Isorhamnetin 3-O-neohesperidoside promotes the resorption of crown-covered bone during tooth eruption by osteoclastogenesis

Delayed resorption of crown-covered bone is a critical cause of delayed tooth eruption. Traditional herbal medicines may be good auxiliary treatments to promote the resorption of crown-covered bone. This study was carried out to analyse the effect of isorhamnetin 3-O-neohesperidoside on receptor activator of nuclear factor-kB ligand (RANKL)-induced osteoclastogenesis in vitro and resorption of the crown-covered bone of the lower first molars in mice in vivo. Isorhamnetin 3-O-neohesperidoside promoted osteoclastogenesis and the bone resorption of mouse bone marrow macrophages (BMMs) and upregulated mRNA expression of the osteoclast-specific genes cathepsin K (CTSK), vacuolar-type H + -ATPase d2(V-ATPase d2), tartrate resistant acid phosphatase (TRAP) and nuclear factor of activated T-cells cytoplasmic 1 (NFATc1). NFATc1, p38 and AKT signalling was obviously activated by isorhamnetin 3-O-neohesperidoside in osteoclastogenesis. Isorhamnetin 3-O-neohesperidoside aggravated resorption of crown-covered bone in vivo. In brief, isorhamnetin 3-O-neohesperidoside might be a candidate adjuvant therapy for delayed intraosseous eruption.


Isorhamnetin 3-O-neohesperidoside promoted RANKL-induced osteoclastogenesis, as shown by TRAP staining. Chemical structure of isorhamnetin 3-O-neohesperidoside was shown in
. Only a small number of OCs formed after 4 days of induction in the untreated group, but with increasing isorhamnetin 3-O-neohesperidoside concentrations, the number of OCs and OC area increased gradually (Fig. 2B). Isorhamnetin 3-O-neohesperidoside promoted osteoclastogenesis in a dose-dependent manner (Fig. 2C,D).

Isorhamnetin 3-O-neohesperidoside promoted bone resorption on Osteo Assay Plates.
In the control group, little clearing of the bone biomimetic synthetic surface was observed. However, the resorption area was dose-dependently increased following treatment with isorhamnetin 3-O-neohesperidoside (Fig. 3).
Isorhamnetin 3-O-neohesperidoside promoted the resorption of bone crown-covered resorption in vivo. After the mouse mandibles were separated, fresh crown coverage was collected from the lower first molars for western blotting analysis (Fig. 7A1,A2). Positive RANKL expression was observed in the crown coverage of dental follicle by immunofluorescence (Fig. 7B). More OCs were observed around the crown-covered bone in the groups treated with isorhamnetin 3-O-neohesperidoside (Fig. 7D1,D2) than in the left mandibular first molar, which served as a control (Fig. 7C1,C2,D3) at postnatal day 11. Western blotting showed that isorhamnetin 3-O-neohesperidoside upregulated RANKL protein expression in the crown coverage (Fig. 7E). Crown-covered bone in the isorhamnetin 3-O-neohesperidoside-treated groups was completely resorbed at postnatal day 13 (Fig. 7G1,G2), but some unabsorbed crown-covered bone and several TRAP-positive osteoclasts were still observed in the control groups (Fig. 7F1,F2). These results showed that isorhamnetin 3-O-neohesperidoside can increase osteoclasts and promote the resorption of crown-covered bone, which is an important stage in tooth eruption.

Discussion
Until now, there have been no reports discussing the effect of isorhamnetin 3-O-neohesperidoside on osteoclastogenesis. In this study, we found that isorhamnetin 3-O-neohesperidoside increased RANKL-induced osteoclastogenesis in a dose-dependent manner without cytotoxicity. Isorhamnetin 3-O-neohesperidoside strongly promoted osteoclast formation and function in vitro. The upregulated levels of the osteoclast-specific genes NFATc1, CTSK, V-ATPase d2, and TRAP shown by qPCR further demonstrated the effect of isorhamnetin 3-O-neohesperidoside in aggravating osteoclastogenesis.
We further investigated the molecular mechanisms by which RANKL-induced osteoclastogenesis is increased by isorhamnetin 3-O-neohesperidoside. NFATc1 is a master transcription factor that regulates osteoclastogenesis 21,22 ; NFATc1-deficient osteoclast precursor cells failed to differentiate into osteoclasts in response to RANKL stimulation, while NFATc1 caused precursor cells to undergo efficient osteoclast differentiation without RANKL signalling 21,23,24 . Western blotting showed that the expression of NFATc1 increased gradually from day 1 to day 5 after RANKL induction. However, in the isorhamnetin 3-o-neohesperidin-treated group, the expression of NFATc1 was further increased, indicating that isorhamnetin 3-O-neohesperidoside could upregulate the expression of NFATc1 and then promotes RANKL-induced osteoclastogenesis. Increased NFAT1 also activated the TRAP, CTSK, and VATPase-d2 gene promoters 21,25 , which was consistent with our qPCR results.
As RANKL also plays an important role in activating the downstream NF -κ B, p38, AKT and c-Jun N-terminal kinase (JNK) pathways 25,26 , we explored the effect of isorhamnetin 3-O-neohesperidoside on these osteoclast-related pathways downstream of RANKL. The phosphorylation levels of p38 and AKT but not P65 and JNK were enhanced by isorhamnetin 3-o-neohesperidin. In brief, isorhamnetin 3-O-neohesperidoside promoted RANKL-induced osteogenesis in a multitargeted manner, targeting the NFATc1, p38 and AKT pathways.
Tooth eruption can be divided into 5 stages: pre-eruptive movement, intra-osseous eruption, mucosal penetration, pre-occlusal eruption, and post-occlusal eruption 1 . The resorption of crown-covered bone is essential for the establishment of intraosseous eruption. Consistent with its pro-osteoclastogenic and pro-resorptive properties in vitro, isorhamnetin 3-O-neohesperidoside promoted osteoclast differentiation and crown-covered bone resorption in vivo. More TRAP-positive osteoclasts formed in the isorhamnetin 3-o-neohesperidin-treated www.nature.com/scientificreports www.nature.com/scientificreports/ groups. The resorption of crown-covered bone was faster in the isorhamnetin 3-o-neohesperidin-treated groups than that in the control groups.
Interestingly, western blotting showed that isorhamnetin 3-O-neohesperidoside upregulated RANKL protein expression in crown coverage of dental follicle. Osteoclastogenesis in the coronal alveolar bone, which is essential to create an eruption pathway, was reported to be mediated by RANKL signaling 2,27,28 . Mouse tooth germ development is suppressed by exogenous osteoprotegerin (OPG), an inhibitor of RANK-RANKL signalling that acts as a decoy receptor of RANKL. RANKL-deficient mice developed severe osteopetrosis as well as tooth eruption defects 29 . Immunofluorescence showed positive RANKL expressions in the crown coverage of dental follicles. In vitro results confirmed that Isorhamnetin 3-O-neohesperidoside could promoted RANKL-induced osteogenesis by the NFATc1, p38 and AKT pathway. Increased RANKL in the coronal dental follicle induced by isorhamnetin 3-o-neohesperid further promoted crown-covered bone resorption in vivo. The dental follicle is essential for tooth eruption 30,31 . Disturbance in the functions of dental follicles results in delayed tooth eruption in cleidocranial dysplasia (CCD) patients 32,33 . However, the regulatory mechanisms of dental follicles in tooth eruption are still unclear 34,35 . RANKL can be secreted by osteocytes 36,37 and dental follicle cells 38,39 . To elaborate the mechanisms by which RANKL expression in the coronal dental follicle is increased by isorhamnetin 3-O-neohesperid, RANKL-related signalling pathways and transcriptional factors in dental follicle cells and osteocytes are worthy of further study in future.
Taken together, these results demonstrate isorhamnetin 3-O-neohesperidoside promoted the RANKL-induced osteogenesis of BMMs by NFATc1, p38 and AKT pathways in vitro and aggravated crown-covered bone resorption in vivo. If tooth eruption delayed, active treatment is recommended 40    Bone resorption assay. Corning Osteo Assay plates (Corning, NY, USA) with a bone biomimetic synthetic surface were used. BMMs (2 × 10 4 cells/well) were cultured in complete α-MEM (10% FBS, 30 ng/ml M-CSF and 50 ng/mL RANKL) with isorhamnetin 3-O-neohesperidoside at a concentration gradient (0, 1, 5, 25 and 50 μM) for 9 days. The osteoclasts were then removed by incubation with 5% sodium hypochlorite for 5 min. The total resorption area was analysed using Image J software 25,42 .
Bovine bone slices in 96-well plates were used for an improved bone resorption assay. BMMs (2 × 10 4 cells/ well) were cultured in complete α-MEM (10% FBS, 30 ng/ml M-CSF and 50 ng/mL RANKL) with isorhamnetin 3-O-neohesperidoside at two concentrations (0 and 50 μM) for 9 days. The OCs were then removed by incubation with 5% sodium hypochlorite for 5 min. Resorption was visualized under a scanning electron microscope at 5.0 kV. Five viewing fields from each bone slice were randomly selected for further analysis. Resorption areas were quantified using ImageJ software, as reported previously 43 .
Immunofluorescence. Immunofluorescence was performed as previously described 16,18 . Polyclonal antibody against RANKL (dilution 1:500, Abcam, UK) was applied. The sections were incubated with rhodamine (TRI-TC)-conjugated goat anti-rabbit IgG (Sigma, USA) for 1 h at room temperature. Nuclei were stained with a DAPI solution (Sigma, USA) for 5 min. PBS was used as a control.
Statistical analysis. All data are expressed as the mean ± standard deviation. Student's t-tests, one-way analysis of variance and the Newman-Keuls test were conducted with GraphPad Prism 5 software. Differences with a p-value of less than 0.05 were considered to be statistically significant.