then cultured in complete a-MEM supplemented
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with or without 0.5 µg/ml rifampin for 4h followed by 50 ng/mL RANKL as indicated. The protein samples were extracted from cells and fractionated by SDS-PAGE (10 % polyacrylamide gels). Separated proteins were blot transferred onto a nitrocellulose
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membrane. After blocking with 0.1 % Tween 20 and 5 % nonfat dry milk in Tris-buffered saline at room temperature for 1 h, the membrane was incubated over- night at 4 °C in one of the following primary antibodies: p38 (1:1000), p-p38 (1:1000), JNK
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(1:1000), p-JNK (1:1000), ERK (1:1000), p-ERK (1:1000), p65 (1:1000), p-p65 (1:1000) and β-actin (1:1000) as an internal control (Cell signaling technology, Beverly, MA,
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USA). The membrane was incubated with horseradish peroxidase-conjµgated secondary antibody (1:5000) for 2 h and detected using the Enhanced Chemiluminescence Western blot System (Amersham Biosciences).
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Bone absorption assay BMM cells were seeded at a density of 2.4×104 cells/cm2 onto bovine bone slices. Forty-eight hours later, cells were treated with 50 ng/mL RANKL, 30 ng/mL M-CSF, and 0, 0.5, 5, or 50 µg/ml rifampin until mature osteoclasts formed. After
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fixation, the bone slices were then dehydrated throµgh an ethanol gradient and stained with 1 % toluidine blue and Mayer's hematoxylin. Positively stained areas were counted
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as bone resorption pits and the resorption area was calculated with Image Pro-Plus 5.0 (Media Cybernetics, Bethesda, MD, USA). Statistical analysis
Results presented are representative of at least three independent experiments and are expressed as the means±sem. Differences between treated and untreated groups were assessed by Student’s t-test. One-way ANOVA models were used to compare multiple
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comparisons with Tukey’s studentized range test as post hoc analysis. The significance level was set at 0.05.
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Results Rifampin prevented wear-particle-induced bone loss in vivo To explore the effects of rifampin on pathological osteolysis, we used a mouse calvaria model of Ti
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particle-induced osteolysis. Micro-computed tomography (CT) with three-dimensional reconstruction revealed that extensive bone resorption was observed in the Ti group. In
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the groups with rifampin treatment, particle-induced osteolysis was suppressed, where bone resorption in mice treated with a high concentration was much lower than that in the low concentration (Figure. 1B). Compared with the sham control, the presence of Ti particles induced a significant osteolysis in the calvaria. Quantification of bone
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parameters confirmed that high-rifampin group (50 mg/kg/day) significantly increased the BV/TV and decreased the percentage of total porosity (%) (Figure. 1D). Further in histological assessment and histomorphometric analysis confirmed the attenuation of
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wear particle-induced bone erosion by rifampin (Figure. 1E). The presence of Ti particles induced the inflammatory infiltration of lymphocytes and macrophages. TRAP staining
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revealed that multiple osteoclasts lined along the eroded bone surface in Ti group. Consistent with the micro-CT quantitation, the TRAP-positive cells were reduced in the rifampin groups (Figure. 1F). Rifampin inhibited RANKL-induced osteoclast formation without producing cytotoxicity To examine the potential cytotoxicity of rifampin, CCK8 assays were performed. BMMs were treated with various concentrations of rifampin from day 1 to
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day 3 (time interval 24h). Rifampin had no cytotoxic effects on BMMs at concentrations less than 80 µg/ml compared with the control treatment (Figure. 2 A-C). A slight improvement of proliferation was found at rifampin concentration from 5 to 40µg/ml,
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especially in 48h and 72h time-point. This indicated that rifampin may partially suppress the proliferation of BMMs, but only at high concentrations (>80 µg/ml). We then investigated the effect of rifampin on osteoclast formation. The tartrate-resistant acid
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phosphatase (TRAP) activity in BMMs was significantly reduced since the concentration of rifampin reached to 20 µg/ml at 72h (Figure. 2 D). In contrast, osteoclast formation
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was inhibited by rifampin treatment in a dose-dependent manner. Osteoclast formation was reduced to approximately 10% of control levels by treatment with 50 µg/ml at 5 days culture (Figure. 2 E). Due to non-cytotoxic effects of rifampin at the low concentrations (<80 µg/ml), decreased number of TRAP-positive cells suggested that rifampin inhibited
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osteoclast formation.
Effect of Rifampin on osteoclast bone resorption We next investigated whether rifampin could inhibit osteoclastic bone resorption in vitro. Osteoclast precursors were
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plated on bone slices and treated with rifampin or not. As shown in Figure. 2H/J, bone
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resorption area substantially reduced in the rifampin-treated group. These findings suggested that rifampin impaired osteoclast bone resorption in vitro. Rifampin suppressed RANKL-induced gene expression To further confirm the inhibitory effect of rifampin on osteoclast differentiation, quantitative PCR (qPCR) was used
to
analyze
and
quantify
the
RANKL-induced
mRNA
expression
of
osteoclast-related genes (including TRAP, cathepsin K, c-Fos, V-ATPase d3, V-ATPase d2 and NFATc1). However, the RANKL-induced upregulation of these genes was
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strongly suppressed in the presence of rifampin in a dose-dependent manner (Figure. 3). Therefore, these data further confirmed that rifampin suppressed osteoclast formation and
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osteoclast-specific genes expression. Suppression of rifampin on RANKL-induced p38 and NF-κB signaling In order to elucidate the mechanism underlying rifampin’s inhibition of osteoclast formation and
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function. BMMs were stimulated with RANKL (50 ng/mL) for 0, 5, 15, 30, 60, or 90 min to investigate short term signaling pathways (p-p38, p38, p-ERK1/2, ERK1/2, JNK,
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p-JNK, p65 and p-p65). The results showed that the phosphorylation of p38, JNK1/2, and ERK1/2 peaked within 15 min of RANKL stimulation. We found that rifampin attenuated p38 phosphorylation. However, there were no obvious effect on ERK and JNK activation.
However,
pretreatment
with
rifampin
significantly
decreased
p38
phosphorylation, activated by RANKL. Collectively, these data suggested that rifampin
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inhibited p38 MAPK signaling pathways during osteoclastogenesis. In addition, in the control group with the RANKL’s stimulation, p-p65 was upgraded within 5 min and this trend was rescued in the following 30–90 min (Figure. 4A). When the cells were
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pretreated with rifampin (5 µg/ml), RANKL-induced p65 phosphorylation and
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degradation were both significantly suppressed. These results sµggested that rifampin might participate in the regulation of RANKL-activated NF-κB signaling, and could thus contribute to the inhibition of osteoclast formation. Discussion
THA is an effective treatment for severe trauma and arthritic joint diseases. However, periprosthetic osteolysis and subsequent aseptic loosening remains the major cause of
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arthroplasty failure. Excessive bone resorption plays a critical role in pathologic bone diseases. To our knowledge, the extent of osteolysis is dependent on particle composition, size, and amount, as well as duration of implantation. Though the mechanism of wear
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debris-induced osteolysis is not clear, it is widely accepted that wear particles could increase the secretion of pro-inflammatory cytokines, thereby inducing osteolysis through the dysregulation of osteoclast and osteoblast activity[17] and, eventually, contribute to
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the osteolytic process. Some antibiotic, such as doxycycline[18], erythromycin[19] and enoxacin[20] have an inhibitory effect of wear particle-induced osteolysis. Here, just as
Ti particle-induced osteolysis.
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we hypothesized, the antibiotic rifampin functions as an osteoclast inhibitor could prevent
As adherent endotoxins on Ti particles are involved in many of the biological responses induced by wear particles, including osteoclast formation[21]. A series of
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procedures were used to remove adherent endotoxins, according to Liu et al.’s report[15]. In our research, obvious osteolysis was seen in Ti group compared with the sham group, whereas rifampin-treated groups significantly suppressed particle-induced osteolysis.
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These results were further supported by histological analysis of tissues stained with H&E and TRAP. We proposed that rifampin, a classic drug commonly used in clinics, should
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have great potential and value for the treatment of particle-induced periprosthesic osteolysis. Mature osteoclasts are characterized by the specific phenotypic markers expression, as well as multinucleated cells and the capability of bone resorption[10]. Our research demonstrated that the inhibitory effect of rifampin on osteoclastogenesis involved inhibition of RANKL-induced mRNA-expression of TRAP, cathepsin K, c-Fos, V-ATPase a3, V-ATPase d2 and NFATc1. Bone resorption and osteoclasts formation
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assays revealed that the number and area of bone resorption pits observed in vitro were dramatically decreased after rifampin exposure (Figure. 5A), due to the inhibition of osteoclast formation in a dose-dependent manner. Moreover, this inhibitory effect was
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observed at low concentrations of rifampin (0.5-50 µg/ml), without cytotoxic effects.
RANKL signaling is essential for particle-induced osteoclastic bone resorption[22]. Once upon the RANKL signaling, it is mediated by cytoplasmic factors that activate at
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least five distinct signaling cascades downstream signaling pathways, including MAPKs (ERK, p38, and JNK)[10, 23, 24]. Stimulation of p38 results in the downstream
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activation of the transcriptional regulator mi/Mitf, which controls the expression of the genes encoding TRAP and cathepsin K[25]. Similar result was confirmed in our study. Rifampin has been used for the treatment of bacterial infections for many years. Clinically, rifampin has been found to possess immunomodulatory effects and suggested
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to be repositioned as a Toll-like receptor 4 (TLR4) inhibitor to attenuate allodynia in a preclinical model of neuropathic pain[26]. TLR4 is the principal cell surface receptor for the LPS component of antitoxin, playing key roles in activating a cascade of
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pro-inflammatory events in response to pathogens[27]. Previous studies have suggested that rifampin may inhibit lipopolysaccharide (LPS)-stimulated NF-κB, p38, JNK, and
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MAPK activation through inhibition of the TLR4 pathway[28, 29]. As shown in the in vitro experiment, rifampin inhibited osteoclastogenesis without affecting ERK and JNK. Therefore, RANKL-induced p38 and NF-κB signaling may be involved behind the effects of rifampin treatment on osteoclastogenesis. In conclusion, Rifampin has unexpected properties that inhibit osteoclast formation and function both in vitro and in vivo. Rifampin also reduced the RANKL-induced
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osteoclastic marker genes expression. Furthermore, rifampin attenuated RANKL-induced NF-κB and MAPKs (p38) activation. These results suggest that it may represent a promising agent for the treatment or prevention of osteoporosis, periprosthetic loosening
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after THA, and other bone destructive diseases. Acknowledgements
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This research was financially supported by the National Natural Science Foundation of China (no. 81672157).
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[23] M. Asagiri, H. Takayanagi, The molecular understanding of osteoclast differentiation, Bone, 40 (2007) 251-264.
[24] J.P. David, K. Sabapathy, O. Hoffmann, M.H. Idarraga, E.F. Wagner, JNK1 modulates osteoclastogenesis through both c-Jun phosphorylation-dependent and
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-independent mechanisms, Journal of cell science, 115 (2002) 4317-4325. [25] K.C. Mansky, U. Sankar, J. Han, M.C. Ostrowski, Microphthalmia transcription factor is a target of the p38 MAPK pathway in response to receptor activator of
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11077-11083.
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NF-kappa B ligand signaling, The Journal of biological chemistry, 277 (2002)
[26] X. Wang, P.M. Grace, M.N. Pham, K. Cheng, K.A. Strand, C. Smith, J. Li, L.R. Watkins, H. Yin, Rifampin inhibits Toll-like receptor 4 signaling by targeting myeloid differentiation protein 2 and attenuates neuropathic pain, FASEB journal : official publication of the Federation of American Societies for Experimental Biology, 27 (2013) 2713-2722. [27] K. Ozato, H. Tsujimura, T. Tamura, Toll-like receptor signaling and regulation of
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cytokine gene expression in the immune system, BioTechniques, Suppl (2002) 66-68, 70, 72 passim. [28] W. Bi, L. Zhu, X. Jing, Z. Zeng, Y. Liang, A. Xu, J. Liu, S. Xiao, L. Yang, Q. Shi, L. Guo,
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E. Tao, Rifampicin improves neuronal apoptosis in LPS-stimulated cocultured BV2 cells through inhibition of the TLR-4 pathway, Mol Med Rep, 10 (2014) 1793-1799. [29] W. Bi, L. Zhu, C. Wang, Y. Liang, J. Liu, Q. Shi, E. Tao, Rifampicin inhibits
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microglial inflammation and improves neuron survival against inflammation, Brain
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research, 1395 (2011) 12-20.
Figure. 1. Rifampin prevented titanium particle-induced mouse calvarial osteolysis. (A) Scanning electron micrograph of Ti particles and (C) the high filed for the particles; (B) Representative micro-computed tomography (CT) three-dimensional reconstructed
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images from each group. (D) Bone volume against tissue volume (BV/TV), BMD and the percentage of total porosity of each sample was measured (*P < 0.05; **P < 0.01); (E) Hematoxylin and eosin and (F) TRAP staining were performed from at least three
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sections per group. The black arrows represent osteoclasts. Scale bar are 200µm.
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Figure. 2. Rifampin inhibited RANKL-induced osteoclastogenesis and bone resorption without cytotoxic effects. BMMs were treated with 50 ng/mL RANKL and various concentrations of rifampin, as indicated, for 24h (A), 48h (B) and 72 h (C). (D) After 72h indicated treatment, TRAP activity was measured by assessing optical density at 405 nm. (E) TRAP staining after induction with 50 ng/mL RANKL in the presence or absence of different concentrations of rifampin for 7days. Scale bar are 50µm. The area (F) and number (G) of TRAP-positive cells was counted. (J) 1 % toluidine blue and Mayer's
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hematoxylin staining showed the formation of a lot resorption in control group, rifampin treatment significantly reduced the number of resorption pits. Scale bar are 20µm. (H) Resorption pit areas were measured using Image J software. All experiments were
methods (*p < 0.05, **p < 0.01 versus the control).
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performed at least three times, and the significance was determined as indicated in
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Figure. 3. Rifampin suppressed RANKL-induced expression of osteoclast-specific genes. BMMs were cultured with 50 ng/mL RANKL with different rifampin concentrations, as
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indicated for 72h and osteoclast-specific gene expression (Cathepsin k, TRAP, VATPs-a3, VATPs-d2, c-fos, and NFATc1) was analyzed by real-time PCR, and results were normalized to the expression of GAPDH. All experiments were performed at least three times, and the significance was determined as indicated in methods (*p < 0.05, **p<0.01
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versus the control).
Figure. 4. Rifampin specifically impaired the RANKL-induced p38 and NFκB signalling. (A) BMMs were pretreated with or without 5µg/ml rifampin for 4 h followed by 50
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ng/mL RANKL as indicated. Cell lysates were then analyzed using Western blotting with specific antibodies against phospho-JNK, JNK, phosphor- P38, P38, phosphor-ERK,
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ERK, phospho-p65, P65 and GAPDH. Average ratio of phospho-p38 (B), phospho- p65 (C) relative to GAPDH. The bands’ intensity analysis was quantified by image J software. Each ratio was normalized to 0 min (**p < 0.01 versus the control).
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ACCEPTED MANUSCRIPT 1. Rifampin inhibited Ti-induced osteolysis and osteoclastogenesis in vivo. 2. rifampin suppressed osteoclast differentiation and bone resorption in a dose-dependent manner.
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3. rifampin significantly reduced the expression of osteoclast-specific markers,
factor of activated T cells (NFAT) c1.
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including TRAP, cathepsin K, V-ATPase d2, V-ATPase a3, c-Fos, and nuclear
4. RANKL-induced p38 and NF-κB signaling may be involved behind the effects of
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rifampin treatment on osteoclastogenesis
S0006-291X(17)30105-5
DOI:
10.1016/j.bbrc.2017.01.071
Reference:
YBBRC 37133
To appear in:
Biochemical and Biophysical Research Communications
Received Date: 3 January 2017 Accepted Date: 15 January 2017
Please cite this article as: L. Zhu, H. Kang, C.-a. Guo, W.-s. Fan, Y.-m. Wang, L.-f. Deng, Z.-q. Yan, Rifampin suppresses osteoclastogenesis and titanium particle-induced osteolysis via modulating RANKL signaling pathways, Biochemical and Biophysical Research Communications (2017), doi: 10.1016/ j.bbrc.2017.01.071. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Title page
Rifampin suppresses osteoclastogenesis and titanium
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particle-induced osteolysis via modulating RANKL
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signaling pathways
Liang zhu1,2*; Hui Kang2*; Chang-an Guo1; Wen-shuai Fan1; Yi-ming Wang1; Lian-fu Deng2; Zuo-qin Yan1#
1 Zhongshan Hospital of Fudan University, 180 Fenglin Road; Shanghai 200032, China
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2 Shanghai Key Laboratory for Bone and Joint Diseases, Shanghai Institute of Orthopaedics and Traumatology, Shanghai Ruijin Hospital, Shanghai Jiaotong University
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School of Medicine, Shanghai, China
# Corresponding author: Prof. Zuoqin Yan
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E-mail: [email protected], Tel: +86-13818009668, Fax: 86-021-64037269 Address: Department of Orthopedic Surgery, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, China * These authors contributed equally to this work.
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Abstract Background: Wear particles liberated from the surface of prostheses are considered to be
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main reason for osteoclast bone resorption and that extensive osteoclastogenesis leads to peri-implant osteolysis and subsequent prosthetic loosening. The aim of this study was to assess the effect of rifampin on osteoclastogenesis and titanium (Ti) particle-induced
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osteolysis. Methods: The Ti particle-induced osteolysis mouse calvarial model and bone marrow-derived macrophages (BMMs) were used. Rifampin, at dose of 10 or 50
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mg/kg/day, was respectively given intraperitoneally for 14 days in vivo. The calvariae were removed and processed for Further histological analysis. In vitro, osteoclasts were generated from mouse BMMs with receptor activator of nuclear factor-κB ligand (RANKL) and the macrophage colony stimulating factor. Rifampin at different concentrations was added to the medium. The cell viability, tartrate-resistant acid
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phosphatase (TRAP) staining, TRAP activity and resorption on bone slices were analysis. Osteoclast-specific genes and RANKL-induced MAPKs signaling were tested for further study of the mechanism. Results: Rifampin inhibited Ti-induced osteolysis and
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osteoclastogenesis in vivo. In vitro data indicated that rifampin suppressed osteoclast
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differentiation and bone resorption in a dose-dependent manner. Moreover, rifampin significantly reduced the expression of osteoclast-specific markers, including TRAP, cathepsin K, V-ATPase d2, V-ATPase a3, c-Fos, and nuclear factor of activated T cells (NFAT) c1. Further investigation revealed that rifampin inhibited osteoclast formation by specifically abrogating RANKL-induced p38 and NF-κB signaling. Conclusion: Rifampin had significant potential for the treatment of particle-induced peri-implant osteolysis and other diseases caused by excessive osteoclast formation and function.
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Keywords: Osteolysis; Rifampin; NF-κB; P38; Wear particles Background
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Total hip arthroplasty (THA) is thought to be the highly successful treatment for severe trauma, osteoporotic fracture, and other arthritic joint diseases. However, particle-induced periprosthetic osteolysis and subsequent aseptic loosening remain the
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major causes of arthroplasty failure, especially in long-term complication[1]. Particulate wear particles from the interface between implant component and surrounding bone are
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thought to play a central role in osteolysis and aseptic loosening[2]. The osteoclast is the specialized cell responsible for the removal of bone in normal metabolism and various pathological processes. It is likely that a significant amount of the bone loss occurring in the osteolysis associated with the aseptic loosening of an implant is mediated by the
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classical bone resorptive activity of osteoclasts[3]. Although the pathophysiology mechanisms of particle-induced osteolysis remains unclear, it was generally accepted that the wear particles stimulate macrophages, phagocytes, fibroblasts, and T lymphocytes to
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secrete high concentrations of cytokines and chemokines[4-6]. This can accelerate osteoclastic bone resorption[7].
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Rifampin, an important antibiotic agent, is widely used as a major drug for the
treatment of mycobacterial infections. Unexpected properties of rifampin were reported that a subset of pregnane X receptor (PXR) activators, such as rifampin, vitamin K2, might function as effective therapeutic agents for the management of osteoporosis[8]. In PXR knockout
(PXRKO) mice,
the bone turnover of
the trabecular
bones, bone formation is reduced, whereas bone resorption is enhanced compare with the WT mice[9]. However, the beneficial effects of rifampin on bone osteoylsis have not
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previously been evaluated. Three major subfamilies of the MAPK signaling pathways[10] (p38, JNK 1/2, ERK1/2) and NF-κB pathway were crucial for osteoclast formation and development[11, 12]. Due to the importance of osteoclasts in abnormal osteolytic disease,
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inhibitors that can suppress osteoclast formation and/or function are candidates for the prevention and treatment of wear particle-induced osteolysis and pathological bone
rifampin treatment on osteoclastogenesis.
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Methods
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loss[13]. We hypothesized that NF-κB and MAPK may be invovled behind the effects of
Media and reagents Commercial pure titanium (Ti) particles, with average diameter of 5.3 mm, were purchased from Johnson Matthey (Catalog #00681;Ward Hill, MA, USA) (Figure. 1A/C). The cell counting kit (CCK-8) was obtained from Dojindo (Kumamoto, Japan). Recombinant soluble human M-CSF and mouse RANKL were obtained from
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R&D (R&D Systems, Minne-apolis, MN, USA). Rifampin was purchased from Sigma Aldrich (St Louis, MO, USA). The TRAP staining kit and all other reagents were purchased from Sigma Aldrich, unless stated otherwise.
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Titanium particle-induced calvarial osteolysis model The mouse calvarial osteolysis
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model was established according to Jin[14] to determine the preventative effects of rifampin on osteolysis in vivo. All experiment procedures were performed in accordance with the Animal Care Committee of Fundan University. Briefly, 24 healthy 8-week-old C57BL/J6 mice were assigned randomly into four groups: sham PBS control (sham), Ti particles with PBS (vehicle), and Ti particles with low (10 mg/kg/day) and high (50 mg/kg/ day) concentrations of rifampin (low and high rifampin, respectively). The way to remove endotoxins adherent on Ti particles was according to the Liu et al ’s article[15].
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The mice were anesthetized, and the cranial periosteum was separated from the calvarium by sharp dissection. Then, 30 mg of Ti particles were embedded under the periosteum at the middle suture of the calvaria. Mice in the low and high rifampin groups were injected
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intraperitoneally with rifampin at 10 or 50 mg/kg/day, respectively, for 14 days. Mice in the sham and vehicle groups received PBS daily. At the end of the experiment, the mice were sacrificed, and the calvaria were excised and fixed in 4% paraformaldehyde for
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micro-CT analysis. During the whole experiment the animals were housed in stainless steel cages at conventional controlled conditions (temperature 25 ± 2°C, relative humidity
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of 50 ± 10%, 12 h light–dark cycle) and had free access to the standard laboratory food and tap water.
Micro CT scanning The fixed calvaria samples were analyzed using a high-resolution micro-CT (Skyscan 1072; Skyscan, Aartselaar, Belgium). The scanning protocol was set
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at an isometric resolution at 8.3 µm, and X-ray energy settings of 80 kV and 80 mA. After reconstruction, a ROI around the midline suture was selected for further qualitative and quantitative analysis. BV/TV, Bone mineral density (BMD) and percentage of total
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porosity of each sample measured.
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Histological and histomorphometric analysis After micro-CT scanning, the calvaria samples were decalcified in 10% EDTA for 3 weeks, and then embedded in paraffin. Histological sections were prepared for TRAP and H&E staining. The specimens were then examined and photographed under a high quality microscope. The percentage of TRAP-positive multinucleated osteoclasts area pre bone area was assessed in each sample.
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Cell culture, cell viability and osteoclast differentiation Bone marrow-derived macrophages (BMMs) were isolated from the femur and tibiae bone marrow as previously described[16] and cultured in a-MEM supplemented with 10% FBS, 1%
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penicillin/streptomycin, and 10 ng/mL M-CSF for 24 h. Non-adherent cells were then removed, and the adherent cells were cultured in a 37°C, 5% CO2 incubator for a further 3-4 days until cells were confluent. For cell viability assay, the BMMs were then seeded
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into a 96-well plate at a density of 8×103 cells/well with 30 ng/mL M-CSF for 24 h. Cells were then treated with different concentrations of rifampin (0, 1.25, 2.5, 5, 10, 20, 40, 80,
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160 or 320 µg/ml). 10 µl of CCK-8 reagent were added to each well 1 h before the end of incubation. The optical density (OD) value of each sample was measured at a wavelength of 450 nm on a microplate reader. The result of cell viability measurement is expressed as the absorbance at OD450. For osteoclast differentiation, cells were cultured in complete
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a-MEM supplemented with 30 ng/mL M-CSF, 50 ng/mL RANKL, and different concentrations of rifampin (0, 0.5, 5 or 50 µg/ml). Culture media were replaced every 2 days until mature osteoclasts had formed. Cells were then washed twice with PBS, fixed
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with 4% paraformaldehyde for 20 min, and stained for TRAP according to the manufacturer’s instruction. TRAP-positive cells with more than three nuclei were
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counted under a microscope and the area of TRAP-positive cells was quantified using Image Pro-Plus 5.0 (Media Cybernetics, Bethesda, MD, USA). The supernatant of the cell lysates was tested for the TRAP activity by using Tartrate Resistant Acid Phosphatase Assay Kit (Beyotime Institute of Biotechnology, Hai men, China). RNA extraction and quantitative PCR assay BMMs were seeded in 6-well plates a density of 1×105 cells/well with 30 ng/mL M-CSF for 24 h. Cells were then cultured in
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complete a-MEM supplemented with 30 ng/mL M-CSF, 50 ng/mL RANKL, and different concentrations of rifampin (0, 5 or 50 µg/ml) for 3 days. Total RNA was extracted using TRIzol reagent (Invitrogen, Life Technologies; Carlsbad, CA, USA)
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following the manufacturer’s instructions. Total RNA (2 µg) was used to generate single-stranded cDNA, which was then used as template in real-time PCR. PCR was performed on an ABI 7500 Real-Time PCR System (Applied Biosystems 7500 System,
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Foster City, CA, USA) using SYBR Green PCR Master Mix (Applied Bio-systems). GAPDH was used as the housekeeping gene, and all reactions were run in triplicate. The
NFATc1
and
cathepsin
K
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mouse primer sequences of GAPDH, TRAP, V-ATPase a3, V-ATPase d2, c-fos, were
as
follows:
GAPDH
forward
5'-ACCCAGAAGACTGTGGATGG-3'
and
reverse
5'-CACATTGGGGGTAGGAACAC-3';
TRAP
forward
V-ATPase
a3
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5'-TCCCCAATGCCCCATTC-3' and reverse 5'-CGGTTCTGGCGATCTCTTTG-3'; forward
5'-GCCTCAGGGGAAGGCCAGATCG-3'
and
reverse
5'-GGCCACCTCTTCACTCCGGAA-3'; V-ATPase d2 forward 5'-AAGCCTTTGTTT-
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GACGCTGT-3' and reverse 5'-TTCGATGCCTCTGTGAGATG-3'; c-Fos forward 5'-AGGCCCAGTGGCTCAGAGA-3'
and
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5'-GCTCCCAGTCTGCTGCATAGA-3'; 5'-ACCACCTTTCCGCAACCA-3' cathepsin
K
forward
and
reverse
NFATc1 reverse
forward
5'-TTCCGTTTCCCGTTGCA-3';
5'-GGCTGTGGAGGCGGCTAT-3'
and
reverse
5'-AGAGTCAATGCCTCCGTTCTG-3'. Western blotting BMMs were seeded in 6-well plates a density of 1×105 cells/well with 30 ng/mL M-CSF for 24 h. Cells were then cultured in complete a-MEM supplemented
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with or without 0.5 µg/ml rifampin for 4h followed by 50 ng/mL RANKL as indicated. The protein samples were extracted from cells and fractionated by SDS-PAGE (10 % polyacrylamide gels). Separated proteins were blot transferred onto a nitrocellulose
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membrane. After blocking with 0.1 % Tween 20 and 5 % nonfat dry milk in Tris-buffered saline at room temperature for 1 h, the membrane was incubated over- night at 4 °C in one of the following primary antibodies: p38 (1:1000), p-p38 (1:1000), JNK
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(1:1000), p-JNK (1:1000), ERK (1:1000), p-ERK (1:1000), p65 (1:1000), p-p65 (1:1000) and β-actin (1:1000) as an internal control (Cell signaling technology, Beverly, MA,
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USA). The membrane was incubated with horseradish peroxidase-conjµgated secondary antibody (1:5000) for 2 h and detected using the Enhanced Chemiluminescence Western blot System (Amersham Biosciences).
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Bone absorption assay BMM cells were seeded at a density of 2.4×104 cells/cm2 onto bovine bone slices. Forty-eight hours later, cells were treated with 50 ng/mL RANKL, 30 ng/mL M-CSF, and 0, 0.5, 5, or 50 µg/ml rifampin until mature osteoclasts formed. After
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fixation, the bone slices were then dehydrated throµgh an ethanol gradient and stained with 1 % toluidine blue and Mayer's hematoxylin. Positively stained areas were counted
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as bone resorption pits and the resorption area was calculated with Image Pro-Plus 5.0 (Media Cybernetics, Bethesda, MD, USA). Statistical analysis
Results presented are representative of at least three independent experiments and are expressed as the means±sem. Differences between treated and untreated groups were assessed by Student’s t-test. One-way ANOVA models were used to compare multiple
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comparisons with Tukey’s studentized range test as post hoc analysis. The significance level was set at 0.05.
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Results Rifampin prevented wear-particle-induced bone loss in vivo To explore the effects of rifampin on pathological osteolysis, we used a mouse calvaria model of Ti
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particle-induced osteolysis. Micro-computed tomography (CT) with three-dimensional reconstruction revealed that extensive bone resorption was observed in the Ti group. In
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the groups with rifampin treatment, particle-induced osteolysis was suppressed, where bone resorption in mice treated with a high concentration was much lower than that in the low concentration (Figure. 1B). Compared with the sham control, the presence of Ti particles induced a significant osteolysis in the calvaria. Quantification of bone
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parameters confirmed that high-rifampin group (50 mg/kg/day) significantly increased the BV/TV and decreased the percentage of total porosity (%) (Figure. 1D). Further in histological assessment and histomorphometric analysis confirmed the attenuation of
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wear particle-induced bone erosion by rifampin (Figure. 1E). The presence of Ti particles induced the inflammatory infiltration of lymphocytes and macrophages. TRAP staining
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revealed that multiple osteoclasts lined along the eroded bone surface in Ti group. Consistent with the micro-CT quantitation, the TRAP-positive cells were reduced in the rifampin groups (Figure. 1F). Rifampin inhibited RANKL-induced osteoclast formation without producing cytotoxicity To examine the potential cytotoxicity of rifampin, CCK8 assays were performed. BMMs were treated with various concentrations of rifampin from day 1 to
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day 3 (time interval 24h). Rifampin had no cytotoxic effects on BMMs at concentrations less than 80 µg/ml compared with the control treatment (Figure. 2 A-C). A slight improvement of proliferation was found at rifampin concentration from 5 to 40µg/ml,
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especially in 48h and 72h time-point. This indicated that rifampin may partially suppress the proliferation of BMMs, but only at high concentrations (>80 µg/ml). We then investigated the effect of rifampin on osteoclast formation. The tartrate-resistant acid
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phosphatase (TRAP) activity in BMMs was significantly reduced since the concentration of rifampin reached to 20 µg/ml at 72h (Figure. 2 D). In contrast, osteoclast formation
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was inhibited by rifampin treatment in a dose-dependent manner. Osteoclast formation was reduced to approximately 10% of control levels by treatment with 50 µg/ml at 5 days culture (Figure. 2 E). Due to non-cytotoxic effects of rifampin at the low concentrations (<80 µg/ml), decreased number of TRAP-positive cells suggested that rifampin inhibited
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osteoclast formation.
Effect of Rifampin on osteoclast bone resorption We next investigated whether rifampin could inhibit osteoclastic bone resorption in vitro. Osteoclast precursors were
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plated on bone slices and treated with rifampin or not. As shown in Figure. 2H/J, bone
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resorption area substantially reduced in the rifampin-treated group. These findings suggested that rifampin impaired osteoclast bone resorption in vitro. Rifampin suppressed RANKL-induced gene expression To further confirm the inhibitory effect of rifampin on osteoclast differentiation, quantitative PCR (qPCR) was used
to
analyze
and
quantify
the
RANKL-induced
mRNA
expression
of
osteoclast-related genes (including TRAP, cathepsin K, c-Fos, V-ATPase d3, V-ATPase d2 and NFATc1). However, the RANKL-induced upregulation of these genes was
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strongly suppressed in the presence of rifampin in a dose-dependent manner (Figure. 3). Therefore, these data further confirmed that rifampin suppressed osteoclast formation and
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osteoclast-specific genes expression. Suppression of rifampin on RANKL-induced p38 and NF-κB signaling In order to elucidate the mechanism underlying rifampin’s inhibition of osteoclast formation and
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function. BMMs were stimulated with RANKL (50 ng/mL) for 0, 5, 15, 30, 60, or 90 min to investigate short term signaling pathways (p-p38, p38, p-ERK1/2, ERK1/2, JNK,
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p-JNK, p65 and p-p65). The results showed that the phosphorylation of p38, JNK1/2, and ERK1/2 peaked within 15 min of RANKL stimulation. We found that rifampin attenuated p38 phosphorylation. However, there were no obvious effect on ERK and JNK activation.
However,
pretreatment
with
rifampin
significantly
decreased
p38
phosphorylation, activated by RANKL. Collectively, these data suggested that rifampin
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inhibited p38 MAPK signaling pathways during osteoclastogenesis. In addition, in the control group with the RANKL’s stimulation, p-p65 was upgraded within 5 min and this trend was rescued in the following 30–90 min (Figure. 4A). When the cells were
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pretreated with rifampin (5 µg/ml), RANKL-induced p65 phosphorylation and
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degradation were both significantly suppressed. These results sµggested that rifampin might participate in the regulation of RANKL-activated NF-κB signaling, and could thus contribute to the inhibition of osteoclast formation. Discussion
THA is an effective treatment for severe trauma and arthritic joint diseases. However, periprosthetic osteolysis and subsequent aseptic loosening remains the major cause of
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arthroplasty failure. Excessive bone resorption plays a critical role in pathologic bone diseases. To our knowledge, the extent of osteolysis is dependent on particle composition, size, and amount, as well as duration of implantation. Though the mechanism of wear
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debris-induced osteolysis is not clear, it is widely accepted that wear particles could increase the secretion of pro-inflammatory cytokines, thereby inducing osteolysis through the dysregulation of osteoclast and osteoblast activity[17] and, eventually, contribute to
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the osteolytic process. Some antibiotic, such as doxycycline[18], erythromycin[19] and enoxacin[20] have an inhibitory effect of wear particle-induced osteolysis. Here, just as
Ti particle-induced osteolysis.
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we hypothesized, the antibiotic rifampin functions as an osteoclast inhibitor could prevent
As adherent endotoxins on Ti particles are involved in many of the biological responses induced by wear particles, including osteoclast formation[21]. A series of
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procedures were used to remove adherent endotoxins, according to Liu et al.’s report[15]. In our research, obvious osteolysis was seen in Ti group compared with the sham group, whereas rifampin-treated groups significantly suppressed particle-induced osteolysis.
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These results were further supported by histological analysis of tissues stained with H&E and TRAP. We proposed that rifampin, a classic drug commonly used in clinics, should
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have great potential and value for the treatment of particle-induced periprosthesic osteolysis. Mature osteoclasts are characterized by the specific phenotypic markers expression, as well as multinucleated cells and the capability of bone resorption[10]. Our research demonstrated that the inhibitory effect of rifampin on osteoclastogenesis involved inhibition of RANKL-induced mRNA-expression of TRAP, cathepsin K, c-Fos, V-ATPase a3, V-ATPase d2 and NFATc1. Bone resorption and osteoclasts formation
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assays revealed that the number and area of bone resorption pits observed in vitro were dramatically decreased after rifampin exposure (Figure. 5A), due to the inhibition of osteoclast formation in a dose-dependent manner. Moreover, this inhibitory effect was
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observed at low concentrations of rifampin (0.5-50 µg/ml), without cytotoxic effects.
RANKL signaling is essential for particle-induced osteoclastic bone resorption[22]. Once upon the RANKL signaling, it is mediated by cytoplasmic factors that activate at
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least five distinct signaling cascades downstream signaling pathways, including MAPKs (ERK, p38, and JNK)[10, 23, 24]. Stimulation of p38 results in the downstream
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activation of the transcriptional regulator mi/Mitf, which controls the expression of the genes encoding TRAP and cathepsin K[25]. Similar result was confirmed in our study. Rifampin has been used for the treatment of bacterial infections for many years. Clinically, rifampin has been found to possess immunomodulatory effects and suggested
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to be repositioned as a Toll-like receptor 4 (TLR4) inhibitor to attenuate allodynia in a preclinical model of neuropathic pain[26]. TLR4 is the principal cell surface receptor for the LPS component of antitoxin, playing key roles in activating a cascade of
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pro-inflammatory events in response to pathogens[27]. Previous studies have suggested that rifampin may inhibit lipopolysaccharide (LPS)-stimulated NF-κB, p38, JNK, and
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MAPK activation through inhibition of the TLR4 pathway[28, 29]. As shown in the in vitro experiment, rifampin inhibited osteoclastogenesis without affecting ERK and JNK. Therefore, RANKL-induced p38 and NF-κB signaling may be involved behind the effects of rifampin treatment on osteoclastogenesis. In conclusion, Rifampin has unexpected properties that inhibit osteoclast formation and function both in vitro and in vivo. Rifampin also reduced the RANKL-induced
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osteoclastic marker genes expression. Furthermore, rifampin attenuated RANKL-induced NF-κB and MAPKs (p38) activation. These results suggest that it may represent a promising agent for the treatment or prevention of osteoporosis, periprosthetic loosening
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after THA, and other bone destructive diseases. Acknowledgements
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This research was financially supported by the National Natural Science Foundation of China (no. 81672157).
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Reference
[1] P.E. Purdue, P. Koulouvaris, B.J. Nestor, T.P. Sculco, The central role of wear debris in periprosthetic osteolysis, HSS journal : the musculoskeletal journal of Hospital for Special Surgery, 2 (2006) 102-113.
[2] W.J. Maloney, R.L. Smith, T.P. Schmalzried, J. Chiba, D. Huene, H. Rubash, Isolation
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and characterization of wear particles generated in patients who have had failure of a hip arthroplasty without cement, The Journal of bone and joint surgery. American
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volume, 77 (1995) 1301-1310.
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Figure. 1. Rifampin prevented titanium particle-induced mouse calvarial osteolysis. (A) Scanning electron micrograph of Ti particles and (C) the high filed for the particles; (B) Representative micro-computed tomography (CT) three-dimensional reconstructed
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images from each group. (D) Bone volume against tissue volume (BV/TV), BMD and the percentage of total porosity of each sample was measured (*P < 0.05; **P < 0.01); (E) Hematoxylin and eosin and (F) TRAP staining were performed from at least three
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sections per group. The black arrows represent osteoclasts. Scale bar are 200µm.
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Figure. 2. Rifampin inhibited RANKL-induced osteoclastogenesis and bone resorption without cytotoxic effects. BMMs were treated with 50 ng/mL RANKL and various concentrations of rifampin, as indicated, for 24h (A), 48h (B) and 72 h (C). (D) After 72h indicated treatment, TRAP activity was measured by assessing optical density at 405 nm. (E) TRAP staining after induction with 50 ng/mL RANKL in the presence or absence of different concentrations of rifampin for 7days. Scale bar are 50µm. The area (F) and number (G) of TRAP-positive cells was counted. (J) 1 % toluidine blue and Mayer's
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hematoxylin staining showed the formation of a lot resorption in control group, rifampin treatment significantly reduced the number of resorption pits. Scale bar are 20µm. (H) Resorption pit areas were measured using Image J software. All experiments were
methods (*p < 0.05, **p < 0.01 versus the control).
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performed at least three times, and the significance was determined as indicated in
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Figure. 3. Rifampin suppressed RANKL-induced expression of osteoclast-specific genes. BMMs were cultured with 50 ng/mL RANKL with different rifampin concentrations, as
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indicated for 72h and osteoclast-specific gene expression (Cathepsin k, TRAP, VATPs-a3, VATPs-d2, c-fos, and NFATc1) was analyzed by real-time PCR, and results were normalized to the expression of GAPDH. All experiments were performed at least three times, and the significance was determined as indicated in methods (*p < 0.05, **p<0.01
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versus the control).
Figure. 4. Rifampin specifically impaired the RANKL-induced p38 and NFκB signalling. (A) BMMs were pretreated with or without 5µg/ml rifampin for 4 h followed by 50
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ng/mL RANKL as indicated. Cell lysates were then analyzed using Western blotting with specific antibodies against phospho-JNK, JNK, phosphor- P38, P38, phosphor-ERK,
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ERK, phospho-p65, P65 and GAPDH. Average ratio of phospho-p38 (B), phospho- p65 (C) relative to GAPDH. The bands’ intensity analysis was quantified by image J software. Each ratio was normalized to 0 min (**p < 0.01 versus the control).
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ACCEPTED MANUSCRIPT 1. Rifampin inhibited Ti-induced osteolysis and osteoclastogenesis in vivo. 2. rifampin suppressed osteoclast differentiation and bone resorption in a dose-dependent manner.
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3. rifampin significantly reduced the expression of osteoclast-specific markers,
factor of activated T cells (NFAT) c1.
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including TRAP, cathepsin K, V-ATPase d2, V-ATPase a3, c-Fos, and nuclear
4. RANKL-induced p38 and NF-κB signaling may be involved behind the effects of
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rifampin treatment on osteoclastogenesis