Nuciferine prevents bone loss by disrupting multinucleated osteoclast formation and promoting type H vessel formation
Chengchao Song1,2 | Jing Cao2,3 | Yongsheng Lei1,2 | Hui Chi1,2 | Pengyu Kong1 | Guanghua Chen1 | Tailong Yu1 | Jianan Li4 | Ravi Kumar Prajapati1 | Jingjun Xia1 | Jinglong Yan1

Bone homeostasis is orchestrated by osteoblasts and osteo- clasts, and coupling of these cell activities is very important for maintaining skeletal health. Recently, researchers have

identified a new type of vessel, termed type H vessels, which show strong expression of CD31 and endomucin (Emcn) in the endothelium.1-3 These vessels couple the opposing processes of bone absorption and formation.4 Although the relative abundance of these specific vessels is low, many osteoprogenitor cells are distributed around them.1 Some

Abbreviations: ATP6V0D2, ATPase H + Transporting V0 Subunit D2; CCK-8, cell counting kit-8; CM, conditioned media; DC-STAMP, DC specific transmembrane protein; Emcn, endomucin; HE, hematoxylin and eosin; M-CSF, macrophage-colony stimulating factor; NCF, nuciferine; PDGF-BB, platelet-derived growth factor-BB; RankL, receptor activator of nuclear factor κB ligand; Trap, tartrate-resistant acid phosphatase.

Studies have reported that type H vessels are induced by PDGF-BB secreted from preosteoclasts,5,6 and conditional knockout of PDGF-BB in tartrate-resistant acid phosphatase (Trap)+ cells significantly impaired the formation of type H vessels and bone generation.7 However, the number of type H vessels decreases with age, and the degree of this reduction is consistent with the severity of bone loss.8 Thus, reversing this decrease might ameliorate bone loss-associated diseases, such as osteoporosis. Although both mature osteoclasts and immature preosteoclasts are sources of PDGF-BB, the matu- ration of osteoclasts from Trap+ preosteoclasts decreases the secretion of PDGF-BB.5 We hypothesized that interruption of the maturation process may not only inhibit bone resorp- tion but also facilitate the formation of type H vessels and thus prevent osteoporosis.
Osteoclasts differentiate from monocytes/macrophages
after the stimulation of two important factors, RankL and M-CSF.9 Researchers have proven that many compounds can inhibit multinucleated osteoclast formation, but their ef- fects on type H vessels and Trap+ preosteoclasts are unclear. Therefore, exploration of the effects of these compounds on type H vessel formation is needed. Compounds that do not interfere with Trap+ preosteoclast formation but hinder the fusion process and then enhance the formation of type H vessels would be of interest. Nuciferine (NCF) is a bioactive compound that is derived from lotus leaves and shows broad pharmacological effects, such as anti-inflammatory and an- tioxidative activities10-12; this compound notably suppresses the NF-κB signaling pathway, which is very important in osteoclastogenesis.13-15 However, the effects of NCF on the formation of osteoclasts and type H vessels are unclear. In the present study, we investigated the effects of NCF on os- teoclasts and clarified whether this compound could promote PDGF-BB production and type H vessel formation in ova- riectomized mice.

2.1 | Reagents and animals
Nuciferine was purchased from Sigma-Aldrich (Burlington, MA, USA). Recombinant murine RankL and M-CSF were purchased from PeproTech (Rocky Hill, NC, USA). The MAPK family antibody sampler kit, phospho-MAPK family antibody sampler kit, IKBα antibody, p-IKBα an- tibody, p-IKKα/β antibody, and NFATc1 antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). p65, p-p65, PDGF-BB, and Emcn antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). C-Fos, Lamin B1, CD31, osteocalcin (OCN), IKKα, IKKβ, and β-actin antibodies and all secondary antibod- ies were purchased from Abcam (Cambridge, MA, USA).

Human umbilical vein endothelial cells (HUVECs) were obtained from the American Type Culture Collection (ATCC). Balb/c mice were obtained from the Central Animal Laboratory of the Second Affiliated Hospital of Harbin Medical University, and the animal experiments were approved by the ethics committee of Harbin Medical University.

2.2 | Osteoclast differentiation and Trap staining
Monocytes were collected from 5-week-old male Balb/c mice. The cells were flushed from the marrow space of the tibias and femora and cultured in α-MEM with 10% (v/v) FBS and 30 ng/mL M-CSF for 24 hours. Next, the unat- tached cells were collected and cultured in α-MEM with 10% (v/v) FBS and 30 ng/mL M-CSF for another 72 hours. Then, these attached cells were defined as bone marrow-derived macrophages (BMMs). To induce osteoclast differentiation, RankL (100 ng/mL) was added for another 120 hours. NCF was added at the indicated time. For Trap staining, the cells were treated according to the procedures of the Trap staining kit (Sigma, St. Louis, MO, USA).

2.3 | Cell viability test
Cell counting kit-8 (CCK-8) assays (Dojindo, Japan) were used to measure cell viability. BMMs were obtained as described previously and treated with different concentra- tions of NCF for 24, 48, and 72 hours. At the indicated times, the cells were treated according to the product description.

2.4 | Bone pit formation
Bone marrow-derived macrophages were incubated on Osteo Assay 24-well plates (Corning, Corning, NY, USA). The osteoclasts were induced as described previously. When the osteoclasts were mature, the medium was discarded, and 10% bleach solution was added. The bone pits were observed and captured with a microscope.

2.5 | Conditioned medium of osteoclasts and ELISA
Bone marrow-derived macrophages were differentiated into osteoclasts with or without NCF. After 5 days of induction, the culture supernatant was centrifuged to eliminate the cell debris and used as Conditioned medium (CM). HUVECs or

aortic rings were cultured in different CMs. According to their sources, the CMs were described as follows: the control group (CM was collected from BMMs, which were cultured with 30 ng/mL M-CSF), RankL group (CM was collected from BMMs, which were cultured with 30 ng/mL M-CSF and 100 ng/mL RankL), and RankL + NCF group (CM was collected from BMMs, which were cultured with 30 ng/mL M-CSF, 100 ng/mL RankL and 30 μM NCF). To illustrate the role of PDGF-BB, PDGF-BB neutralizing antibody was used when necessary, and IgG was used as a control. To ex- clude the direct effects of NCF on BMMs, we also cultured BMMs with M-CSF and NCF together, and the CM was de- scribed as the NCF group. The concentration of PDGF-BB in CM was determined by a commercial ELISA kit (R&D Systems, Minneapolis, MN, USA).

2.6 | Wound healing and transwell assays
Human umbilical vein endothelial cells were seeded on 6-well plates and cultured in endothelial cell medium (ECM) (1001, ScienCell Research Laboratories) until the single cell layer reached approximately 90% confluence. The cell monolayer was scratched with a sterile pipette tip and washed with PBS to remove cell debris and non-ad- herent cells. Then, the culture medium was replaced with a mixed medium containing 50% ECM and 50% CM. The scratch wound areas were recorded at 0 and 12 hours. For the transwell assay, HUVECs were loaded on the upper chamber of a 24-well transwell plate (Corning, Corning, NY, USA). The lower chambers were filled with mixed medium. Twenty-four hours later, the HUVECs on the upper surface of the filter were scratched. The migrated HUVECs were fixed in 4% paraformaldehyde and stained with 0.5% crystal violet.

2.7 | Tube formation and aortic ring sprouting assays
Human umbilical vein endothelial cells were seeded on Matrigel-coated 48-well plates and cultured in mixed me- dium. Six hours later, the HUVECs were stained with cal- cein-AM for 15 minutes at 37°C. The plates were observed and photographed with a fluorescence microscope.
Thoracic aortas were obtained from 6-week-old Sprague-Dawley rats. The connective tissues outside the aortas were discarded, and the aortas were cut into 2-mm- thick rings. These rings were plated on Matrigel-coated 48-well plates and cultured with mixed medium containing 50% H-DMEM containing 10% FBS and 50% CM. Five days later, the mesh structures were photographed and counted.

2.8 | Quantitative real-time PCR
Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA), and cDNA was synthesized from 2 µg of total RNA using a Transcriptor First Strand cDNA Synthesis Kit (Roche Diagnostics, Basel, Switzerland). mRNA expression was quantitatively detected by Quantitative real-time PCR (qRT-PCR) using a FastStart Universal SYBR Green Master Kit (Roche Diagnostics, Basel, Switzerland). The relative mRNA expression was normalized to β-actin expression and calculated by the 2–△△CT method. The primer sequences used were as follows:
Trap: forward, 5′-GCAACATCCCCTGGTATGTG-3′, and reverse, 5′-GCAAACGGTAGTAAGGGCTG-3′; MMP-9:
ward, 5′-CAGGATGGCTGCGGTGTTGAC-3′, and reverse, 5′-GCTGGTGGTGGTGGTGTAATATGG-3′; β-actin: for-

2.9 | Western blot analysis
The cell lysates were extracted using RIPA lysis buffer (Beyotime Biotechnology, Beijing, China) containing 1 mM PMSF (Beyotime Biotechnology, Beijing, China) and 1× protease inhibitor (Beyotime Biotechnology, Beijing, China) and centrifuged to discard the cell debris. Protein (30 µg) was run on a 10% SDS-PAGE gel and transferred to PVDF membranes. The membranes were blocked with 5% nonfat milk in TBST solution for 1 hour and incubated with pri- mary antibodies overnight at 4°C. Before incubation with the secondary antibodies, the membranes were washed in TBST solution three times.

2.10 | Animal groups and treatments
For analysis of the effects of NCF on osteoclastogenesis and type H vessel formation in vivo, 5-week-old Balb/c mice were grouped as follows: (i) the sham group (mice treated with a sham operation); (ii) the OVX group [mice underwent bilat- eral ovariectomy (OVX) and were treated with DMSO]; and
(iii) the NCF group (mice underwent bilateral OVX and were

treated with NCF). NCF (40 mg/kg) was intragastrically admin- istered to the mice in the NCF group every day until 2 months after the operation. The OVX group was treated with the same volume of DMSO. All mice were sacrificed after 2 months, and whole blood and bone marrow were collected for the detec- tion of PDGF-BB, OCN, and CTX-I with commercial ELISA kits (OCN, Novus Biologicals, CO, USA; CTX-I, Novus Biologicals, CO, USA). To assess bone formation, 0.1% calcein (w/v) was injected intraperitoneally 10 and 3 days before sac- rifice. Calcein double labeling was observed in undecalcified bone slices under a fluorescence microscope.

2.11 | Micro-CT analysis

a fluorescein-conjugated secondary antibody for 2 hours. Nuclei were stained with DAPI.

2.15 | Statistical analysis
All experiments were performed at least three times, and data are expressed as the mean ± standard deviation. The data were analyzed by unpaired Student’s t test or one-way ANOVA using SPSS 19.0 software (IBM). *P < .05, **P < .01, and ***P < .001 were considered statistically significant. 3 | RESULTS Femora were separated from mice, fixed in 4% paraformalde- 3.1 | +NCF increases the relative quantity hyde for 24 hours, and scanned by micro-CT (90 kV, 88 μA,of Trap preosteoclasts and inhibits osteoclast 10 μm) from the growth plate proximally to 2 mm. For com- parisons of the bone structures after different treatments, the bone parameters, including bone mineral density (BMD, mg/ cm3), trabecular bone volume/total volume (BV/TV, %), tra- becular thickness (Tb.Th, mm), trabecular separation (Tb.Sp, μm), and trabecular number (Tb.N, mm−1), were calculated. 2.12 | Hematoxylin and eosin staining and Trap staining Femora were decalcified in 10% EDTA solution for 21 days. The samples were cut into 5 μm tissue sections and stained with Hematoxylin and eosin (HE) and Trap solution. Osteoclast surface/bone surface (OC.S/BS, %) was calculated. 2.13 | Bone cryosection preparation Briefly, the fresh femora were fixed in 4% paraformalde- hyde for 4 hours and decalcified in 20% EDTA solution for 3-4 days. Before cryosection preparation, the femora were cryoprotected with 20% sucrose and 2% PVP solution and embedded in a mixture composed of 20% sucrose, 2% PVP solution, and 8% gelatin. After preservation in a refrigerator at −80°C overnight, the femora were cut at a thickness of 20 μm. 2.14 | Immunofluorescence staining assay For Immunofluorescence (IF) staining, the sections or cells were incubated in 0.3% Triton X-100 for 30 minutes and blocked with BSA solution. Then, the samples were incu- bated with the primary antibody at 4°C overnight. Finally, the samples were washed three times and incubated with maturation and activity in vitro To identify a noncytotoxic concentration of NCF, we per- formed a CCK-8 assay. The cell viability was determined at 24, 48, and 72 hours. The cell viability was not affected by NCF at concentrations below 30 μM (Figure 1A). In addition, NCF promoted BMM proliferation in a time-dependent man- ner. Therefore, a concentration of 30 μM was chosen in the following experiments. Trap staining and bone pit formation assays were per- formed to observe the effects of NCF on osteoclastogene- sis. The results showed that NCF decreased the number of multinucleated osteoclasts and the bone resorption area but increased the ratio of Trap+ preosteoclasts to multinucleated osteoclasts (Figure 1B-E). These findings indicated that NCF could inhibit the differentiation of Trap+ preosteoclasts into multinucleated osteoclasts. To elucidate the period in which NCF functioned, we added NCF at different times (Figure 1F). The Trap staining results indicated that NCF reduced the number of multinucleated osteoclasts and enhanced the ratio of Trap+ preosteoclasts to multinucleated osteoclasts when the cells were treated with NCF on days 0-1 and 1-2 (Figure 1G,H). However, the effects were significantly attenuated when NCF was added on days 2-3 and 3-4 (Figure 1G,H). These results suggested that NCF inhibited multinucleated osteoclast formation and bone resorption but increased the relative number of Trap+ preosteoclasts. 3.2 | NCF inhibits the mRNA expression and RankL-induced activity of c-Fos and NFATc1 During RankL-induced osteoclastogenesis, the expression of several genes was increased. To test the effects of NCF on these genes, we induced osteoclasts with or without NCF. FIGURE 1 NCF decreases the ratio of multinucleated osteoclasts to Trap+ preosteoclasts and inhibits bone resorption in vitro. A, CCK-8 analysis of BMMs cultured with or without different concentrations of NCF, *P < .05 vs the 0 μM nuciferine group at the indicated time. B, Trap staining of osteoclasts cultured with or without NCF, scale bar = 100 μm. C, Bone pit assay of osteoclasts cultured with or without NCF, scale bar = 100 μm. D, The number of multinucleated osteoclasts and the ratio of Trap+ preosteoclasts to multinucleated osteoclasts, *P < .05; **P < .01 vs the 0 μM NCF group. OC.N (osteoclast number), pOC.N (preosteoclast number). E, Quantification of the bone pit area, **P < .01 vs 0 μM NCF. F, Illustration explaining the addition time of NCF. G, Osteoclasts were induced and stained with Trap solution as in B, except that NCF was added at the indicated time. Scale bar = 100 μm. H, BMMs were exposed to NCF for different durations, and then, the number of multinucleated osteoclasts and the ratio of Trap+ preosteoclasts to multinucleated osteoclasts were calculated. ***P < .001 vs the 0 μM nuciferine group. The data are shown as the mean ± SD and were obtained from at least three repeated experiments RankL enhanced the expression of Trap, MMP-9, DC- STAMP, ATP6V0D2, c-Fos, and NFATc1, whereas NCF attenuated the RankL-mediated increases in these genes (Figure 2A). Then, we examined the protein levels of c-Fos and NFATc1. The Western blot (WB) results showed that the expression levels of c-Fos and NFATc1 were elevated after RankL stimulation, but the levels of both of these pro- teins were decreased by the addition of NCF (Figure 2B,C). To further demonstrate the effects of NCF on c-Fos, we per- formed IF staining. The results revealed that the relative fluo- rescence intensity of c-Fos in the nucleus was increased after 12 hours of stimulation with RankL but decreased after NCF treatment (Figure 2D,E). These results demonstrated that NCF suppressed the expression of osteoclastogenesis-related genes and proteins. 3.3 | NCF promotes the expression of PDGF-BB and angiogenesis in vitro As mentioned above, NCF increased the ratio of Trap+ pre- osteoclasts to multinucleated osteoclasts, but its effect on the production of PDGF-BB was unclear. Thus, we exam- ined the mRNA and protein levels of PDGF-BB. The results FIGURE 2 NCF inhibits the relative expression of osteoclastogenesis-associated genes and proteins. A, The relative mRNA expression levels of Trap, MMP-9, DC-STAMP, ATP6V0D2, c-FOS and NFATc1 were assessed by qRT-PCR n.s., P > .05; *P < .05; **P < .01; ***P < .001 vs the DMSO group at the indicated time. The mRNA expression in each group was normalized to that in the 0 time group. B, The c-Fos and NFATc1 protein levels were analyzed by WBs. C, Quantification of c-Fos and NFATc1 expression was performed. *P < .05; **P < .01; ***P < .001 vs the DMSO group at the indicated time. The relative protein level in each group was normalized to that in the 0 time group. D, IF staining was performed to observe the location of c-Fos (red). Nuclei were stained with DAPI (blue). Scale bar = 100 μm. E, Quantification of the relative c-Fos fluorescence intensity in the nuclei was analyzed. *P < .05; **P < .01; ***P < .001. The data are shown as the mean ± SD and were obtained from at least three repeated experiments showed that RankL-stimulated PDGF-BB expression and that NCF potentiated RankL-induced PDGF-BB expres- sion (Figure 3A,B). As PDGF-BB regulates type H vessels through paracrine action, we tested the PDGF-BB content in the cell culture supernatant. Consistent with the qRT-PCR and WB results, NCF promoted the secretion of PDGF-BB when combined with RankL (Figure 3C). To determine the role of PDGF-BB in the migration and angiogenesis of HUVECs, we cultured HUVECs with different CMs. Compared to that in the control group, HUVEC migra- tion was increased in the RankL group, and the effect was en- hanced when NCF was combined with RankL (Figure 4A,B). To confirm the role of PDGF-BB, PDGF-BB neutralizing an- tibody was added to the RankL + NCF group, and cell migra- tion was decreased significantly (Figure 4A,B). Similar results were observed in tube formation and aortic ring assays. There were more tube structures and meshes in the RankL group than in the control group, and the increase was larger in the FIGURE 3 NCF promotes the RankL-induced production of PDGF-BB in vitro. A, Quantitative analysis of PDGF-BB mRNA expression was performed by qRT-PCR. B, The protein level of PDGF-BB was analyzed by WBs. C, The concentration of PDGF-BB in CM was analyzed by ELISA. The data are shown as the mean ± SD and were obtained from at least three repeated experiments, **P < .01; ***P < .001 RankL + NCF group (Figure 4C,D). However, the increase in the RankL + NCF group was decreased when PDGF-BB neu- tralizing antibody was added (Figure 4C,D). To exclude a di- rect effect of NCF on BMMs, we cultured HUVECs and aortic rings with CM from the NCF group. Interestingly, compared to those in the control group, the cell migration of HUVECs and the meshes of aortic rings were decreased in the NCF group (Figures 4-7A,B,D). Although the tube structures formed by HUVECs were increased in the NCF group, the increase was not significant (Figure 4C). These results indicated that NCF could enhance RankL-stimulated PDGF-BB secretion and pro- mote angiogenesis-related activities. 3.4 | NCF suppresses RankL-induced MAPK and NF-κB signaling activities To elucidate the mechanisms underlying the effects of NCF on osteoclastogenesis and angiogenesis, protein levels were analyzed by WB at the indicated times. The results showed that NCF significantly inhibited the RankL-stimulated phos- phorylation of p38 and JNK at 15 and 30 minutes and sup- pressed the increased phosphorylation of ERK1/2 stimulated by RankL at 30 minutes (Figure 5A,B). Although the relative expression of p-ERK1/2 was not significantly different be- tween the RankL group and RankL + NCF group, the level of p-ERK1/2 in the RankL + NCF group was lower than that in the RankL group (Figure 5A,B). To confirm whether the NF-κB signaling pathway is involved in the inhibitory mechanism, we first checked the phosphorylation of IKK and IKBα. Our results showed that NCF repressed the RankL- induced phosphorylation of IKKα/β and inhibited the phos- phorylation and degradation of IKBα (Figure 5C,D). In the canonical NF-κB signaling pathway, the activation of p65 is controlled by its dormant complex with IKBα. In response to stimulation, IKBα is phosphorylated and degraded by ac- tivated IKK, and then p65 is released and translocated into the nucleus to initiate the transcription of downstream genes. As expected, RankL boosted the nuclear translocation and phosphorylation of p65, but NCF depressed the activation of p65 (Figure 5E,F). To confirm that the NF-κB signaling path- way is involved in the inhibitory mechanism, we performed p65 IF staining. IF staining showed that RankL treatment fa- cilitated NF-κB translocation from the cytosol to the nucleus (Figure 5G,H). However, when BMMs were cultured with RankL and NCF together, NF-κB was primarily located in the cytosol, which verified the NCF-mediated inhibition of NF-κB nuclear translocation (Figure 5G,H). These results showed that the MAPK and NF-κB signaling pathways par- ticipated in the inhibitory effects of NCF on RankL-induced osteoclastogenesis and angiogenesis. 3.5 | NCF administration promotes type H vessel formation and prevents OVX-induced bone loss in vivo To determine whether NCF could prevent osteoclast matura- tion and promote type H vessel formation in vivo, we utilized an OVX-induced bone destruction mouse model. As type H vessels are characterized by strong expression of CD31 and Emcn in the endothelium, CD31 and Emcn double IF stain- ing was performed. OVX decreased the quantity of type H vessels in the metaphysis of the femur, but NCF administra- tion enhanced the number of type H vessels in OVX mice (Figure 6A,B). Since type H vessels are surrounded by os- teoprogenitor cells, we performed osteocalcin IF staining to observe the influence of NCF on these cells. The results showed that NCF treatment partially reversed the decrease in osteocalcin+ cells in the OVX group (Figure 6C,D). Calcein double labeling also confirmed that the OVX mice treated with NCF had more bone formation than those not treated with NCF (Figure 6E,F). Then, we performed a micro-CT scan to verify changes in BMD in different groups. Images FIGURE 4 NCF potentiates the angiogenesis-related activities of Trap+ preosteoclasts. A and B, The mobility of HUVECs in different CMs was assessed by transwell and wound healing assays. Scale bar in D = 200 μm; scale bar in E = 100 μm; quantification of the number and area of migrated HUVECs was performed. C and D, The effects of different CMs on angiogenesis were assessed by tube formation and aortic ring sprouting assays. Scale bar = 100 μm; The total number of tube structures and meshes was calculated to analyze the effects on angiogenesis. The data are shown as the mean ± SD and were obtained from at least three repeated experiments. n.s., P > .05; *P < .05; **P < .01; ***P < .001 from the micro-CT scan clearly showed that OVX impaired the bone structure and that NCF partially relieved this bone destruction (Figure 6G). The values of BMD, BV/TV, Tb.N, and Tb.Th were significantly decreased in OVX mice, and NCF treatment attenuated these reductions (Figure 6H). Correspondingly, the value of Tb.Sp was significantly in- creased in the OVX group, and this increase was smaller in the NCF treatment group (Figure 6H). HE staining results also confirmed the therapeutic effects of NCF on OVX-induced bone loss (Figure 7A). To assess the inhibitory effects of NCF on osteoclasts in vivo, we stained the metaphysis of the femur with Trap solution. The Trap- stained sections showed that many osteoclasts accumulated in trabecular bone near the metaphysis in the OVX group, but this increase was suppressed in the NCF group (Figure 7B). In addition, the osteoclast surface/bone surface ratio was strongly decreased in the NCF group compared to that in the OVX group (Figure 7C). As PDGF-BB contributes to the formation of type H vessels, we detected the PDGF-BB con- centration in the bone marrow and peripheral blood. Notably, OVX decreased the concentration of PDGF-BB in the bone marrow and circulation, but NCF treatment reversed the de- crease in PDGF-BB in OVX mice (Figure 7D). To confirm the rate of bone formation and resorption, circulatory mark- ers were investigated. NCF treatment increased the serum level of OCN and decreased the serum level of CTX-I in OVX mice (Figure 7E). 4 | DISCUSSION Osteoporosis is a common skeletal disease that results in in- creased fractures and is an economic burden in aging popu- lations.16,17 Extensive efforts have been made to identify effective treatments to prevent the development of osteopo- rosis. Because the relative overactivation of osteoclasts plays FIGURE 5 NCF inhibits the MAPK and NF-κB signaling pathways. A and B, The protein levels of the MAPK family and phospho-MAPK family were analyzed and quantified by WBs. The value in each group was normalized to that in the 0 time group. C and D, The levels of NF- κB upstream cascade proteins were analyzed and quantified. The value in each group was normalized to that in the 0 time group. E and F, The phosphorylation and nuclear translocation of p65 was analyzed and quantified in the cytosol and nucleus separately. The value in each group was normalized to that in the 0 time group. G and H, The location of NF-κB p-65 (green) was observed with IF, and the nuclei (blue) were stained with DAPI. Scale bar = 25 μm. The relative IF intensity of p65 in the nucleus was quantified. The data are shown as the mean ± SD and were obtained from at least three repeated experiments. n.s., P > .05, *P < .05, **P < .01 a major role in bone destruction,18 osteoclasts are believed to be a major therapeutic target.19,20 Although related drugs show excellent inhibitory effects on bone resorption, some of them can also disrupt ossification6 and PDGF-BB secre- tion.21 Recently, researchers have demonstrated that type H vessels may have major effects on bone development1 and are mostly regulated by PDGF-BB.5 Despite their limited quantity, many osteoprogenitor cells remain near type H vessels, and disruption of type H vessels can influence bone growth.1,22 Although complete inhibition of osteoclasts can reduce aging-related bone loss, this strategy ignores the posi- tive role of Trap+ preosteoclasts in the coupling of osteogen- esis and angiogenesis.5 Trap+ preosteoclasts can secrete high levels of PDGF-BB and promote type H vessel formation, and some studies have shown that PDGF-BB treatment or a genetic increase in Trap+ preosteoclasts can prevent OVX- induced osteoporosis.5 Thus, ideal anti-osteoporotic com- pounds that not only inhibit bone resorption but also couple type H vessels and osteogenesis should be explored.13 In the present study, we demonstrated that NCF is a candidate for osteoporotic treatment. NCF did not inhibit macrophage dif- ferentiation into Trap+ preosteoclasts, but it blocked Trap+ preosteoclast maturation and fusion into multinucleated os- teoclasts. The relatively increased Trap+ preosteoclasts pro- moted the secretion of PDGF-BB and the formation of type H vessels and prevented OVX-induced osteoporosis in vivo. Osteoclasts differentiate from macrophages after RankL stimulation. FIGURE 6 NCF promotes type H vessel formation and prevents OVX-induced bone loss. A, Type H vessels were observed by double IF of CD31 (green) and Emcn (red). Nuclei (blue) were stained with DAPI. GP: growth plate; BM: bone marrow. Scale bar = 100 μm. B, Quantification of type H vessels. C, Representative images of IF staining of OCNs (green) and nuclei with DAPI (blue), scale bar = 50 μm. BM, bone marrow; TB, trabecular bone. D, The number of OCN+ cell surfaces/bone surfaces was calculated. E, Representative images of calcein double labeling, scale bar = 20 μm; F, Quantification of mineral apposition rate (MAR) and bone formation rate/bone surface (BFR/BS). G, Representative micro-CT images of the femur, scale bar = 400 μm; H, Quantitative analysis of bone mineral density (BMD), bone volume/total volume (BV/TV), trabecular number (Tb.N), trabecular separation (Tb.sp), and trabecular thickness (Tb.Th). The data are shown as the mean ± SD and were obtained from six repeated experiments. *P < .05, **P < .01 differentiation into Trap+ preosteoclasts and is required for osteoclasts to mediate bone resorption.23 Therefore, inhibition of cell-cell fusion can inhibit the bone resorption of osteo- clasts but may not influence preosteoclast formation. A se- ries of processes are involved in osteoclast formation.9 Trap+ preosteoclasts form in the early stage, and multinucleated os- teoclasts form in the later stage. We found that NCF prevented the formation of multinucleated cells and bone resorption and increased the ratio of Trap+ preosteoclasts to multinucleated osteoclasts when it was added in the first 2 days after RankL stimulation. This indicated that NCF might have the ability to inhibit the differentiation of Trap+ preosteoclasts into multi- nucleated osteoclasts. The inhibitory effects were obvious in the early period and weak in the late period. Then, we tested the role of NCF in osteoclast function. The bone pit forma- tion assay confirmed that NCF inhibited the bone resorption of osteoclasts. FIGURE 7 NCF represses bone destruction and promotes PDGF-BB secretion in OVX mice. A, Representative hematoxylin and eosin images of femora, scale bar = 500 μm. B, Representative Trap staining images of femora, scale bar = 500 μm. C, Quantitative analysis of osteoclast surface (OC. S)/bone surface (BS). D, The levels of CTX-I and OCN in the serum were detected by ELISA. E, The levels of PDGF-BB in the serum and in bone marrow were detected by ELISA. The data are shown as the mean ± SD and were obtained from six repeated experiments. *P < .05, **P < .01, ***P < .001 the fusion process,23-25 and blockage of its expression can interrupt preosteoclast fusion.26 Therefore, the depression of DC-STAMP might be responsible for the inhibitory ef- fects of NCF on cell-cell fusion. In addition, NCF treatment decreased the osteoclast surface/bone surface ratio and pre- served bone destruction in OVX mice. These results indicate that NCF can inhibit multinucleated osteoclast formation and bone destruction in vitro and in vivo. Previous studies have shown that deficiency of Trap+ preosteoclasts significantly impaired bone formation, and the disruption of type H vessels was one of the main rea- sons for this result.5,7,27 Angiogenesis plays an important role in sustaining the normal bone structure, and osteogenesis is based on the nutrient supply of blood vessels and requires molecular stimulation from endothelial cells.1-3,28,29 The con- tribution of type H vessels to bone formation has been ver- ified,1-3,5,7,22,30,31 and its quantity is relevant to bone mass.8 Although we proved that NCF could inhibit osteoclast for- mation and increase the quantity of Trap+ preosteoclasts, the influence of NCF on the function of endothelial cells and the formation of type H vessels was not clear. In this study, we confirmed that RankL could increase PDGF-BB secretion, and the increase was potentiated by the addition of NCF. In addition, compared to other groups, in the RankL + NCF group, the migratory ability of HUVECs was enhanced, and tube structures of HUVECs and meshes of aortic rings were increased. However, these tube structures and meshes were decreased by the addition of a PDGF-BB neutralizing an- tibody. Consistent with the in vitro studies, NCF prevented bone destruction in OVX mice, promoted bone formation, and increased the concentration of PDGF-BB in the bone marrow and circulation. In addition, it also promoted the formation of type H vessels and the increased number of osteoprogenitor cells in the femora. These results were consistent with previ- ous reports that PDGF-BB secreted by Trap+ preosteoclasts was greater than that secreted by multinucleated osteoclasts and that PDGF-BB contributed to type H vessel formation.5 Then, we investigated the underlying mechanism by which NCF inhibited osteoclast formation and promoted an- giogenesis. RankL-induced osteoclast formation requires the involvement of intracellular factors to mediate osteoclast dif- ferentiation and function, mainly including the MAPK and NF-κB signaling pathways.32 Once RankL initiates osteoclas- togenesis, the phosphorylation of MAPK subfamilies, includ- ing p38, ERK1/2, and JNK, is activated,14 and NF-κB, which is located in the cytosol, translocates into the nucleus to ac- tivate downstream gene expression.33 ERK and JNK partic- ipate in terminal preosteoclast fusion.34,35 p38 also plays an important role in osteoclast differentiation and partially reg- ulates the NF-κB signaling pathway.36 Although the MAPK subfamilies and NF-κB have different effects, they all influ- ence the transcriptional activities of c-Fos and NFATc1.13 The importance of c-Fos has been proven in c-Fos-deficient mice with severe bone developmental obstacles resulting from functional defects in osteoclasts.37 In addition, a binding site of c-Fos was identified in the NFATc1 promoter.38 Therefore, c-Fos is very important for the function of NFATc1. Once NFATc1 is activated, it rapidly induces self-amplification to enhance osteoclastogenesis-associated genes, such as Trap, MMP-9, and DC-STAMP.15 In this study, we found that RankL stimulation promoted the phosphorylation of MAPK subfamilies, but NCF treatment suppressed phosphorylation. In addition, in the NF-κB signaling pathway, NCF inhibited IKKα/β activity and suppressed the phosphorylation and deg- radation of IKBα. In the canonical NF-κB signaling pathway, IKBα binds with p65 and suppresses its activity and nuclear translocation. Therefore, the suppression of IKBα indicated the limitation of NF-κB. To verify our hypothesis, we per- formed WB and IF staining, and all of the results reflected that NCF inhibited the RankL-induced activity and phos- phorylation of p65. To confirm the effects of NCF on c-Fos and NFATc1, we checked their gene and protein levels. The results showed that NCF inhibited the RankL-induced expres- sion of c-Fos and NFATc1, and IF staining also demonstrated that the c-Fos fluorescence intensity in the nucleus was de- creased in the NCF treatment group. Based on these findings, we concluded that the inhibition of the MAPK/NF-κB/c-Fos/ NFATc1 signaling axis may explain the inhibitory effects of NCF on osteoclastogenesis and angiogenesis. Although the novel effects of NCF on osteoclastogenesis and angiogenesis were demonstrated in this study, there are still some issues that need discussion. In the bone remodeling process, both bone resorption and bone formation play vital roles, and the interaction between osteoclasts and osteoblasts is quite complex.15 In this work, we discussed only the direct effects of NCF on osteoclasts, but we ignored the effects of NCF on osteoblasts. However, it is worth noting that NCF treatment could increase the number of osteocalcin+ cells in OVX mice, suggesting the possible positive effects of NCF on osteoblasts, which is worth in-depth study. Furthermore, compared to bisphosphonates, which almost completely re- verse bone loss and even increase BMD,39 the recovery of bone loss in OVX mice was not satisfactory after NCF treat- ment at 40 mg/kg. Although the prevention of osteoporosis was undesirable for NCF, the values of BV/TV, Tb.N, TB.Sp, Tb.Th, and BMD were partially rescued, and the number of osteoclasts on the bone surface was also partially decreased. These results suggest that an NCF of 40 mg/kg might not be adequate to prevent osteoporosis in OVX mice. Even for bisphosphonates, the effect in treating osteoporosis is dose dependent.40 In addition, there are some significant disad- vantages to using NCF orally, such as poor absorption, rapid metabolism and rapid systemic elimination.41 Poor bioavail- ability may also contribute to the unsatisfactory anti-osteo- porosis effects of NCF. Therefore, to achieve a potent effect of NCF in vivo, a suitable dose, an appropriate method of administration and excellent drug carriers should be further investigated. 5 | CONCLUSIONS In conclusion, NCF not only inhibits multinucleated osteo- clast formation but also enhances the formation of type H vessels. These dual effects facilitate the anti-osteoporotic ef- fects of NCF in OVX mice. Mechanistically, NCF inhibits the fusion of Trap+ preosteoclasts into multinucleated osteo- clasts and promotes the production of PDGF-BB from Trap+ preosteoclasts, and the inhibition of MAPK/NF-κB/c-Fos/ NFATc1 signaling pathways may be involved in this process. 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