Thymoquinone prevents RANKL-induced osteoclastogenesis activation and osteolysis in an in vivo model of inflammation by suppressing NF-KB and MAPK Signalling

Pharmacological Research 99 (2015) 63–73

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Thymoquinone prevents RANKL-induced osteoclastogenesis activation and osteolysis in an in vivo model of inflammation by suppressing NF-KB and MAPK Signalling Dinesh Thummuri a , Manish Kumar Jeengar a , Shweta Shrivastava a , Harishankar Nemani b , Ravindar Naik Ramavat b , Pradip Chaudhari c , V.G.M. Naidu a,∗ a

Department of Pharmacology & Toxicology, National Institute of Pharmaceutical Education & Research, Balanagar, Hyderabad, Telengana 500 037, India National Centre for Laboratory Animal Sciences, National Institute of Nutrition, Habsiguda, Hyderabad, Telengana 500 037, India c Comparative Oncology Program & Small Animal Imaging Facility, Advanced Center for Treatment, Research & Education in Cancer (ACTREC), Tata Memorial Centre, Kharghar, Navi Mumbai 410 210, India b

a r t i c l e

i n f o

Article history: Received 23 January 2015 Received in revised form 16 May 2015 Accepted 17 May 2015 Available online 28 May 2015 Keywords: Thymoquinone Osteoclastogenesis Osteolysis RANKL NF-KB Chemical compounds studied in this article: Thymoquinone (PubChem CID: 10281) Lipopolysaccharide (PubChem CID: 11970143) Tamoxifen (PubChem CID: 2733526) Dimethyl sulfoxide (PubChem CID: 2733526) Thiazolyl blue tetrazolium bromide (PubChem CID: 64965) Hydrogen peroxide (PubChem CID: 784) DC-FDA (PubChem CID: 104913) Ketamine (PubChem CID: 3821) Xylazine (PubChem CID: 5707)

a b s t r a c t Osteoclasts are multinuclear giant cells responsible for bone resorption in inflammatory bone diseases such as osteoporosis, rheumatoid arthritis and periodontitis. Because of deleterious side effects with currently available drugs the search continues for novel effective and safe therapies. Thymoquinone (TQ), the major bioactive component of Nigella sativa has been investigated for its anti-inflammatory, antioxidant and anticancer activities. However, its effects in osteoclastogenesis have not been reported. In the present study we show for the first time that TQ inhibits nuclear factor-KB ligand (RANKL) induced osteoclastogenesis in RAW 264.7 and primary bone marrow derived macrophages (BMMs) cells. RANKL induced osteoclastogenesis is associated with increased expression of multiple transcription factors via activation of NF-KB, MAPKs signalling and reactive oxygen species (ROS). Mechanistically TQ blocked the RANKL induced NF-KB activation by attenuating the phosphorylation of IkB kinase (IKK␣/␤). Interestingly, in RAW 264.7 cells TQ inhibited the RANKL induced phosphorylation of MAPKs and mRNA expression of osteoclastic specific genes such as TRAP, DC-STAMP, NFATc1 and c-Fos. In addition, TQ also decreased the RANKL stimulated ROS generation in macropahges (RAW 264.7) and H2 O2 induced ROS generation in osteoblasts (MC-3T3-E1). Consistent with in vitro results, TQ inhibited lipopolysaccharide (LPS) induced bone resorption by suppressing the osteoclastogenesis. Indeed, micro-CT analysis showed that bone mineral density (BMD) and bone architecture parameters were positively modulated by TQ. Taken together our data demonstrate that TQ has antiosteoclastogenic effect by inhibiting inflammation induced activation of MAPKs, NF-KB and ROS generation followed by suppressing the gene expression of c-Fos and NFATc1 in osteoclast precursors. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction

Abbreviations: TQ, thymoquinone; RANKL, receptor activator of nuclear factor kappa B ligand, receptor activated nuclear factor kappa B (NF-KB); TRAF6, TNF receptor-associated factor 6; LPS, lipopolysaccharide; DC-STAMP, dendritic cell-specific transmembrane protein; BMC, bone mineral content; BMD, bone mineral density; Tb.th., trabecular thickness; Tb.sp., trabecular space; TRAP, tartrateresistant acid phosphatase. ∗ Corresponding author at: Department of Pharmacology & Toxicology, NIPERHyderabad, A.P., India. Tel.: +91 40 23073740; fax: +91 40 23073751. E-mail addresses: [email protected], [email protected] (V.G.M. Naidu). http://dx.doi.org/10.1016/j.phrs.2015.05.006 1043-6618/© 2015 Elsevier Ltd. All rights reserved.

Bone is a central dynamic element in skeletal tissues that is constantly being remodelled to maintain healthy skeleton for efficient and lifelong execution of important skeletal functions in vertebrates. The strength and integrity of the bone are tightly regulated by the bone forming osteoblasts and bone resorpting osteoclasts. Increased bone resorption by osteoclasts is a manifestation of several lytic bone diseases such as osteoporosis, rheumatoid arthritis, periodontitis, Paget’s disease and malignant bone diseases [1]. Osteoclasts are multinucleated giant cells originated from haematopoietic progenitors through differentiation process mainly

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governed by two key cytokines: macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL). The Binding of RANKL to its receptor, RANK leads to recruitment of TNF receptor-associated factor 6 (TRAF6) to the cytoplasmic domain of RANK leading to activation of TRAF6. TRAF6 activation in turn triggers various downstream signalling pathways such as the nuclear factor KB (NF-KB) as well as three mitogen activated protein kinases including p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) and c-jun-N-terminal kinase (JNK). Recent reports have shown that higher concentration of reactive oxygen species (ROS) has deleterious effects but small amounts of ROS acts as secondary messengers and activates signalling pathways such as JNK and p38. RANKL stimulation in osteoclast precursors increases the ROS generation mediated through TRAF6, Rac1, and NADPH (nicotinamide adenine dinucleotide phosphate) oxidase 1 (Nox1) and further enhances the osteoclasts differentiation. Activation of MAPK pathway up regulates the expression of c-Fos. Increase in expression of c-Fos further up regulates the expression of NFATc1 [2,3]. NFATc1 fosters its transcriptional targets such as TRAP, Cathepsin-K, DC-STAMP [4,5]. Hence targeting NF-KB and MAPK signalling may be better alternative strategy for the treatment of bone destructive diseases by inhibiting the osteoclastogenesis. To date, the Food and Drug Administration approved treatment strategies include the anti-resorptive agents bisphosphonates and the bone-forming agent parathyroid hormone (use of PTH) [6]. Parathyroid hormone (PTH) therapy has shown increased bone formation and mineralization by up regulating the osteoblast differentiation but it’s use is limited by cost and long-term safety issues [7]. Therefore, the anti-resorptive agents bisphosphonates remain as therapeutic mainstay treatment to prevent bone loss by inhibiting the differentiation and enhancing the apoptosis of osteoclasts. Prolonged use of anti-resorptive therapy, however, is limited due to renal toxicity and jaw necrosis [8,9]. Hence, there is an emerging need for the development of better and alternate treatment strategies to maintain skeletal health. Thymoquinone (TQ), the main bioactive component of the black seed oil has been reported to have antioxidant, chemo preventive and anti-inflammatory effects. For instance, TQ suppressed the adjuvant induced arthritis and also decreased the periodontitis in rat periodontitis model [10,11]. However the effect of TQ on RANKL induced osteoclastogenesis has not been investigated. In light of the anabolic effect of TQ on osteoblasts [12], in the present study we showed that TQ suppressed RANKL induced osteoclastogenesis (in vitro) and LPS induced bone loss (in vivo) models. In vitro studies for molecular mechanisms established that TQ decreases RANKL induced osteoclastogenesis by inhibiting the NF-KB and MAPK signalling. From the in vivo data obtained by micro-CT analysis, we further confirmed that TQ significantly prevents LPS induced severe bone loss.

␤-actin was obtained from Santa Cruz biotechnology (Santa Cruz, CA, USA). The anti-mouse HRP conjugated secondary antibody and anti-rabbit HRP conjugated secondary antibody were purchased from Santa Cruz biotechnology (Santa Cruz, CA, USA).

2. Materials and methods

2.5. Assay of intracellular ROS

2.1. Materials and reagents

Intracellular ROS generation was measured by DCF-DA method given by Shrivastava et al. with slight modifications [15]. The RAW 264.7 and MC-3T3-E1 cells were seeded into a 96-blackwell plate and allowed to grow for 24 h. After pretreatment with the indicated concentrations of TQ (2.5 and 5 ␮M) for 6 h, RAW 264.7 and MC-3T3-E1 cells were stimulated with RANKL (50 ng/ml for 10 min) and H2 O2 (250 ␮M for 2 h) respectively. After treatment, cells were incubated with 10 ␮M of cell permeant reagent 2 ,7 dichlorofluorescein diacetate (DCF-DA) at 37 ◦ C for 15 min. The intracellular ROS mediated oxidation of DCF-DA to the fluorescent compound 2,7-dichlorofluorescein (DCF) was monitored by fluorescence microscopy and Multimode Plate Reader (Spectra Max

Recombinant human soluble RANKL was purchased from Invitrogen (CA, USA). LPS (serotype 026:B6), Thymoquinone (TQ), M-CSF and other reagents were purchased from Sigma–Aldrich (St. Louis, MO, USA). 20 mM stock solution of TQ was prepared in DMSO and the final concentration of DMSO in all the treatment groups is less than 0.2%. Primary antibodies to phospho-p38 MAPK, p38 MAPK, phospho-ERK, ERK, phosphoJNK, JNK, phospho-IKK␣/␤, IKK␣, phospho-NF-KB (ser 536), NF-KB, NFATc1, were purchased from cell signalling technology (Danvers, MA). Primary antibody to Cathepsin-K and

2.2. Cell lines RAW 264.7 (mouse macrophage cells), MC-3T3-E1 (murine preosteoblast) cells were obtained from American Type Culture Collection (Manassas, VA). RAW 264.7 cells were cultured in DMEM and MC-3T3-E1 cells were cultured in ␣-MEM. Medias were supplemented with 10% foetal bovine serum, penicillin and streptomycin. 2.3. Cell viability assay MTT assay was performed according to the method given by Naidu et al. [13]. Briefly, RAW 264.7 and BMM cells were seeded at a density of 1 × 103 cells per well in 96-well plate. After 24 h cells were treated with different concentrations of TQ and further cell viability was determined by MTT method. At the end of the experiment 10 ␮l of MTT (5 mg/ml) in 100 ␮l medium was added and incubated at 37 ◦ C for 4 h. Then the media with MTT was removed and the purple formazan crystals formed were dissolved in 200 ␮l of dimethyl sulphoxide and read at 570 nm using multidetection plate reader (Spectramax M4, Molecular devices, USA). 2.4. In vitro osteoclastogenesis assay RAW 264.7 cells were seeded in 24-well plates at a density of 10 × 103 cells per well and allowed to adhere overnight. The next day medium was replaced and then cells were treated with 100 ng/ml RANKL, TQ and both for 5 days. Osteoclastogenesis was confirmed by tartrate-resistant acid phosphatase (TRAP) staining with leucocyte acid phosphatase kit from sigma (St. Louis, MO, USA). The multinucleated cells (more than 3 nuclei) were counted using phage contrast microscope at 100× magnification. Bone marrow macrophage cells (BMMs) were prepared as described previously [14]. Briefly, cells extracted from the femur and tibiae of a 6-week-old C57/BL6 mouse were incubated in ␣-MEM medium containing 30 ng/mL M-CSF in a T-25 cm2 flask. While changing the medium, the cells were washed in order to deplete residual stromal cells. After reaching 90% confluence, cells were washed with PBS three times and trypsinised to harvest BMMs. Cells adhering to the bottom of the flask were considered as BMMs, these BMMs were plated in 24-well plates at a density of 8 × 105 cells per well in triplicate and incubated in a humidified incubator containing 5% CO2 at 37 ◦ C. Then cells were treated with various concentrations of TQ (2.5, 5, or 7.5 ␮M) in the presence of M-CSF (30 ng/mL) and RANKL (100 ng/ml). After 7 days of treatment, cells were stained for TRAP and scored as described above.

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M4, Molecular devices, USA) at excitation (498 nm) and emission (530 nm). 2.6. Assay of glutathione and catalase GSH levels were measured by an enzymatic method as described by Ellman with slight modifications [16]. Briefly, RAW 264.7 and MC-3T3-E1 cells seeded in 6 well plates were treated with TQ at 2.5 and 5 ␮M. After TQ pretreatment for 6 h, RAW 264.7 and MC3T3-E1 cells were stimulated with RANKL (50 ng/ml for 10 min) and H2 O2 (250 ␮M for 2 h) respectively. After stimulation cells were suspended in ice-cold extraction buffer (1% Triton X-100 and 0.6% sulfosalicylic acid). The extracts were centrifuged (10,000 × g) for 5 min at 4 ◦ C and supernatants used to estimate GSH levels. In a 96well plate, 0.03 ml of the above supernatant was added to 0.06 ml of 0.05 mM 5 ,5 -dithio-bis (2-nitrobenzoic acid) solution and 0.12 ml of 0.1 M potassium phosphate buffer (pH 8.4) to reach a final volume of 0.21 ml. In these conditions, DTNB is reduced by GSH and the thiolate anion production was measured at 412 nm. A standard curve was constructed with known GSH concentrations and results were expressed as GSH nM/mg protein. Catalase activity in cells was determined by measuring the rate of decomposition of H2 O2 at 240 nm. To 15 ␮l of sample, 2 ml of phosphate buffer (50 mM, pH 7) containing H2 O2 was added. The catalase activity was expressed as U/mg protein. 2.7. RT-PCR studies For real time PCR, RAW 264.7 cells were seeded at 5 × 105 cells per well into 6-well plates and pretreated with TQ for 6 h before RANKL stimulation (100 ng/ml) and cultured for 48 h. Total RNA was isolated using RNeasy Mini kit (Qiagen, Valencia, CA, USA). cDNA was prepared by reverse transcription with 2 ␮g of total RNA using Verso cDNA synthesis kit (Thermo Scientific). Primers express software (version 3.0.17) was employed for designing and synthesizing primers for RT-PCR. The sequence of primers are as follow; mouse NFATc1: forward, 5 -GTCTCTTTCCCCGACATCAT3 and reverse, 5 -TCTCCAAGTAACCGTGTAGC-3 ; mouse c-Fos: forward, 5 -CCTGGTGCTGGATTGTATCT-3 and reverse, 5 -CACCTCGACAATGCATGATC-3 ; mouse DC-STAMP: forward, 5 -CTGTGTTACTGAGGGCTCTTTG-3 and reverse, 5 -CAGAGAAGTCTTCCAGGATCTC-3 ; mouse TRAP: forward, 5 -GCCCCAAAGAAATGACCATC3 and reverse, 5 -CTGTAAGTAAGCCCCTTGGT-3 ; mouse ␤-actin: forward, 5 -AGACCTCTATGCCAACACAG-3 and reverse, 5 -ACTCATCGTACTCCTGCTTG-3 . RT-PCR was performed on the Light Cycler (ABi-7900HT fast light cycler, AB applied biosystem, CA, USA) using SYBR green master mix. The following reaction components were prepared to the indicated end concentration: 1 ␮l of primer mix, 5 ␮l of 2× SYBR green master mix (Invitrogen, Life Technologies), 2 ␮l of cDNA and nuclease free double distilled water were added to a final volume of 10 ␮l. All samples were run in triplicate. Realtime PCR was run with an initial denaturation step for 5 min at 95 ◦ C, followed by 4◦ cycles of 15 s at 94 ◦ C, 20 s at 48 ◦ C as annealing temperature and extension step for 25 s at 72 ◦ C. Relative gene expression was assessed using the comparative Ct (Ct ) method and normalized to ␤-actin.

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membranes (Millipore, Bedford, MA, USA). Non-specific interactions were blocked with 5% bovine serum albumin for 1 h and then membranes were probed with the indicated primary antibodies for 12 h at 4 ◦ C as indicated. Then membranes were incubated with the appropriate secondary antibodies conjugated with HRP. The antigen-antibody complex was visualized with an ECL detection kit (Amersham Bioscience). For subsequent antibody treatment, membranes were stripped in stripping buffer and re-probed with another antibody. The immune blots were quantified by densitometry scanning with NIH Image J software. 2.9. NF-KB nuclear translocation assay The translocation of NF-KB (p65) was assessed by an enzymatic immunoassay. According to the manufacturer’s instructions the nuclear extract samples were used for analysis of NF-KB DNAbinding using NF-KB p65/p50 EZ-TFA Transcription Factor Assay Colorimetric Kit (Merck Millipore, U.S.A.). Raw 264.7 cells were pretreated with TQ (2.5–15 ␮M) for 6 h and then stimulated with RANKL for 30 min. After treatment cytoplasmic and nuclear extracts were prepared by using NE-PER (Pierce). Briefly, nuclear extracts were incubated in the oligonucleotide-coated wells where the oligonucleotide sequence contains the NF-KB response element consensus binding site. After washing, samples were incubated by addition of the specific primary antibody directed against NF-KB (p65). A secondary antibody conjugated to horseradish peroxidase (HRP) was added to provide a sensitive colorimetric readout at 450 nm. 2.10. Animal studies C57/BL6 male mice (8 weeks old) were divided into three groups of seven mice each. Bone resorption was induced by injecting LPS (5 ␮g/g body weight) i.p. on day 1 and day 4. TQ was orally administered at a dose of 5 mg/kg body weight one day prior to LPS injection and continued for 8 days. At the end of the study, blood and femurs were collected for further analysis. Left femurs of all animals were scanned with a high-resolution micro-CT (Tri-Foil imaging, CA, USA) at a resolution of 21 ␮M using the following settings: X-ray voltage, 60 kV; electric current, 130 ␮A. Bone histomorphometric analysis, i.e. bone mineral content (BMC), bone mineral density (BMD), trabecular space (Tb.Sp) and trabecular thickness (Tb.Th) were performed using MicroView v. 2.0 Software. Bone tissues were fixed in 4% formaldehyde solution for 1 day at 4 ◦ C and then decalcified in 12% EDTA. Decalcified bones were paraffin embedded and sectioned. For histological examination sections were stained with haematoxylin and eosin (H&E) and TRAP. Images were viewed under phase contrast microscope (Model: Nikon, Japan) and photographs were taken with the help of a digital camera (Nikon, Inc. Japan) at 40× and 100× magnifications. 2.11. Cytokine analysis Serum cytokines such as TNF-␣ and IL-6 were analyzed by mouse Th1/Th2/Th17 Cytokine Kit (BD Biosciences, San Jose, CA) using BD FACS verse, as per manufacturer’s instructions.

2.8. Western blotting 2.12. Statistical analysis For western blotting, RAW 264.7 cells were pretreated with TQ for 6 h and then stimulated with RANKL (100 ng/ml) for 30 min. Whole cell extracts and nuclear extracts were prepared using RIPA buffer (Sigma) and NE-PER (Pierce) containing phosphate and protease inhibitor cocktails. Protein concentration was measured using Broadford protein assay. For western blotting proteins were resolved by SDS-PAGE then transferred to polyvinylidene difluoride

All results were expressed as mean ± SEM. The inter group variation between various groups was measured by one way analysis of variance (ANOVA) using the Graph Pad Prism, version 5.0 and the comparison between groups was conducted by “Bonferroni’s Multiple Comparison Test”. Results were considered statistically significant when *P < 0.05.

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Fig. 1. TQ inhibits RANKL-induced osteoclastogenesis in RAW 264.7 cells. (A) The structure of TQ. (B) RAW 264.7 cells (10 × 103 per well) were incubated with 100 ng/ml RANKL, 7.5 ␮M of TQ or both RANKL and TQ (2.5, 5 and 7.5 ␮M) for 5 days and then stained for TRAP. Magnification, 100× original. (C) Bar graphs showing quantification of multinucleated osteoclasts. Data expressed as mean ± SEM (n = 3). *P < 0.05 and ***P < 0.001 significant vs RANKL control. (D) RAW 264.7 cells (2 × 103 ) were incubated with medium only (control) or with 2.5–10 ␮M TQ for 2 days. Cell proliferation was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.

3. Results 3.1. TQ inhibits RANKL induced osteoclastogenesis In order to demonstrate the impact of TQ on RANKL induced osteoclastogenesis, we employed two standard

in vitro osteoclast differentiation models. First, RAW 264.7 cells were treated with RANKL. Second, primary BMMs were treated with M-CSF and RANKL. TRAP positive multinucleated cells were formed in 3–5 days for RAW 264.7 cells and 5–7 days for BMMs. TQ treatment significantly decreased

Fig. 2. TQ inhibits RANKL-induced osteoclastogenesis in BMMs. (A) BMM cells (8 × 105 per well) were incubated with 100 ng/ml RANKL, 7.5 ␮M of TQ or both RANKL and TQ (2.5, 5 and 7.5 ␮M) for 7 days and then stained for TRAP. Magnification, 100× original. (B) Bar showing quantification of multinucleated osteoclasts. Data expressed as mean ± SEM (n = 3). *P < 0.05 and ***P < 0.001 significant vs RANKL control. (C) BMM cells (2 × 103 ) were incubated with medium only (Control) or with 2.5–10 ␮M TQ for 2 days. Cell proliferation was assessed with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide method.

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Fig. 3. TQ inhibits RANKL-induced NF-KB activity. (A) Raw 264.7 osteoclast precursors were pretreated for 6 h with increasing doses of TQ (0–15 ␮M), and then stimulated with RANKL (100 ng/ml) for 30 min. Total protein extracts were analyzed by western-blotting using antibodies against phospho-NF-KB (ser536), NF-KB, ␤-actin, phosphoIKK␣/␤ (176/180) and IKK␣. (B) Graphical depiction of western blotting analysis of phospho-NF-KB and phospho-IKK␣/␤ (176/180). (C) Raw264.7 osteoclast precursors were pretreated for 6 h with increasing doses of TQ (0–15 ␮M), and then induced with RANKL (100 ng/ml) for 30 min nuclear extracts were examined for NF-KB nuclear translocation. Data expressed as mean ± SEM (n = 3). *P < 0.05, ***P < 0.001 significant vs RANKL control.

Fig. 4. TQ inhibits RANKL-induced activation of MAPK signalling. (A) Raw 264.7 osteoclast precursors were pretreated for 6 h with increasing doses of TQ (0–15 ␮M), and then stimulated with RANKL (100 ng/ml) for 30 min. Total protein extracts were analyzed by western-blotting for phospho-Erk1/2, Erk1/2, phospho-p38, p38, phospho-JNK and JNK proteins. (B) Graphical depiction of western blotting analysis. Data expressed as mean ± SEM (n = 3). ***P < 0.001 significant vs RANKL control.

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Fig. 5. TQ suppressed the RANKL-induced expression of osteoclast specific genes and NFATC1 and Cathepsin-K protein expression. (A) mRNA expression of TRAP, c-Fos, NFATc1 and DC-STAMP analyzed by real-time PCR. (B) Effect of TQ on protein expression of NFATc1, Cathepsin-K. (C) Graphical representation of fold changes in the NFATc1 and Cathepsin-K protein expression normalized to the expression of ␤-actin. Data expressed as mean ± SEM (n = 3). **P < 0.01, ***P < 0.001 significant vs RANKL control.

osteoclasts number in in vitro osteoclast differentiation models (Figs. 1C and 2B). To determine whether the inhibition of osteoclastogenesis by TQ is associated with cellular toxicity or not, we further analyzed the cell viability by MTT assay. From the results it was found that the antiosteoclastogenic effect of TQ was not associated with cellular toxicity (Fig. 1D and 2C).

3.2. TQ inhibits the RANKL induced phosphorylation of IKK˛/ˇ and p-p65 (ser536) IKK␣/␤ regulates the phosphorylation of IkB␣, which regulates the nuclear translocation of NF-KB. Various kinase signalling pathways regulate the phosphorylation of P-65 (ser536) which increases its transactivational potential; for instance, IKK␣/␤ have

Fig. 6. Effect of TQ on ROS generation, GSH and catalase levels in RAW264.7 cells and MC-3T3-E1 cells. (A) RANKL (50 ng/ml) stimulation for 10 min enhanced the ROS accumulation in RAW 264.7 cells and the effect reversed by 6 h pretreatment of TQ (2.5 ␮M and 5 ␮M). DCF fluorescence was measured with Nikon fluorescence microscope as described in “Assay of intracellular ROS.” Representative microscopic fields are shown at 200× magnification. (B) Stimulation with 250 ␮M of H2 O2 for 2 h enhanced the ROS accumulation in MC-3T3-E1 cells and it was reversed by 6 h pretreatment of TQ (2.5 ␮M and 5 ␮M). The production of ROS was assayed as in panel (A). (C) and (D) Effect of TQ on intracellular GSH and catalase in RANKL and H2 O2 stimulated RAW 264.7 and MC-3T3-E1 cells respectively. Data expressed as mean ± SEM (n = 3). +++ P < 0.01 significant vs control *P < 0.05, ***P < 0.001, significant vs RANKL control, @@@ P < 0.01 significant vs control, # P < 0.05, ### P < 0.001 significant vs H2 O2 control.

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Fig. 7. Administration of TQ prevented LPS induced bone loss in mice. (A) Study design. (B) Images of micro-CT analysis. (C) Graphical depiction of bone mineral density (BMD), bone mineral content (BMC), trabecular space (Tb.Sp.) and trabecular thickness (Tb.Th). Data expressed as mean ± SEM (n = 7 mice). *P < 0.05 significant vs LPS control, # P < 0.05 significant vs control.

been known to mediate the phosphorylation of P-65 (ser536) [17]. Both these processes increase the transcriptional activation of NFKB. To investigate the RANKL augmented NF-KB transcriptional activation, the phosphorylation of IKK␣/␤ and NF-KB (ser536) were studied. It was observed that phosphorylation of IKK␣/␤ and NF-KB was significantly increased upon RANKL stimulation and significantly suppressed by TQ treatment in a way that was concentration dependent (Fig. 3A). We also studied the RANKL mediated nuclear translocation of NF-KB by NF-KB p65/p50 EZ-TFA Transcription Factor Assay Colorimetric Kit. We found that nuclear translocation of NF-KB was significantly increased upon RANKL stimulation and significantly inhibited by TQ in a concentration dependent way (Fig. 3C). From this data it was confirmed that TQ inhibits osteoclastogenesis by inhibiting the RANKL induced NF-KB activation and nuclear translocation.

3.3. TQ inhibited the ERK/MAPK signalling To get further insights into the protective molecular mechanisms by which TQ inhibits osteoclastogenesis, we evaluated the phosphorylation of ERK1/2, P38 and JNK. Consistent with the previous reports, RANKL treatment significantly increased the phosphorylation of ERK1/2, P38 and JNK. Whereas, TQ treatment significantly decreased their phosphorylation in a concentration dependent way (Fig. 4A).

3.4. TQ suppressed RANKL-induced osteoclast-specific gene and protein expression RANKL induced osteoclastogenesis is associated with changes in expression of osteoclast specific genes. We further assessed the effect of TQ on osteoclastogenesis by evaluating the expression of osteoclast specific genes such as TRAP, NFATc1, c-Fos and DCSTAMP. From the results of quantitative real time PCR experiment it was found that RANKL significantly enhanced the expression of above mentioned osteoclast specific genes. Whereas TQ treatment significantly decreased the expression of RANKL induced osteoclastic specific genes (Fig. 5A). Furthermore, we determined the protein expression of NFATc1 and downstream effector Cathepsin-K. It was found that TQ significantly decreased the protein expression of RANKL activated NFATc1 and Cathepsin-K expression (Fig. 5B and C). qRT-PCR and western blotting results clearly showed that TQ inhibits osteoclastogenesis by inhibiting the osteoclast specific gene expression. 3.5. TQ prevents RANKL induced oxidative stress in RAW 264.7 and MC-3T3-E1 cells As oxidative stress plays an important role in bone resorption, we examined ROS production in RANKL stimulated RAW 264.7 and H2 O2 stimulated MC-3T3-E1 cells with the cell permeant oxidation-sensitive dye DCF-DA. TQ pretreatment significantly

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Fig. 8. Administration of TQ suppressed LPS induced histological damage. (A) Histological images of bone tissue stained with H & E at 40×. (B) Histological images of bone tissue stained with H & E at 100×.

inhibited the RANKL and H2 O2 induced ROS production (Fig. 6A and B). Further, we also investigated the effect of TQ on intracellular glutathione-redox balance in macrophages and osteoblasts. It was observed that TQ up regulated the GSH and catalase levels in RANKL stimulated RAW 264.7 and H2 O2 stimulated MC3T3-E1 cells (Fig. 6C and D). These results clearly show that TQ ameliorates the oxidative stress mediated osteoclastogenesis. 3.6. TQ prevented LPS-induced osteolysis After observing the inhibitory effect of TQ in in vitro osteoclastogenesis, we determined whether the protective effect could be observed in in vivo, by using a mouse model of LPS induced osteolysis. Mice challenged with LPS injection showed a marked reduction in the trabecular bone mass assessed by micro-CT analysis. Significant inhibition of bone mass loss was observed in mice treated with TQ at a dose of 5 mg/kg (Fig. 7B). Furthermore, quantitative analysis of trabecular bone parameters confirmed the LPS induced osteolysis with significant reduction in BMC, BMD and Tb.Th and increased Tb.Sp. In contrast bone mineral density and bone architecture parameters were positively modulated by TQ (Fig. 7C). Histological examination confirmed the protective effects of TQ on LPS-induced bone loss. As shown in Figs. 8A and 9B, LPS injection led to inflammatory bone erosion and increased numbers of TRAPpositive osteoclasts. However, inflammatory bone erosion was not observed in TQ treated mouse femur tissue sections, an observation consistent with the decreased number of TRAP-positive osteoclasts (Fig. 9A and B). It was also found that serum TNF-␣ and IL-6 levels were completely decreased in TQ treatment (Fig. 9C). These results imply that TQ prevents the inflammation induced bone resorption, consistently with the decreased number of TRAP-positive

osteoclasts and serum TNF-␣, and IL-6 levels. Taken together our data showed that TQ prevented the inflammation induced bone loss. 4. Discussion Bone is constantly being remodelled by the fine tuning of bone forming osteoblast cells and bone resorpting osteoclasts. Perturbation in this process leads to the development of bone loss and it is associated with over activation of an inflammatory signalling cascade. The current mainstay treatments for bone loss are suffering with side effects such as renal toxicity and osteonecrosis with bisphosphonates, and endometrial cancer risk with selective oestrogen receptor modulators [8,18]. F. Rossi et al. have shown that 17-␤-oestradiol inhibits osteoclast activity by increasing the cannabinoid CB2 receptor expression. So, natural or synthetic selective CB2 receptor agonists may help to prevent the postmenopausal bone loss in women [19]. Recently Cathepsin-k inhibitors came in to light due to the crucial role of this protein in bone resorption and in clinical trials they showed promising results. However, small increases in numbers of serious adverse effects, including stroke, atrial fibrillation, and atypical fractures, remain a concern [20]. Recently P38, ERK MAP kinases have been established as significant participants of inflammation mediated osteoclastogenesis. Taken together MAP kinases have been considered as potential targets for their key role in RANKL mediated osteoclast formation [21–23]. Support for this notion comes from other studies in which natural products namely androgophalide, apigenin and sauchinone used to prevent inflammation mediated bone loss by suppressing MAP kinase signalling [14,24,25]. In this regard, natural compounds endowed with positive effects on bone health gained more importance as an alternative medicine. TQ, the main

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Fig. 9. Administration of TQ suppressed LPS induced activation of osteoclasts and serum IL-6 and TNF-␣ level. (A) Images of TRAP staining at 100×. (B) Percentage of osteoclast surface per bone surface. (C) Serum cytokine analysis of IL-6 and TNF-␣. Data expressed as mean ± SEM (n = 7 mice). *P < 0.05, and **P < 0.01 significant vs LPS control, ## P < 0.01 and ### P < 0.001 significant vs control.

bioactive component of Nigella sativa has gained more importance due its wide pharmacological activities with proven clinical safety dose up to 2600 mg/day [26]. A randomized, double-blind, and placebo-controlled trial to check the efficacy of oral N. sativa (NS) seed extract supplement in patients with mild hypertension (HT) showed that daily use of NS seed extracts for 2 months had a blood pressure-lowering effect in patients with mild HT [27]. Another interesting study showed that N. sativa powder administered to the hypercholesterolemia patients for 2 months reduced the total cholesterol and triglycerides [28]. In the preclinical studies, TQ has been proven for its anti-inflammatory, antioxidant and antineoplastic remedy [29] by inhibiting the Akt, ERK, NF-KB and NF-KB regulated anti-apoptotic proteins expression [30,31]. TQ has also been shown to enhance the antitumor activity of tamoxifen in oestrogen negative breast cancer cells by inactivation of NF-KB and XIAP [32]. Numerous reports have shed light on TQ inhibitory activity on MAPK signalling, ROS formation and NF-KB. Hence, the current study was undertaken to investigate the effect of TQ in preventing bone osteolysis and whether this was due to inhibition of osteoclastogensis via inhibition of oxidative stress, MAPK signalling and inflammation. Both in vitro and in vivo results demonstrated that TQ exhibits anti-inflammatory activities. After stimulation of macrophages with RANKL, it binds to RANK receptor and causes trimerization of TRAF6 leading to activation of NF-KB and MAPKs (ERK, JNK and p38), which induces the expression of osteoclast specific genes [2,3,33]. NF-KB, the major mediator of RANKL induced osteoclastogenesis plays a vital role in osteoclast activation. The importance of NF-KB in osteoclast formation is shown by genetic studies. Genetic disruption of NF-KB subunits p50 and p52 and IKK␤ showed osteopetrosis caused by failure of osteoclast

formation [34,35]. Our study show that TQ inhibited the RANKL mediated activation of NF-KB by inhibiting phosphorylation of IKK␣/␤ (ser 176/180) without effecting the expression of IKK␣. Transcription factor assay results have also shown that TQ inhibited the RANKL induced NF-KB nuclear translocation. It was also observed that pretreatment with TQ to osteoclast precursors decreased the RANKL induced phosphorylation of three MAPKs such as ERK ½, p38 and JNK. Taken together our results clearly showed that TQ inhibits RANKL induced osteoclastogenesis by inhibiting the NF-KB and MAPK signalling. It has been well established that ROS are involved in osteoclast activation and bone loss mediated by increased expression of RANKL from stromal/osteoblast cells. Bai et al. have shown that oxidative stress increases RANKL secretion from osteoblasts and alters OPG/RANKL ratio [36]. Another interesting study by Lee et al. has shown that RANKL increases oxidative stress in macrophages and aggravate the bone loss [37]. Recently L. Gambari et al. have shown that sodium hydrosulphide (NaHS) a common H2 S donor dose dependently decreased the osteoclast formation by upregulating the Nrf2 protein expression and its nuclear translocation. Furthermore, Nrf2 silencing in human pre osteoclasts completely abolished NaHS mediated inhibition of osteoclastogenesis which further strengthens the implication of ROS in osteoclast activation [38]. Antioxidant treatments have been proved to be effective to rescue the bone loss induced by oxidative stress by decreasing the NF-KB activation in osteoclasts and RANKL expression in osteoblasts. Owing to the antioxidant property of TQ, next we addressed its role on oxidative stress induced osteoclastogenesis. TQ have multiple actions in addition to NF-KB inhibition, for instance TQ up regulates the expression of haeme oxygenase-1 via increased nuclear translocation

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of nuclear factor (NF)-erythroid2-(E2)-related factor-2 (Nrf2) and increased the antioxidant response element (ARE)-reporter gene activity [39]. In the present study we have evaluated the effects of TQ on antioxidant system in RANKL stimulated macrophages and H2 O2 stimulated osteoblasts. We found that TQ significantly decreased ROS generation and up regulated the antioxidant defence system such as GSH and catalase. These findings provide additional explanation for the therapeutic efficacy of TQ in osteoporosis models. The inhibitory effect of TQ on osteoclastogenesis was also corroborated by evaluating the RANKL-induced mRNA expression levels of osteoclast specific genes. Activation of the MAPK and NFKB signalling induces the formation of mature and functionally active osteoclasts by upregulating the osteoclastogenic transcription factors such as c-Fos and NFATc1. The c-Fos/c Jun/NFATc1 pathway plays crucial role in osteoclast development, and the lack of any of these three components arrests osteoclastogenesis [40]. The role of c-Fos in osteoclastogenesis has been confirmed by c-Fos knockout mouse, where it produced osteopetrosis due to impaired osteoclast formation [41]. NFATc1, a master transcription factor involved in the terminal differentiation of osteoclasts functions as downstream effector of RANKL [42]. Our results clearly show that TQ has an antiosteoclastogenic potential by reducing RANKL induced activation of c-Fos and NFATc1 in osteoclast precursors. NFATc1 regulates the expression of genes involved in the osteoclast differentiation and function such as TRAP, Cathepsin-K and DC-STAMP [4]. We found that mRNA levels of TRAP, DC-STAMP and protein expression of Cathepsin-K were significantly decreased by TQ. This clearly shows that TQ not only suppressed the NFATc1 but it also suppressed the downstream effectors such as TRAP, CathepsinK and DC-STAMP. LPS, an amphiphilic molecule in the outer membrane of gramnegative bacteria, is involved in bone resorption in inflammatory diseases by MAPK, NF-KB and TLR mediated signalling pathway. In response to LPS cells such as macrophages, lymphocytes, gingival fibroblasts, and osteoblasts, express TLR4 and produce PGE2 and proinflammatory cytokines such as TNF-␣ and IL-6. IL-6, TNF-␣ in turn enhance osteoclastogenesis by promoting RANKL production from osteoblast precursors (bone marrow stromal cells (BMSC)) and/or mature osteoblasts [43–45] and/or by reducing OPG production and/or by upregulating the receptor RANK on osteoclast precursors. Taken together the pro inflammatory cytokines appear to play key role in induction of osteoclast differentiation, recruitment, and survival. Apart from the increase in RANKL production from osteoblasts, TNF␣ also synergises with RANKL to amplify osteoclastogenesis and to intensify osteoclastic resorption by integrating with RANKL-induced signal transduction pathways [46]. In the present study we have thus relied on the LPS induced osteolysis model as in vivo model to assess the protective effect of TQ on bone loss. We found that an oral administration of TQ at a dose of 5 mg/kg suppressed the bone resorption in LPS induced bone osteolysis model. Micro-CT analysis clearly showed significant increases in BMD and BMC in the animals treated with TQ associated with decreased number of TRAP-positive osteoclasts, along with a significant reduction in the elevated levels of serum IL6 and TNF-␣ levels. These results clearly confirm that TQ prevents the bone loss by inhibiting the inflammation mediated osteoclastogenesis process. Based on our finding, a clinical trial assessing the therapeutic potential of TQ for the prevention of inflammation mediated bone osteolysis appears in order. 5. Conclusion We conclude that TQ prevents RANKL induced osteoclastogenesis in in vitro and LPS induced inflammatory bone loss in in vivo

mice model. We also demonstrated that the inhibitory effect of TQ occurs via inhibition of MAPKs and NF-KB and subsequent inhibition of c-FOS and NFATc1. Therefore, TQ could be developed as a novel treatment for bone lytic disorders. Conflict of interest Authors state no conflict of interest. Authors’ contributions Dinesh Thummuri: carried out all aspects of the study, experimental work, data analyses, graphics, and wrote the manuscript. V.G.M. Naidu: contributed to conception, design of the project, analyzing the data, and organized for collaborative research with Harishankar and Pradip Chaudhari discussed the data with the first author Dinesh Thummuri, and provided intellectual contributions. Manish kumar Jeengar and Shweta Shrivastava: involved in animal experiments, real-time PCR, and reviewing of the manuscript. Harishankar Nemani and Ravindar Naik Ramavat: provided facility for Histopathology and data analysis. Pradip Chaudhari: provided Tri-Foil imaging system for performing micro-CT and data analysis. Acknowledgments The authors are thankful to Department of Pharmaceuticals, Ministry of Chemicals and Fertilizers, Government of India and Project Director, NIPER Hyderabad for providing fellowship and research activity. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phrs.2015.05.006 References [1] T.C.A. Phan, J. Xu, M.H. Zheng, Interaction between osteoblast and osteoclast: impact in bone disease, Histol. Histopathol. 19 (2004) 1325–1344. [2] Z.H. Lee, H.-H. Kim, Signal transduction by receptor activator of nuclear factor kappa B in osteoclasts, Biochem. Biophys. Res. Commun. 305 (2003) 211–214. [3] W.J. Boyle, W.S. Simonet, D.L. Lacey, Osteoclast differentiation and activation, Nature 423 (2003) 337–342. [4] H. Takayanagi, The role of NFAT in osteoclast formation, Ann. N. Y. Acad. Sci. 1116 (2007) 227–237. [5] F. Ikeda, R. Nishimura, T. Matsubara, S. Tanaka, J.-I. Inoue, S.V. Reddy, et al., Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation, J. Clin. Investig. 114 (2004) 475–484. [6] J. Jeon, D. Puleo, Formulations for intermittent release of parathyroid hormone (1–34) and local enhancement of osteoblast activities, Pharm. Dev. Technol. 13 (2008) 505–512. [7] P. Morley, J.F. Whitfield, G.E. Willick, Parathyroid hormone an anabolic treatment for osteoporosis, Curr. Pharm. Des. 7 (2001) 671–687. [8] M.A. Perazella, G.S. Markowitz, Bisphosphonate nephrotoxicity, Kidney Int. 74 (2008) 1385–1393. [9] C. Dannemann, K.W. Graitz, M.O. Riener, R.A. Zwahlen, Jaw osteonecrosis related to bisphosphonate therapy: a severe secondary disorder, Bone 40 (2007) 828–834. [10] F. Vaillancourt, P. Silva, Q. Shi, H. Fahmi, J.C. Fernandes, M. Benderdour, Elucidation of molecular mechanisms underlying the protective effects of thymoquinone against rheumatoid arthritis, J. Cell. Biochem. 112 (2011) 107–117. [11] H. Ozdemir, M.I. Kara, K. Erciyas, H. Ozer, S. Ay, Preventive effects of thymoquinone in a rat periodontitis model: a morphometric and histopathological study, J. Periodontal Res. 47 (2012) 74–80. [12] A. Wirries, A.-K. Schubert, R. Zimmermann, S. Jabari, S. Ruchholtz, N. ElNajjar, Thymoquinone accelerates osteoblast differentiation and activates bone morphogenetic protein-2 and ERK pathway, Int. Immunopharm. 15 (2013) 381–386. [13] V. Naidu, U.M. Bandari, A.K. Giddam, K.R.D. Babu, J. Ding, K.S. Babu, et al., Apoptogenic activity of ethyl acetate extract of leaves of Memecylon edule on human gastric carcinoma cells via mitochondrial dependent pathway, Asian Pac. J. Trop. Med. 6 (2013) 337–345.

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