5.1 Reagents and antibodies

CK was solubilized in dimethyl sulfoxide (DMSO) and diluted in culture medium or PBS. Fetal bovine serum, Modified Eagle’s Medium of alpha, penicillin, and streptomycin were obtained from PeproTech (New Jersey, USA). Recombinant Murine sRANK Ligand (RANKL) was purchased from R&D (Minnesota, USA). NaOAc3H2O, Sodium Tartrate Dihydrate, Ethylene Glycol Monoethyl Ether, Naphthol AS-MX phosphate, and Fast Red Violet LB salt were obtained from Sigma (St. Louis, Missouri, USA). TRIzol reagent and SYBR-Green RT-PCR kit were provided by Takara (Osaka, Japan). Evo M-MLV One Step RT-PCR Kit was supplied by Accurate Biotechnology (Hunan) Co., Ltd, (Changsha, China)CCK-8, RIPA Lysis Buffer, and NcmECL Ultra kit were purchased from NCM Biotech (Suzhou, China). Bone Resorption Assay Kit (CSR-BRA-48KIT) was provided by Cosmo Bio (Tokyo, Japan). Primary antibodies anti-ALP, OPN, GAPDH and OCN were purchased by Santa Cruz Biotechnology (Texas, USA); Primary antibodies anti-MMP9, anti-p-NFκB p65, anti-p-IκB, anti-Nrf2, and anti-HO-1 were purchased from Bioss (Beijing, China); Anti-CTSK was purchased from Sigma (St. Louis, Missouri, USA); and DAPI were obtained from Sigma (St. Louis, Missouri, USA). Secondary antibodies HRP-conjugated Goat Anti-Rabbit IgG, Goat anti-Mouse IgG, Rabbit anti-Goat IgG (H + L) were purchased from Affinity (Jiangsu, China). Mouse Anti-Rabbit IgM/Cy3, Rabbit Anti-Mouse IgM/FITC were supplied from Bioss (Beijing, China). The RAW264.7 cells were obtained fromThermo Scientific (Massachusetts, USA).

5.2 Cytotoxicity assay

The cell viability of RAW 264.7 cells was measured by a CCK-8 kit. RAW 264.7 cells were cultured without or with CK (1, 5, 10, 20, 40 μM) for 24 h, 48 h, and 72 h. And CCK-8 reagent was incubated for 1 h at 37℃. The Optical density (OD) of the samples was detected at 450 nm.

5.3 RNA extraction and RT-PCR

After stimulation with 50 ng/ml RANKL for 3 days in 24-well plates, cells were collected. Total RNA was extracted by TRIzol. Next, Reverse Transcription-Polymerase Chain Reaction (RT-PCR) was performed using Evo M-MLV One Step RT-PCR Kit. The gene expression level was calculated by the 2–ΔCT or 2–ΔΔCT method, and GAPDH was used as the reference for standardization. The primer sequences were shown in Additional file 2: Table S1.

5.4 Bone resorption assay

Osteoclast activity was determined with the Bone Resorption Assay Kit (Cosmo Bio, CSR-BRA-48KIT), according to the manufacturer's instructions. Briefly, add Fluoresceinamine Labeled Sodium Chondroitin Sulfate (FACS) to each well of a 48-well plate and incubate at 37 °C for 1–2 h. After rinsing with PBS and medium, macrophage bone marrow-derived macrophages (BMMs) are added and incubated. The cultured cells were stimulated with 50 ng/ml RANKL and 50 ng/ml M-CSF to form osteoclasts. With or without CK for 6 days in culture, cells can be removed by treatment with 5% sodium hypochlorite, and after rinsing, photographed with a microscope.

5.5 ROS measurements

Intracellular ROS activity was examined using 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate (H2DCFDA) dye. RAW 264.7 cells were inoculated in 12-well plates. After cell attachment, the medium was exchanged for medium containing RANKL (50 ng/ml), with or without CK (10 μm) for 72 h. Cells were incubated with 5 µM staining solution for 30 min at room temperature avoid light and washed with PBS. H2DCFDA staining images were then taken by a fluorescence microscope (Olympus, IX73L, USA). Fluorescence intensity was measured with Image J software.

5.6 Protein extraction and Western blot analysis

Cells were washed twice with pre-cooling of PBS and then solubilized in RIPA lysis buffer. After freeze–thaw cycling at 4 °C for 0.5 h, the lysate was clarified by centrifugation at 4 °C for 15 min. Total protein from RAW 264.7 cells or BMMs was transferred to PVDF membrane by SDS-PAGE. The PVDF membranes were immersed in a containment solution for 1 h at 37 ℃. The membrane was then incubated with primary antibodies at 4 °C overnight. The primary antibodies included anti-GAPDH, anti-p-IκB, anti-p-p65, anti-MMP9, and anti-CTSK. The membrane was washed and incubated with a secondary antibody for 1 h. The immunoreactions were visualized with BeyoECL Moon.

5.7 In vitro osteoclastogenesis assay

RAW264.7 cells were seeded in 96-well plates at 5 × 103 cells/well. RAW264.7 cells were induced to osteoclasts with RANKL (50 ng/mL), without or with CK (1, 10 μM) for 5 days. After fixing the cells with 10% neutral formalin, the osteoclasts were washed with PBS. Tartrate-resistant acidic phosphatase (TRAP) staining solution 70 μl/well (96-well plate) or 150 μl/well (48-well plate) 10 -30 min at room temp or 37 °C, and wash with H2O and air dry. Multi-nucleated (nuclei ≥ 3) TRAP + cells were counted as osteoclasts (OCs).

5.8 Animal experiments

Eight-week-old Balb/C female mice (19 ± 4.45 g) were obtained from the Center of Experiment Animal of Guangzhou University of Chinese medicine. The study was approved by the Academic Committee on the Ethics of Animal Experiments of the Guangzhou University of Chinese medicine. The mice were randomly divided into the following three groups: Sham (n = 10), OVX + PBS (n = 10), OVX + CK (n = 10, 10 mg/kg). The duration of intraperitoneal injection was 4 and 8 weeks after OVX surgery, respectively. The right tibia and femur were decalcified for histology analysis, while the undecalcified samples on the left were analyzed by bone micro-architecture analysis.

5.9 Micro-CT analysis

The bone trabecular microstructure of the proximal cancellous bone of the right femur was determined using a Skyscan 1176 micro-CT scanner (Bruker micro-CT, Kontich, Belgium). Briefly, the designated scanned femoral region (5 μm/slice, 1 K) was located 1–5 mm from the growth plate-epiphysis junction. Bone mineral density (BMD), trabecular thickness (Tb. Th), trabecular number (Tb. N), bone volume fraction (BV/TV), trabecular separation (Tb. Sp), structural model index (SMI), and femoral 3D images were then obtained from the system software.

5.10 Histology

The tibias and femurs were fixated in 10% buffer formalin overnight, decalcified in 10% EDTA for 21 days, dehydrated, and then paraffin-embedded. Sections were cut into 5 μm thick and the samples were subjected for immunohistochemical (IHC), and TRAP staining. For IHC staining, sections were incubated with 10% BSA at 37 ℃ for 1 h after peroxidase inactivation and antigen retrieval, then incubated with primary antibody overnight at 4 °C and secondary antibody for 1 h at 37 °C. After counterstaining with hematoxylin, image acquisition was performed. The primary antibodies used in this study included anti-MMP9 (1:200), anti-CTSK (1:200), anti-Nrf2 (1:200), anti-HO-1 (1:200) and anti-p-p65(1:100) antibodies. The relative intensity was analyzed by Image J software.

5.11 Immunofluorescence staining

For paraffin section, the slides were dewaxed to distilled water and then performed antigen retrieval. For RAW264.7 cells, cells were seeded in 24-well plates (with coverslips) with 5 × 103 cells /ml and were collected after 48 h without or with 10 μM CK treatment. Fix cells with 4% paraformaldehyde for 15 min. Add 5% BSA to the slides, seal at room temperature for 1 h, and then incubate with primary antibody at 4 °C overnight. Then incubate with fluorescent secondary antibody at 37 °C for 1 h. The primary antibodies used in this study included anti-p-p65 (1:200), and anti-Nrf2 (1:200). DAPI staining was used to visualize the cell nuclei. Images were captured with a fluorescence microscope (Olympus, IX73 L, USA).

5.12 Statistical analysis

Data were analyzed by using GraphPad Prism 7. Quantitative data were represented as mean ± standard deviation (S.D.). Multiple comparisons between groups were statistically analyzed using one-way or two-way analysis of variance (ANOVA). P < 0.05 was regarded as a statistically significant difference.