A Novel Inhibitor INF 39 Promotes Osteogenesis via Blocking the NLRP3/IL-1β Axis

Purpose A balance between osteoblasts and osteoclasts is essential to maintain skeletal integrity, regulating bone metabolism and bone remodeling. The nucleotide binding oligomerization domain, leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome is known as a cytosolic complex involved in producing proinflammatory cytokines consisting of interleukin- (IL-) 1β, which accelerates the occurrence of osteoporosis. Therefore, we aimed to investigate the effect of a novel NLRP3 inhibitor INF 39 on bone formation and bone resorption. Material and Methods. Cell viability of INF 39-treated osteoclasts and calvarial osteoblasts was tested by CCK-8 assays. Quantitative RT-PCR (qRT-PCR) was used to evaluate gene expression level during osteoblast and osteoclast formation. Western blot analysis was used to determine the effect of INF 39 on osteogenic and osteoclast-related proteins. Result It was shown that INF 39 promotes osteoblast differentiation via inhibiting NLRP3, thereby reducing the production of IL-1β dependent on NLRP3 in vitro. However, RANKL-induced osteoclast differentiation is not influenced by INF 39 in vitro. Conclusion Our study suggests that NLRP3 could be a new target and INF 39 may be a potential option for prevention and treatment of osteoporosis.


Introduction
Tight interplay between osteoblasts and osteoclasts regulates bone metabolism and bone remodeling, which is the process of removing older bone and replacing it with a new one [1]. A balance between the two cells is essential to maintain skeletal integrity, and any disturbance in this balance will result in the generation of bone pathology, the most common being postmenopausal osteoporosis in aging women leading to increased fracture incidence by impairment of bone strength [2]. There are considerable issues with the use of currently available drugs for the treatment of osteoporosis. Antiresorptive drugs, such as diphosphonate, are very efficient at reducing osteoclast numbers and activity, resulting in markedly decreased bone resorption, while they also profoundly inhibit bone formation [3,4]. Bone formation-acceleration drugs, such as teriparatide, can promote osteoblast proliferation and increase bone mass effectively, but they are coupled with increased bone resorption.
Studies have demonstrated that the proinflammatory cytokine interleukin-(IL-) 1β inhibits mineralization and osteogenesis [5,6] and decreased IL-1β attenuates bone loss and represses osteoclast formation and activity [7][8][9]. The nucleotide binding oligomerization domain, leucine-rich repeat and pyrin domain containing protein 3 (NLRP3) inflammasome is known as a cytosolic complex involved in producing proinflammatory cytokines consisting of IL-1β and IL-18. Considerable models driving activation of the NLRP3 inflammasome have been proposed, including K + efflux, ROS production, mitochondrial dysfunction, and lysosomal rupture [10]. The NLRP3 protein is assembled and activated in response to the process wherein Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) to activate the nuclear factor-κB (NF-κB) signaling pathway. Then, procaspase-1 is recruited and autocleaves itself by releasing active caspase-1 to cleave pro-IL-1β and pro-IL-18 to promote interleukin maturation and subsequent release, which sets up the inflammation [11][12][13].
There is increasing evidence implicating that NLRP3 inflammasome is a potential target for therapy because of its involvement in several diseases, such as osteoporosis, osteoarthritis, and rheumatoid arthritis [14][15][16][17]. INF 39 is a novel acrylate compound inhibiting NLRP3 ATPase activity. van der Schot and Bonvin [18] found that INF 39 was a nontoxic, irreversible NLRP3 inhibitor exerting a role in decreasing IL-1β release from macrophage in vitro. Meanwhile, the in vivo study confirmed that the compound was able to alleviate the effect of dinitrobenzenesulfonic acid-induced colitis in rats. It is also corroborated that direct inhibition of NLRP3 inflammasome with INF 39 is effective for the treatment of bowel inflammation and has more advantages than caspase-1 inhibition or IL-1β receptor blockade [19]. However, the role of INF 39 in bone homeostasis is unclear. Here, we aimed to investigate the effect of INF 39 on bone homeostasis and explore the underlying molecular mechanism.

Mice.
All experimental procedures were approved by the Ethics Review Committee for Animal Experimentation of Sir Run Run Shaw Hospital. The mice were from C57BL/6 background and maintained at the specific pathogen-free animal care facility and housed in a room at 24 ± 2°C, with 50 ± 5% humidity and a 12-hour light/ dark cycle, with lights on from 7:00 a.m. to 7:00 p.m. All mice were allowed access to drinking water and regular rodent chow ad libitum.
2.3. Cell Culture, Osteoblast, and Osteoclast Differentiation. Primary calvarial osteoblasts were obtained as described previously [4,20]. Calvariae were from 2-to 4-day-old C57BL/6 mice, which were digested overnight with collagenase type II in Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS while being incubated at 37°C. After centrifugation, the cells were collected and seeded into culture dishes in DMEM containing 10% FBS. After 4 days, cells were reseeded in 12-well dishes and cultured in osteogenic medium (1 mM β-glycerophosphate and 5 μM L-ascorbic acid 2-phosphate) with different concentrations of INF 39 (0, 25, 50, and 100 nM). After 7 days, ALP staining was performed, and the level of ALP in the cells and cell culture medium was determined according to the manufacturer's instructions. Following the treatment with osteogenic medium for 21 days using INF 39 at concentrations of 0, 25, 50, and 100 nM, cells were gently washed twice with PBS, fixed in 4% paraformaldehyde for 20 min, and stained with Alizarin Red S solution (ARS) (ScienCell, San Diego, CA, USA). Untreated cells were considered as a control. The images of extracellular matrix mineralization nodules were obtained using an inverted microscope with a digital camera. Quantification was performed according to the manufacturer's instructions (ScienCell, San Diego, CA, USA) [21].

Cytotoxicity
Assay. The cytotoxic effects of INF 39 on primary calvarial osteoblasts and BMMs were determined using the CCK-8 assay. BMMs were seeded in 96-well plates at a density of 2 × 10 4 cells per well in triplicate in the presence of 25 ng/mL M-CSF for 24 hours. Similarly, calvarial osteoblasts were seeded in 96-well plates at a density of 2 × 10 4 cells per well in triplicate with DMEM medium. Cells were then treated with different concentrations of INF 39 (0, 12.5, 25, 50, 100, and 200 nM) for 48 or 96 hours. Then, 10 μL of CCK-8 buffer was added to each well, and the plates were incubated for an additional 2 hours. Afterward, the absorbance was measured at 450 nm wavelength (650 nm reference) on an ELX800 absorbance microplate reader (Bio-Tek Instruments, Winooski, VT, USA).  3 BioMed Research International volume of 10 μL. RT-PCR was performed using an ABI Prism 7500 system (Applied Biosystems, Foster City, CA, USA) with SYBR Green QPCR Master Mix (Takara Bio, Otsu, Japan). The total volume (10 μL) of each PCR comprised 5 μL SYBR Green QPCR Master Mix, 2 μL doubledistilled water (ddH 2 O), 2 μL cDNA, and 1 μL (10 μM) each of forward and reverse primers. RT-PCR was performed at 95°C for 10 min (activation), followed by 45 cycles at 95°C for 10 s, 60°C for 20 s, and 72°C for 20 s (amplification) and a final extension at 72°C for 90 s. Specificity of amplification was verified by performing RT-PCR and analyzing the melting curves [23,24]. Gene expression assay was normalized to GAPDH. Mouse GAPDH, cathepsin K (CTSK), c-Fos, NFATc1, Runx2, ALP, COL1a, OCN, and NLRP3 primer sequences are presented in Supplemental Table S1. To determine the effect of INF 39 on Runx2 and ALP, primary calvarial osteoblasts were treated with 25, 50, or 100 nM INF 39 or without it, for 7 days. Total protein was extracted from the cultured cells using radioimmunoprecipitation assay (RIPA) lysis buffer (Sigma-Aldrich). Lysates were centrifuged at 12,000 g for 10 min, and the supernatants were collected. Proteins were separated by 10% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Bio-Rad, Hercules, CA, USA). The membranes were blocked in 5% nonfat dry milk in TBST at room temperature for 1 hour and incubated with the primary antibodies overnight at 4°C. Protein bands were visualized using LAS-4000 Science Imaging System (Fujifilm, Tokyo, Japan), and the obtained images were analyzed with ImageJ software (NIH, Bethesda, MD, USA) [25,26].
2.8. ELISA. The whole blood was collected by orbital venous of the mice at the end of the treatment period. Serum was prepared and stored at -80°C until used. ELISA was used to measure serum level of IL-1β (mouse IL-1β ELISA kit;  2.9. Statistical Analysis. Statistical analyses were performed using Prism 7 (GraphPad Software, Inc., San Diego, CA, USA). The results were expressed as mean ± SD. Statistical difference was assessed by Student's t test or one-way ANOVA. Values of P < 0:05 were regarded as statistically significant.

INF 39 Positively Regulates Osteoblast Activity via
Repressing NLRP3 In Vitro. To determine the potential role of INF 39 in osteoblasts, we assessed the proliferation and differentiation of primary calvarial osteoblasts harvested from neonatal mice. Cell viability of INF 39-treated calvarial osteoblasts was tested by CCK-8 assays at 48 and 96 hours. It was shown that no cytotoxicity was at the dose of 200 nM (Figure 1(b)). Next, we investigated the effect of INF 39 in osteoblast differentiation from preosteoblasts to mature osteoblasts. Calvarial osteoblasts treated with different concentrations of INF 39 (25, 50, and 100 nM) were exhibited to induce an increase of the ALP activity in the culture medium and cell (Figure 1(c)). In addition, we explored the effect of INF 39 on the deposition of minerals in the extracellular matrix by using Alizarin Red S staining. More calcified nodules were examined on day 21 following the treatment with 25, 50, and 100 nM INF 39, compared with those in the controls. Therefore, the mineralizing capacity was strengthened, which was consistent with the change of ALP activity in the prior experiment (Figure 1(e)). Furthermore, we compared the expression of ALP and the deposition of minerals in the extracellular matrix between controls and NLRP3 knockdown osteoblasts and found that both the ALP activity and the deposition of minerals in the The gray level of Runx2 and NLRP3 was quantified and normalized to GAPDH using ImageJ (n = 3). Data are shown as the mean ± SD. * P < 0:05, * * P < 0:01, and * * * P < 0:005, compared with the controls. 6 BioMed Research International extracellular matrix were significantly increased after NLRP3 knockdown (Figures 1(d) and 1(f)).
To further evaluate the effect of INF 39 on specific gene expressions of mouse calvarial osteoblasts, we first examined the mRNA expression of osteoblast-specific genes Runx2, COL1a, ALP, and OCN by qRT-PCR analysis (Figures 2(a)-2(d)). It was shown that the expression of Runx2, COL1a, and ALP mRNA in osteoblasts was remarkably increased after treatment with 25, 50, and 100 nM INF 39 compared with that in the controls. However, the mRNA    (Figure 2(e)). The results of the western blot analysis were consistent with those of the qRT-PCR analysis. Furthermore, we found that Runx2 mRNA significantly increased and OCN mRNA was slightly elevated after knockdown of NLRP3 using siRNA against mouse osteoblast NLRP3 (Figures 2(f) and 2(g)). Moreover, the expression of Runx2 was also upregulated after knockdown of NLRP3 compared with that of the controls (Figure 2(h)). These results indicate that INF 39 upregulates osteoblast-related gene expression via inhibiting NLRP3 in vitro.

INF 39 Promotes Osteogenesis via
Blocking the NLRP3/ IL-1β Axis In Vitro. To further clarify the underlying mechanisms, we tested the concentration of IL-1β in the osteoblast medium. We detected that the concentration of IL-1β was remarkably reduced in the NLRP3 knockdown group compared with the negative control (NC) group (Figure 3(a)). Afterward, we found that the expression of IL-1β was downregulated in NLRP3 knockdown osteoblasts compared with the NC osteoblasts (Figures 3(b) and 3(c)). Similarly, the level of IL-1β in osteoblasts was significantly decreased with various concentrations of INF 39 (0, 50, and 100 nM) for 7 d (Figures 3(d) and 3(e)). These results suggest that osteogenesis is increased via blocking the NLRP3/IL-1β axis in vitro.

Discussions
As the osteoporotic fracture brings a heavy burden on the family and society, prevention and therapy of osteoporosis become essential specifically for the aging population. At present, therapeutic methods for osteoporosis are limited, and the effect of the available drugs is not that desirable. Therefore, we focus on the research of the novel compound, INF 39, which potentially targets osteoporosis. We investigated the effect of INF 39 on bone formation through performing a series of experiments in vitro. Our results showed that INF 39 promoted osteoblast differentiation via inhibiting NLRP3, thereby reducing the production of IL-1β dependent on NLRP3 in vitro ( Figure 5). At the beginning, we performed ALP and ARS staining [27,28] to determine that INF 39 accelerated osteogenesis in both the early and late stages in a dose-dependent manner. The osteoblast-related genes, including ALP, COL1a, Runx2, and OCN [29,30], had a higher expression in the INF 39-treated groups than in the control group. Accordingly, the expression of specific proteins, such as ALP and Runx2, was elevated in the INF 39-treated groups than in the control group. All these observations collectively suggested that INF 39 had a positive effect on osteoblast formation. Subsequently, we investigated the underlying mechanism of the effect of INF 39 on osteogenesis. Primary calvarial osteoblasts treated with NLRP3 siRNA showed that the ALP expression and calcium deposits were significantly increased compared with those in the normal control group. Moreover, the key transcription factor of osteoblast formation Runx2 also exhibited an apparent rise in the NLPR3 knockdown group in contrast to the normal control group. The NLRP3 inflammasome is known as a major producer of cleaved IL-1 family cytokines, and activation of NLRP3 induces NLRP3-dependent IL-1β secretion, leading to inflammatory disease via the NF-κB pathway, leading to inflammatory disease via NF-κB and p38/MAPK pathways [31][32][33][34][35]. Interleukin-1β, a prototypic inflammatory cytokine, accelerates acute or chronic inflammation, resulting in the initiation and progress of osteopenia and osteoporosis [36]. Therefore, the expression of IL-1β in the osteoblasts was examined and was shown to decline with NLRP3 knockdown. To verify whether osteoblast formation was influenced by the variation of the expression of IL-1β, we tested the concentrations of IL-1β in the osteoblast medium, and it was shown that the level of IL-1β was lower in the osteoblast NLRP3 knockdown group than in the normal group.
Our study first investigated the effect of the novel compound INF 39 on bone metabolism and found that INF 39 plays an important role in increasing bone formation. Thus, this novel compound may have more potential advantages than other drugs already on the market. However, some limitations could not be avoided in our study. For instance, we have not investigated the effect of INF 39 on skeletal homeo-stasis in vivo. Other mechanisms of INF 39 regulating osteogenesis and osteoclastogenesis remain to be further explored in the future.

Conclusions
Our study reveals that INF 39 promotes osteoblast formation via blocking NLRP3 leading to downregulation of the expression of IL-1β in vitro. Our study suggests that NLRP3 could be a new target and INF 39 may be a potential option for prevention and treatment of osteoporosis.

Data Availability
The data used to support the findings of this study are included within the article.