Oxidized Black Phosphorus Nanosheets as an Inorganic Antiresorptive Agent

Open AccessCCS ChemistryRESEARCH ARTICLE1 Apr 2021Oxidized Black Phosphorus Nanosheets as an Inorganic Antiresorptive Agent Yun Xu†, Jianbin Mo†, Wei Wei and Jing Zhao Yun Xu† State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 †Y. Xu and J. Mo contributed equally to this work.Google Scholar More articles by this author , Jianbin Mo† State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 †Y. Xu and J. Mo contributed equally to this work.Google Scholar More articles by this author , Wei Wei *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing 210093 Google Scholar More articles by this author and Jing Zhao *Corresponding authors: E-mail Address: [email protected] E-mail Address: [email protected] State Key Laboratory of Coordination Chemistry, Chemistry and Biomedicine Innovation Center (ChemBIC), School of Chemistry and Chemical Engineering, Nanjing University, Nanjing 210093 Google Scholar More articles by this author https://doi.org/10.31635/ccschem.020.202000212 SectionsSupplemental MaterialAboutAbstractPDF ToolsAdd to favoritesTrack Citations ShareFacebookTwitterLinked InEmail Black phosphorus (BP) is a newly discovered two-dimensional material that has promising applications from bioelectronics to biomedicine. However, facile oxidation of BP leads to changes in surface chemical composition and physical properties, often being referred to as the degradation process of BP. Degradation products of BP nanosheets, namely, oxidized BP nanosheets (oBPNSs), are routinely considered as by-products without many uses. Herein, we found that oBPNSs displayed excellent osteoclastogenesis inhibition effects without impairing cell viability. In contrast to the classic antiresorptive bisphosphonate drugs, oBPNSs showed a different mode of action by suppressing the maturation of osteoclasts. Bone resorption assays, osteoclast actin ring analysis, and tartrate-resistant acid phosphate activity assay results indicated that oBPNSs suppressed receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced osteoclastogenesis in a dose- and oxidation-dependent manner. Transcriptomic and proteomic analyses indicated that oBPNSs inhibited the activation of the NF-κB signaling pathway and phosphorylation of mitogen-activated protein kinase (MAPK) in differentiated osteoclasts, as confirmed by Western blot analysis. Our results suggest that oBPNSs might be potential antiresorptive nanomaterials to treat osteoporosis. Download figure Download PowerPoint Introduction Black phosphorus (BP), an inorganic analog of graphene (Gra), has attracted enormous attention due to its exciting lamellar structure possibilities.1 Considering its weak interlayer vander Waals forces, bulk orthorhombic BP can be exfoliated mechanically down to ultrathin BP nanosheets (BPNSs) based on solution separation methods.2,3 BPNSs have been used in a wide variety of biomedical applications, such as imaging and sensing, drug carriage, cell targeting, photothermal therapy, and medical diagnosis.4–7 For example, Tao et al.8 creatively conjugated polyethylene glycol-amine to BPNSs to construct an effective theranostic delivery platform with higher biocompatibility and physiological stability. Zeng et al.9 fabricated a novel multifunctional codelivery system of BPNSs, and they encapsulated BPNSs in a pH-sensitive polydopamine film to enhance its photothermal performance and stability in the physical environment for the drug delivery systems. From these studies, substantial strategies have been devoted to preventing BP from rapid degradation under ambient conditions, whereas the degradation of BP is inevitable. The instability of bare BP prompts a challenge to its potential biomedical applications based on oxidized BP. The structure of phosphorus oxide requires the dissociation of O2 or O2− and H2O to remove the connected P atoms from the BP.10,11 The oxidation process of the BPNSs is the insertion of oxygen atoms into P–P bonds, which are cleaved directly and readily by solvents and finally degraded.12,13 According to a previous report, the decomposition obtained from oxidative BPNSs (oBPNSs) in oxygenated water is variable and complicated.12,14,15 In the presence of oxidized H2O, POx species most likely to occur at the defects and plane edges.14–16 The partially oxidized BP has multiple phosphate groups on its surface. Inorganic pyrophosphate is a natural regulator of bone metabolism,17,18 and bisphosphonate (Bip) is a metabolically stable pyrophosphate-like substance, which is currently the most widely used drug for the treatment of bone resorption and calcification-related diseases such as osteoporosis.19–21 Multiple phosphate groups formed on the edge or surface site of oBPNSs could be reminiscent of Bip drugs. We hypothesized that oBPNSs might be able to accumulate in bone minerals and bind rapidly to the surface of the bone in a similar way as Bip drugs. Bone homeostasis is tightly regulated by coordinated resorption and formation; this process involves osteoclasts for resorbing pre-existing bone matrix and osteoblasts for rebuilding new bone.21,22 When both cell lineages fall out of balance, skeletal and systemic diseases, including myeloma bone disease and osteoporosis, might occur.23 Osteoporosis is a skeletal disorder that places millions of patients, especially older and postmenopausal women, at high risk.24 To tackle the imbalance of bone remodeling, enhancing the formation and activity of osteoblasts and efficiently inhibiting the function of osteoclasts are two main therapeutic strategies.21,25 As a conventional antiresorptive drug, Bip is useful for a broad spectrum of osteoporosis conditions and acts by reducing osteoclast viability and directly adsorbing on bone minerals. The side effects of Bip include osteonecrosis of the jaw (ONJ), severe suppression of bone turnover, and prolonged half-life.20,26 Therefore, the development of new agents or approaches with highly selective chemotherapeutic effects is highly desired. Chemically, the Bip is characterized by two phosphate groups, namely the P–C–P backbone, which exhibits a strong affinity for bone minerals (hydroxyapatite [HAP]), thereby promoting effective inhibition of bone metabolism in vitro and in vivo. Modification of one or both groups would reduce the affinity for bone minerals, so both phosphonate groups are required to maintain their medicinal effectiveness.19,27 Due to partial oxidation, multiple phosphate groups exist on the surface edge of BP. We speculate that oBPNSs might be an effective analog of Bip. Recently, Yuan et al.28 made pioneering progress in bioinspired extracellular vesicles embedded with BP quantum dots that displayed bone regeneration activity. The oBPNSs showed good biocompatibility, while self-produced inorganic phosphates were potentially involved in matrix mineralization by generating calcium phosphate. In our research, we found that oBPNSs might act as efficient analogs of Bip therapeutic agents, as shown in Scheme 1. The more oxidized BPNSs displayed an increased affinity for the bone matrix, restraining receptor activator of nuclear factor-κB-ligand (RANKL)-induced osteoclastogenesis and resorption. Numerous studies have demonstrated that nanomaterials, such as gold nanoparticles and lipid nanoparticles, are highly efficacious in treating bone-related diseases.29,30 Our results suggest that the effect of oBPNSs on osteoclasts might have immense implications in nanomedicine. Scheme 1 | Overview of oBPNSs and their function in osteoclast formation. Download figure Download PowerPoint Experimental Method Characterization of nanomaterials Morphology of BPNSs was determined by transmission electron microscopy (TEM; JEOL JEM-1200EX; Shanghai, China), atomic force microscope (AFM; Bruker MultiMode 8), and scanning electron microscopy (SEM; NOVA NANOSEM430; Beijing, China). The zeta potential of nanomaterials in phosphate buffer saline (PBS) was measured on a Nano-ZS instrument (Malvern Instruments Ltd., Shanghai, China). The size of the nanomaterials in PBS was detected with a dynamic light scattering (DLS) instrument (Brookhaven BI-200SM; New York, NY). The Raman spectrum of BPNSs was obtained from Horiba Jobin-Yvon LabRam HR VIS high-resolution confocal Raman microscope with the 633 nm laser as the excitation source (Horiba, Shanghai, China). The X-ray photoelectron spectroscopy (XPS) spectrum was analyzed with an X-ray photoelectron spectrometer (Thermo escalab 250Xi; Waltham, MA). The Super-Bradford Protein Assay Kit of Western blot analysis was purchased from Beyotime in Supporting Information. Bone resorption assay Bone-marrow-derived macrophages (BMMs) were seeded on bovine bone slides in 12-well plates and induced by 30 ng/mL M-CSF (PeProTech, Jiangsu Province, China), 50 ng/mL RANKL (PeProTech) and treated with different concentrates of BPNSs and Gra until osteoclasts formed. The culture plates were filled with 10% bleaching agent for 10 min to detach the osteoclasts. The remaining resorption pits were visualized under a phase microscope, and the regions resorted to the osteoclasts appearing white and gray. Tartrate-resistant acid phosphate activity assay BMMs were seeded in 24-well plates and incubated with complete medium containing essential cytokines. Cells were fixed by incubating with 4.2% formaldehyde for 15 min and washed three times with PBS. The osteoclasts were stained by a commercially available staining kit (Sigma, Shanghai, China), which were observed with more than three nuclei per cell. Tartrate-resistant acid phosphate (TRAP) staining was carried out as suggested by the instruction of the manufacturer. The nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) dye (KeyGEN, Nanjing, China) to make single osteoclasts more visible. The multinucleated cells were counted for quantitative assessment of the formation of osteoclasts and were viewed by a light microscope. The TRAP activity of the osteoclasts was measured employing the TRAP activity assay kit, following the manufacturer’s instruction. Fluorescence image BMMs were seeded in a 35 mm glass-bottom dish at appropriate density in the presence of a complete culture medium. After osteoclasts were induced, cells were fixed in 4% formaldehyde about 15 min. Subsequently, osteoclasts were permeabilized by 0.1% Triton X-100 and washed using PBS three times. The actin ring of osteoclasts was stained with phalloidin (ThermoFisher, Shanghai, China) for 30 min, and multiple nuclei were counterstained using DAPI (KeyGEN) for 10 min marked blue. After washing, the actin ring formation of osteoclasts was observed by fluorescence microscopy (ZEISS, Shanghai, China). Degradation analysis of BPNSs in vitro To detect the degradation of BPNSs, the N-Methyl pyrrolidone (NMP) solution of BPNSs was centrifuged, and then the nanoparticles were resuspended with deionized water. Supernatants of centrifuged BPNSs were taken at the indicated time points for further analysis. The phosphate and total phosphorus (TP) of supernatants were quantified by standard methods.31 Furthermore, the variation trend in Raman intensity of individual BPNSs sample was measured with time under ambient conditions, which performed to gain Raman signatures in oxidized states. The Raman spectral were collected on a confocal Raman spectroscope (Horiba, LabRAM HR Evolution) using a laser of 633nm with a spot size of about 3 μm. The power of the laser was limited to 0.5 mW to avoid the oxidation of black phosphorus caused by laser radiation. During the tests, the objective lens, acquisition time, accumulation time and grating was 50×, 2 s, 5 s and 600 lines mm–1, respectively. The binding affinity of oBPNS with HAP in vitro BiP-targeted bone diseases show a unique binding affinity toward inorganic bone mineral HAP. About 0.3 mg BPNSs were resuspended in deionized water for different time to obtain different oxidation levels of oBPNSs. Then the suspensions were mixed with 4 mg HAP (J&K Scientific, Beijing, China) at room temperature for 3 h. After centrifuged for 10 min at 2000 rpm, the P content in the precipitate and supernatant was determined using inductively coupled plasma mass spectrometry (ICP-MS). Alizarin red staining of osteoblasts Mouse calvaria osteoblastic MC3T3-E1 cells were cultured in 24-well plates at an appropriate density. After induced in the osteogenic medium for 21 days, osteoblast cells were fixed with 4.2% formaldehyde for 10–15 min and washed with PBS and stained for mineralization with Alizarin red (KeyGEN), rinsed with PBS, and photographed. Also, the chromogen was dissolved with 10% (w/v) hexadecyl pyridinium chloride monohydrate for 30 min and quantified by colorimetric analysis at OD562. Results and Discussion Morphology and structure of oBPNSs BPNSs were prepared through modified liquid exfoliation according to reported methods.2 The TEM and AFM, shown in Figures 1a and 1b, reveal that the morphology of the isolated BP corresponds to that of structures such as nanoflakes. The average thickness of the oBPNSs was ∼1.5 nm, measured by AFM topographic imaging. DLS was employed to analyze the zeta potential; the surface charge and the average hydrodynamic radius were –18 mV and 203 nm, respectively ( Supporting Information Figure S1). Furthermore, the composition of the oBPNSs was analyzed by Raman spectroscopy and XPS. Three characteristic Raman peaks at 358, 432, and 460 cm−1 were observed (Figure 1c), corresponding to the three vibrational modes of BP of A1g, B2g, and A g 1 , B 2 g and A g 2 . The XPS spectra shown in Figure 1d demonstrated the presence of oxygen, indicating local surface oxidation of BPNSs. To evaluate the binding affinity of BPNSs for bone matrix, HAP, the main compound of inorganic bone, was used as the model substrate in binding affinity assays. The apparent adsorbability of HAP by oBPNSs was observed by SEM (Figure 1e), and the magnified view of the materials bound to the HAP displayed increased detail (Figure 1f). In contrast, the binding affinity of Gra was not as efficient as revealed by parallel two-dimensional materials in the same experiment ( Supporting Information Figure S2). In addition, to estimate the dose-dependent binding, oBPNSs were prepared by centrifugation, dispersed in oxygenated water, and preserved at room temperature for different periods. After continuous stirring with HAP for several hours, the content of P in the supernatant and precipitate was determined and is shown in Figure 1g. Clearly, the bone-targeting nanomaterials showed evident absorption of HAP during the incubation test, like that of Bip. In addition, the data showed that the nonspecific adsorption was proportional to the extent of BPNS oxidation. Figure 1 | Morphology and structural characterization of oBPNSs. (a) TEM image of BPNSs. Scale bar: 200 nm. (b) AFM image of BPNSs. Right: height profiles along the colored lines. Scale bar: 200 nm. (c) Raman spectra of BPNSs. (d) XPS survey spectrum of BPNSs. (e) SEM image of oBPNS-bound hydroxyapatite. Scale bar: 800 nm. Magnified SEM images of the marked region are shown on the right (f). Scale bar: 400 nm. (g) HAP binding affinity of oBPNSs oxidized for 10 and 30 min and 1, 2, 4, and 8 h acquired by measuring the P content of the supernatant after several hours of mixing. Values are presented as the mean ± SD of triplicates. TEM, transmission electron microscopy; AFM, atomic force microscopy; XPS, X-ray photoelectron spectroscopy; SEM, scanning electron microscopy. Download figure Download PowerPoint oBPNSs inhibit RANKL-induced osteoclastogenesis To assess the biocompatibility of oBPNSs, the viability of osteoclast precursors (Raw264.7) and preosteoblast cell lines (MC3T3-E1) were incubated with oBPNSs at different concentrations was and detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The data showed that the oBPNSs did not induce cell death at concentrations as high as 12.5 µg/mL and 25 µg/mL ( Supporting Information Figure S3). Therefore, appropriate concentrations of oBPNSs were selected for subsequent experiments. The osteoclast differentiation assay was performed by adding oBPNSs or other drugs dispersed in complete induced culture medium supplemented with 30 ng/mL macrophage colony-stimulating factor (M-CSF) and 50 ng/mL RANKL, a stimulator of osteoclast maturation and function. Next, cellular uptake of oBPNSs was compared, which was determined by ICP-MS at a series of incubation time intervals in RANKL-treated osteoclasts and osteoblasts. As shown in Figure 2a, the uptake of oBPNS by osteoclasts was significantly higher than that of osteoblasts, especially after 4–6 h, indicating that oBPNSs might enter osteoclasts readily and influence cellular physiological or pathological events. The oBPNSs had no conspicuous effect on cellular uptake by osteoblasts during the entire incubation period. We tested the osteoclast differentiation by TRAP staining, wherein positive cells stain purple, and found that compared with Bip- and Gra-treated groups, oBPNSs-treated groups expectedly displayed dose-dependent precursor cell fusion inhibition and decreased TRAP-positive cell numbers (Figures 2b–2d and Supporting Information Figures S4 and S5). Furthermore, the mRNA expression of cathepsin K, nuclear factor of activated T cells 1 (NFATc1), TRAP, dendritic cell-specific transmembrane protein (DC-STAMP), vacuolated H+ triphosphate transporter-d2 (V-ATPase d2), and c-Fos, a participant in osteoclast-specific differentiation, were significantly downregulated by oBPNS inhibition, as assessed by real-time polymerase chain reaction (PCR; Figure 2e). Accordingly, Western blotting assays indicated that the oBPNS-treated group displayed a lower expression level of TRAP than the RANKL stimulation alone control group (Figures 2f and 2g). Then we performed bone resorption assays using an Osteo Assayplate (Corning® Osteo Assay Surface, Lowell, MA) to analyze the resorbed areas. The osteoclasts dissolved and resorbed the mineral matrix, turning the resorption pits into light-gray and white colors. As shown in Figure 2h, resorption pits in the oBPNS group were smaller than those in other groups ( Supporting Information Figures S12 and S13b). Furthermore, we confirmed that the nanomaterial suppressed the maturation of osteoclasts by performing fluorescence staining on the cytoskeleton of osteoclasts differentiated from Raw264.7 with or without oBPNS treatment. We found that actin rings of osteoclast cytoskeletal structures, which play a critical role in bone resorption, were reduced, compared with those in cells treated with RANKL alone ( Supporting Information Figure S7). In addition to osteoclasts derived from Raw264.7 cells, we also conducted a series of parallel experiments with BMM-derived osteoclasts and found that oBPNSs inhibited osteoclast formation and function ( Supporting Information Figures S6–S10). Finally, the effect of oBPNSs on osteoblast differentiation was investigated by Alizarin red staining (PromoCell, Heidelberg, Germany) to assess calcium deposition by cells cultured for 21 days. Images and quantitation of mineralization indicated that oBPNSs and Gra had no noticeable effect on osteogenesis, consistent with the results of the cell uptake experiments (Figures 2i and 2j and Supporting Information Figure S11). From these findings, we inferred that oBPNSs might be useful as antiresorptive agents for osteoporosis and that oBPNSs inhibit osteoclast formation without the perturbation of cell viability. Figure 2 | oBPNSs inhibit RANKL-induced osteoclastogenesis. (a) Cellular uptake of oBPNSs by osteoclasts and osteoblasts. Values are expressed as the mean ± SD of triplicate. (b) Representative images of TRAP staining. Osteoclasts are stained in purple. Scale bar 100 µm. (c) Statistical analysis of the osteoclast number. TRAP-positive cells with at least three nuclei were deemed osteoclasts. (d) The number of nuclei per osteoclast-like cell (n ≥ 3) was counted. (e) Osteoclast marker gene expression was measured in osteoclasts treated with or without oBPNSs by quantitative RT-PCR. (f) The TRAP expression level of osteoclasts treated with materials was analyzed by Western blot. (g) Quantitative analysis of Western blots. (h) Representative images of resorption pits on the Osteo Assay plate surface. Scale bar 100 µm. (i) Alizarin red staining to assess the mineralization of osteoblasts cultured with oBPNSs and Gra. Scale bar 100 µm. (j) Quantification of mineralization after osteogenic culture for 21 days. All values are expressed as the mean ± SD of triplicates. Statistical significance is assessed by Student’s t test; *P < 0.05, **P < 0.01 Download figure Download PowerPoint The oBPNSs with various oxidation levels inhibit osteoclastogenesis The oxidative of the structure of BPNSs generates the major final products PO23−, PO33−, and PO43−. Wan et al.14 reported that the oxidation and structural degradation of BPNSs are most likely to occur at BPNS defects and plane edges. To characterize the oxidation levels of oBPNSs, we monitored the concentration of POx3− ions obtained from the degradation products. We found that the concentration of phosphate in aqueous solution gradually increased as the oxidation time increased (Figure 3a). After dissolving in aqueous solution, and prolonged, 24 h of air exposure, the concentration of the oxidation products did not change significantly, which might be ascribed to the formation of an oxide layer passivates when the material and the solution had reached equilibrium. Raman spectra of the oBPNSs were recorded at a specific point in time. Compared with those of the pristine sample, the position and full width of the degraded sample at the half-maximal value showed slight changes, but the degradation of the sample led directly to quenching of the Raman signal intensity (Figure 3b), which was dependent on the oxidation time, as shown in Figure 3c. We explored the effects of different oBPNS oxidation levels on RANKL-induced osteoclast formation. High levels of oBPNS oxidation indeed inhibited osteoclastogenesis, as evidenced by a decrease in the number of TRAP-positive multinucleated cells after 2 days of treatment (Figure 3d), the effect of which is similar to that of high-dose Bip ( Supporting Information Figure S13a). Notably, oBPNSs treatment resulted in the decrease in the formation of osteoclasts with less nuclei and thin cytoplasmic septum compartments (Figure 3e and Supporting Information Figure S13b). In contrast, numerous large osteoclasts were generated in the control medium during the induction period. Next, the contribution of oBPNSs to osteoclast bone resorption was assessed using an Osteo Assay plate. As expected, treatment with highly oxidized oBPNSs reduced the overall area of the bone resorption pits, demonstrating that a high concentration of phosphate in BPNSs inhibited osteoclast differentiation in vitro. Figure 3 | The degree of osteoclastogenesis inhibition by BPNSs with various oxidation levels. (a) The concentration of POx3− measured at different oxidation times by molybdenum blue photometry. Values are expressed as the mean ± SD of triplicate. (b) Raman spectra of oBPNSs with varying levels of oxidation in deionized water after preparation under ambient light conditions (red arrow indicates time progression). (c) Time evolution of Ag2 mode intensity obtained from the analysis of the Raman spectra of the same sample. (d) TRAP staining to detect osteoclast formation by Raw264.7 cells treated with oBPNSs with different levels of oxidation for 2 days. Bip as a positive control. Osteoclasts are stained in purple. Scale bar 100 μm. (e) Representative images of resorption pits on the Osteo Assay plate surface. Scale bar 100 μm. Bip as a positive control. Download figure Download PowerPoint Multiomics analysis of the mechanism by which oBPNSs suppress osteoclastogenesis To clarify the potential mechanism of oBPNSs in a precise method, transcriptomic and proteomic analyses were performed. oBPNSs were found to suppress the expression of 956 RANKL-induced genes by 49.4% in three independent experiments (total influenced gene number: 1937; Figure 4a). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, oBPNSs had a significant impact on 31 pathways, including nine osteoclastogenesis-related pathways (Figure 4b), specifically, NF-κB, mitogen-activated protein kinase (MAPK), and forkhead box O (FoxO) signaling pathways, which are transcriptional regulatory signaling pathways closely associated with osteoclast differentiation. Then, we focused on understanding how oBPNSs affect osteoclasts at the protein level according to label-free quantitative proteomics. As shown in Figure 4c, quantitative proteomic screening results showed 80 proteins with significant expression differences, including 54 proteins with upregulated expression and 26 proteins with downregulated expression. Furthermore, 35% of the proteins with significantly altered expression were localized in the nucleus, indicating that oBPNSs mainly disturbed the transcription process ( Supporting Information Figure S14). By searching the KEGG pathway database, we found that oBPNSs could significantly interfere with three osteoclast-related pathways ( Supporting Information Figure S15). Due to posttranscriptional regulation, protein translation, and protein degradation, proteomics analysis was not reliably consistent with the transcriptomic analysis. Thus, we conducted a joint pathway annotation to better understand the effect of oBPNSs ( Supporting Information Figure S16). Interestingly, joint KEGG analysis of the proteomic and transcriptomic data further confirmed that oBPNSs could suppress osteoclastogenesis via different pathways, particularly the NF-κB signaling pathway (Figure 4d), reported previously to be a key signaling pathway in osteoclast differentiation, and regulated by MAPK signaling.32,33 Phosphorylation plays a crucial role in regulating the NF-κB and MAPK pathways, but this process could not be detected by the label-free quantitative proteomics. To this end, we focused on MAPK and NF-κB signaling molecules to reveal the detailed mechanism through Western blotting (Figure 4e and Supporting Information Figure S17). By searching MAPK members, we found that the phosphorylation of only p38 was suppressed in oBPNS-treated osteoclasts. The less p38 is phosphorylated, the less inhibitor of kappa B (I-κB) is degraded. Moreover, the existence of I-κB inhibited the expression of p65 further, implying that oBPNSs could suppress the classical NF-κB signaling pathway. Finally, after osteoclasts treatment with oBPNS, cell extracts were prepared, and the expression of specific alternative NF-κB signaling pathway genes of interest was analyzed by Western blotting using their respective antiphosphorylated antibodies (Figure 4e). We observed that after oBPNS-treatment, the phosphorylation of inhibitory kappa B kinase (IKK) and p100 was weakened in succession. Moreover, NFATc1, a pivotal member of the NFAT transcription super family, played an indispensable role in regulating osteoclastogenic and osteoclast-specific gene expression induced by RANKL. We found that treatment with oBPNSs down