2.1. BMMs and Culture System
BMMs were obtained and cultured according to former relative research 20-22. Primary BMMs were isolated from mice (five-week-old C57/BL6 male) femurs and tiviae. All process were under the supervision of Shanghai General Hospital Animal Center Committee of Animal Care and Use (2018371A218). All procedures were performed under the guidelines of the Ethical Conduct in the Care and Use of Nonhuman Animals in Research. To select primary BMMs, Cell were then cultured in complete α-MEM (10% Gibco FBS ) with 33.3 ng/mL M-CSF (R&D Systems, Inc.).
2.2. Cell Cytotoxicity Assay
Cytotoxicity of LY900009 (Selleck, Inc.) was determined by MTT method. The BMMs were seeded into 96-well plates(1 × 104 cells per well) in complete medium with different concentrations of LY900009 (0, 0.1, 0.2, 0.4, 0.8, 1.6, 3.2, 6.4, 12.8, and 25.6 μM). Following LY900009 treatment, the cells were incubated for different timepoint(24,48,96h). At the end of the process, CCK-8 solution (Dojindo Molecular Technologies, Inc) was used according to specification.
2.3. Osteoclast Formation and TRAP Staining Assay
The function of LY900009 on osteoclast formation was assessed. The BMMs were seeded into 96-well plates (1 × 104 cells per well) and stimulated by RANKL (100 ng/mL, R&D Systems, Inc. ), M-CSF (33.3 ng/mL) and different concentrations of LY900009 (0, 100, 200, and 400 nM). The culture medium was replaced every 48 h for 6 days. Afer the osteoclast was observed in control group, 4% paraformaldehyde was used for OC fixation. TRAP ( Sigma-aldrich; Merck, Inc.) staining solution was added into each of the wells at 37C for 1 h. TRAP+ cells with at least three nuclei were considered to be osteoclasts. Image J software (National Institutes of Health) was used for cell enumeration.
2.4. Bone Resorption Assay
M-CSF-dependent BMMs were incubated in six-well plates (20 × 105 cells/well). After 24 h, the cells were supplied with stimulated by RANKL (100 ng/mL), M-CSF (33.3 ng/mL) until OC formation was observed. The OCs (1 × 104 cells/well ) were then incubated on Corning Osteo Assay Surface plates (Corning, Inc.) and cultured with M-CSF (33.3 ng/mL), RANKL (100 ng/mL)and LY900009 (0, 100, 200, and 400 nM) for four days. A BioTek Cytation 3 Cell Imaging Reader (BioTek, Winooski, VT) and Image J software were used to photograph and analyze the total resorption pits.
2.5 Immunofluorescence analysis of the podosomal actin belt
OCs derived from BMMs were generated and processed. On days 5 - 7, pancake-like osteoclasts were observed in the RANKL-treated control group, fixed with 0.1% Triton X-100 (Sigma-Aldrich; Merck, Inc.), and permeabilized for 5 min. After blocking with 1% BSA-PBS for 1 h, the cytoskeletal actin structure was stained with rhodamine-conjugated phalloidin. The immunofluorescence images was obtained by using BioTek Cytation 3 Cell Imaging Reader. Image J software was used to analyze the size (diffusion area) and number of the dental actin bands.
2.6. Real-time PCR Analysis
Cell total RNA (2×106 cells) was extracted using the TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. The NanoDrop 2000 spectrophotometer (NanoDrop Technologies; Thermo Fisher Scientific, Inc.) was used to assess concentration of the total RNA. The following process was performed according to former research.16 The following mouse primer sets were used: mouse NFATc1: forward, 5’-TGCTCCTCCTCCTGCTGCTC-3’ and reverse, 5’-GCAGAAGGTGGAGGTGCAGC-3’; mouse C-fos: forward, 5’-CCAGTCAAGAGCATCAGCAA-3’ and reverse, 5’-AAGTAGTGCAGCCCGGAGTA-3’; mouse Cath-K: forward, 5’-CTTCCAATACGTGCAGCAGA-3’ and reverse, 5’-TCTTCAGGGCTTTCTCGTTC-3’; mouse Trap: forward, 5’-CAAAGAGATCGCCAGAACCG-3’ and reverse, 5’-GAGACGTTGCCAAGGTGATC-3’; mouse GAPDH: forward, 5’-CACCATGGGAGAAGGCCGGGG-3’ and reverse, 3’-GACGGACACATTGGGGGTAG-5’.
2.7. Western Blotting Analysis
The total cellular proteins (TCPs) were extracted at different time points. Following treatment with 400 nM LY900009 for 2 h, RANKL (100 ng/mL) was used to stimulate the cells for different periods of time (short time course). The membrane was blocked in 1% TBST (tri-buffered saline, Tween 20) in 5% skim milk at room temperature for 1 h, and subsequently reacted with primary antibodies NFATc1(cat no. 5861S; 1:1,000; Cell Signaling Technology, Inc.) ; Cleaved-Notch1(cat no. 4147S; 1:1,000; Cell Signaling Technology, Inc.) ; HES1(cat no.11988; 1:1,000; Cell Signaling Technology, Inc.) GAPDH(cat no.174S; 1:1,000; Cell Signaling Technology, Inc.); p-Akt (cat no.4060S; 1:1,000; Cell Signaling Technology, Inc.); Akt (cat no.4685S; 1:1,000; Cell Signaling Technology, Inc.); p-ERK (cat no.4370S; 1:1,000; Cell Signaling Technology, Inc.); Erk (cat no.9194S; 1:1,000; Cell Signaling Technology, Inc.) ; p65 (cat no.8242S; 1:1,000; Cell Signaling Technology, Inc.); p-p38 (cat no.4511; 1:1,000; Cell Signaling Technology, Inc.); p38 (cat no.8690; 1:1,000; Cell Signaling Technology, Inc.); p-p65 (cat no.3033; 1:1,000; Cell Signaling Technology, Inc.). After incubating the samples overnight at 4°C, the secondary antibody was incubated at room temperature for 1 h using Odyssey V3 .0 image scan (Li-COR. Inc., Lincoln, NE, USA) to observe antibody reactivity.
2.8. Mouse Model of LPS-Induced Calvarial Bone resorption
All process were was carried out in compliance with the ARRIVE guidelines and under the supervision of Shanghai General Hospital Animal Center Committee of Animal Care and Use. All procedures were performed under the guidelines of the Ethical Conduct in the Care and Use of Nonhuman Animals in Research. C57BL/6 male mice, aged 7 to 8 weeks, were randomly assigned to four groups: LPS (control), and LPS with low(400nM) or high(800nM) concentrations of LY900009. 1% pentobarbital sodium (10 mg/kg) was intraperitoneally injected to anaesthetize the mice. Mice were injected with 100 µl LPS(10 mg/kg), and with or without LY900009, respectively, for two weeks. During the experiment period, no significant adverse effects and toxical effects were observed. The mice showed no significant weight loss and lack of spirit, and no mice died during the experiment period. After the operation, euthanasia was performed using pentobarbital sodium(100 mg/kg)23. The knee joints were resected and fixed in 4% paraformaldehyde for 48h. After that, the specimens were prepared for micro-CT (Skyscan 1072; Skyscan, Inc.) and histological analyses.
2.9. Histological Staining and Histomorphometric Analysis
The 10% EDTA was used to decalcified PFA-fixed mice knees for two weeks and embedded in paraffin. Next, the samples were sliced into specific sections(4um thickness) and subjected to H&E and TRAP staining. Digital images were obtained using Axio ScopeA1 light microscope (ZEISS, Inc.). The osteoclasts amount were calculated by Image J software.
2.11. Statistical Analysis
All result were shown as the mean ± standard deviation (SD).The Student's t-test was used to assess the differences between control and therapeutic group. The results for the multiple group comparisons were analyzed using a Scheffe's test and one-way analysis of variance (ANOVA) with SPSS 22.0 software (SPSS, Inc.). Values were determined to be significant at *P < 0.05; **P < 0.01; and ***P < 0.001.