Inhibition effects of a natural inhibitor on RANKL downstream cellular signalling cascades cross‐talking

Abstract Myricitrin is a natural occurring flavonoid glycoside that possesses effects on inhibiting nitric oxide (NO) transmission and preventing inflammatory reaction. Although previous study showed the myricitrin possesses antibone loss effects via reducing the expression of IL‐6 and partially suppressing reactive oxygen species (ROS) production. However, the effects of myricitrin on nuclear factor‐kappaB ligand (RANKL)‐stimulated osteoclastogenesis have not yet been further investigated. The current study was aimed to demonstrating the inhibitory effects of myricitrin on RANKL‐stimulated osteoclastogenesis and relevant mechanisms. We found myricitrin significantly suppressed osteoclastogenesis suggesting that it may acts on RANKL/RANK induced downstream signal cross cascading in osteoclast precursors. In that, our Western blotting results showed myricitrin significantly attenuated RNAKL/MAPKs (phosphorylation of p38, ERK, JNK) and AKT signal cascading. Complementing previous study, our results suggesting as a natural inhibitor, myricitrin possesses the potential therapeutic effects on inflammatory osteolysis.

reported myricitrin possesses effective antioxidative, 11 anti-inflammatory 12 and can protect a variety of cells from in vitro and in vivo injuries. 13,14 Otherwise, recently studies have showed myricitrin regulates various cellular signalling cascades including STAT3 15 and PI3K/Akt/eNOS. 16 Moreover, myricitrin demonstrated suppression effects on myocardial apoptosis relied on the ERK/p53-mediated mitochondrial apoptosis pathway. 17 Furthermore, the most recently study showed the protective effects of myricitrin against osteoporosis via reducing the expression of IL-6 and partially suppressing ROS production. 18 Nevertheless, as dairy natural occurring inhibitor, deeply exploring the novel effects of myricitrin on osteoclastogenesis still on the way. In our current study, we aimed to demonstrate the cellular effects of myricitrin on osteoclastogenesis and precise mechanisms underlying.

| OCs differentiation
All animal care and experimental protocols were approved by the "Medical Ethics Committee of Hong-Hui Hospital, Xi'an Jiaotong University School of Medicine" (No.1002016011) and performed strictly according to the "Guidelines of Jiaotong university institutes of Health for the care and use of laboratory animals." Bone marrow macrophage cells (BMMs) culture, osteoclast differentiation methods and myricitrin dose treating were following previous studies. 16,19,20 Briefly, by flushing the femurs and tibiae of 5-week-old ICR mice with a-minimum essential medium and suspended in a-MEM supplemented with 10% foetal bovine serum. Nonadherent cells were collected and cultured for 3 days in the presence of M-CSF (20 ng/mL).
Floating cells were discarded, and adherent cells on dish bottoms were classified as BMMs. BMMs were seeded at 3.5 9 10 4 cells/ well in a-MEM/10% FBS and were cultured in the presence of M-CSF (20 ng/mL) and RANKL (40 ng/mL) for 4 days in the presence or absence of myricitrin (0, 0.5, 5 or 20 lmol/L/mL). TRAP-positive multinucleated cells with greater than three nuclei were counted as OCs.

| Cell viability assays and cell apoptosis assay
Cell viability and apoptosis of BMMs after myricitrin (0, 0.5, 5 or

| Bone absorption assay and confocalmicroscopy immunofluorescence
Bone absorption assay and filamentous actin immunofluorescence staining method were following previously described. 22 Briefly, BMMs were seeded onto bovine bone slices with three replicates.
After culturing for 48 hours at 37°C, cells were stimulated with 40 ng/mL RANKL and 20 ng/mL M-CSF with or without myricitrin treatment (0, 0.5, 5 or 20 lmol/L/mL) until mature osteoclasts formed. Cells were removed by mechanical agitation and sonication.

| RT-PCR
Total RNA was isolated with QIAzol reagent (QIAGEN, Valencia, CA, USA) according to the manufacturer's instructions RNA (1 lg) was reverse transcribed using oligo dT primers (10 lg) and dNTPs (10 mmol/L). The mixture was incubated at 65°C for 5 minutes, and cDNA was produced by incubating at 42°C for 50 minutes with first strand buffer (50 mmol/L Tris-HCl, pH 8.3, 75 mmol/L KCl, 3 mmol/L MgCl 2 ), 100 mmol/L DTT, RNase inhibitor, and Superscript II reverse transcriptase (Invitrogen).The mouse GAPDH gene was used as internal control. The amplification parameters consisted of an initial denaturation step at 95°C for 5 minutes followed by 40 cycles of denaturation at 95°C for 1 minute, annealing at 60°C for 30 seconds and extension at 72°C for 1 minute. The specificity of the SYBR green assays was confirmed by melting-point analysis.
Expression data were calculated from the cycle threshold (Ct) value using the Ct. Primers employed for amplification are shown in Table 1.

| Western blotting
Protein lysates from myricitrin treated cells were prepared in a buffer containing 50 mmol/L with 50 lg/mL phenylmethylsulfonyl fluoride (Thermo Scientific, Waltham, MA, USA). Thirty micrograms of total cell proteins was mixed with loading buffer and separated on 10% SDS-PAGE gels, and the proteins in the gels were electro-transferred onto nitrocellulose membranes (GE, Marlborough, MA, USA).
Horseradish peroxidase (HRP) conjugated secondary antibodies were used to visualize bands under an ECL-based imaging system. The membranes were blocked with 5% nonfat milk in Tris-buffered saline contacting 0.1% Tween-20 (TBST) for 1 hour, before blotting with the primary antibodies for 2 hours at room temperature. The membranes were washed in TBST and incubated for 1 hour with HRPconjugated immunoglobulin antibodies. Signals were analysed in Ima-geJ and compared to controls after normalization.

| Statistical analysis
Experiments were conducted separately at least 3 times, and all data are presented as the mean AE standard deviation (SD). All data analysis was performed using SPSS software package ver. 14.0 (SPSS, Chicago, IL); one-way ANOVA was used for comparison among the different groups. Post hoc testing of differences between groups was performed by using Duncan's test when the ANOVA was significant. All results were considered to be significant at the 5% critical level (P < .05).

| Myricitrin inhibits TRAP-positive cells differentiation
Our cellular culture results showed, following M-CSF plus RANKL treatment, myricitrin significantly decreased the TRAP-positive multi nucleated OCs formation in the dose-dependent manner ( Figure 1B).
Otherwise, we performed XTT assay and flow cytometry to investigate the cytotoxicity and cellular apoptosis, respectively. Both

| Myricitrin inhibits osteoclastogenesis via the suppression of MAPKs and AKT phosphorylation
However, during the osteoclastogenesis process, c-fos/NFATc1 axis co-stimulated by various signaling cascades, such as MAPKs and AKT signalling. 23  During the past decade, most large pharmaceutical companies ceased natural products (compounds/agents/inhibitors) screening, which is initial step for drug discovery. 25,26 Despite the lack of effort by most of the large pharmaceutical companies, natural product research has still been active during the past decade. 27,28 Institutions mainly from China, Korea, India, Japan and United States keep engaging in the work of natural products study, for re-establish natural products as a major source for potential drug discovery. 19,26,[29][30][31] Given that only limited biological properties or functional activities have been worked out for each natural product, there still has a "long way to go" for fully identifying the biological activities of natural products. 33,34 Myricitrin is a naturally occurring flavonoid derived from bayberry bark and fruit, which has been reported to exhibit numerous biological activities such as antioxidative, anti-inflammatory and antinociceptive effects. 12,14,35 Despite the biological activities, myricitrin is relatively easy to extract and purify. 15 Documents studies showed myricitrin regulates several extracellular signalling pathways, such as STAT3, PI3K/Akt/eNOS 15  In that, as previous study reported, phosphorylation-ERK is the crucial regulator of AP-1 (c-Fos and c-JUN) activation in BMMs. 38 Phosphorylation of c-Fos is sustained by ERK signalling, impaired ERK activation will indirectly leading the attenuation of c-Fos. 39 p38 is particularly critical for the OCs early stage differentiation, as it promotes the activity of microphthalmia-associated transcription factor (MITF) and TRAP expression. 6 AKT signalling is crucial in OCs survival. 40 Previous studies showed the importance of the AKT/ NFATc1 signalling cascades in OCs differentiation. 41

ACKNOWLEDG EMENTS
The authors wish to thank Wanli Smith (University of Maryland) for overseeing the conduct of the study.

CONFLI CT OF INTEREST
The authors declare that they have no competing interests.

CONSENT FOR PUBLICATION
The manuscript is approved by all authors for publication.

AVAILABILITY OF DATA AND MATERIALS
All data and materials were included in the manuscript.