Reagents
Antibodies against Histone 3 (bs-17422R), C1QC (bs-11336R), H2B (bs-52099R), C3B (bs-4871R), GAPDH (bs-2188R), β-tubulin (bs-0715R) were all purchased from Bioss Antibodies (Beijing, China). Antibody against RANK (#119805) was obtained from BioLegend (California, USA), while antibody against Annexin-V(#AMM01981G) was obtained from BD Biosciences (New Jersey, USA). Fetal bovine serum (FBS) was purchased from CELLCOOK (Guangzhou, China) and penicillin-streptomycin were purchased from Gibco (Thermo Scientific, MA, USA). Recombinant mouse RANKL was purchased from R&D Systems (Minneapolis, MN, USA). TRAP stain kit was obtained from Sigma-Aldrich (NY, USA). Annexin V and PI Apoptosis Kit (F6012) was purchased from US Everbright® Inc. (Suzhou, China). Staurosporine was purchased from Med Chem Express (New Jersey, USA).
BMMs extraction and osteoclastogenesis
We extracted the hind limbs of eight-week-old female BALB/c mice and collected bone marrow cell suspensions by repeated marrow cavity flushing. Subsequently, the bone marrow cells were cultured in DMEM medium supplemented with M-CSF for 24 h, and the non-adherent bone marrow mononuclear cells (BMMs) were obtained. For osteoclastogenesis, BMMs were cultured in induction medium (DMEM, 10% FBS, 50 ng/mL RANKL, 25 ng/mL M-CSF) at 37 °C with 5% CO2 for 120 h to generate osteoclasts.
Animal experiment
8-week-old female mice underwent ovary removal surgery. In brief, mice were anesthetized with 4% bromoethane-oxygen gas mixture and maintained at 2% concentration. A 1 cm incision was made in the middle of the abdomen of the mouse to expose the lower poles of the kidneys on both sides. The ovarian glands were determined according to the position of the end of the fallopian tube, and they were removed and ligated to stop the bleeding. Then, 100 μL of 1% penicillin-streptomycin solution was dripped into the abdominal cavity, and the peritoneal layer and the skin layer were sutured. BALB/c used in the experiment were obtained from the Laboratory Animal Center of Third Military Medical University (Chongqing, China) and MRL/lpr mice were purchased from the Jackson Laboratory (Maine, USA). In the experiment to observe the progress of osteoporosis, 45 BALB/c mice were randomly divided into sham operation group (N = 27) and Operation Group (N = 18). In an experiment to investigate the effect of OC-ABs deficiency on bone mass in mice, 8 BALB/c mice served as wild-type controls, and 16 female MRL/lpr mice were randomly divided into MRL/lpr Group and MRL/lpr + OC-ABs Group. In an experiment to investigate the effect of OC-ABs on delaying the progression of osteoporosis, 25 BALB/c mice were randomly divided into OVX group (N = 15) and OVX + OC-ABs Group (N = 10), 5 BALB/c mice in OVX group were killed at 1 day after operation and taken as blank control. In the animal experiment, each mouse was numbered to achieve single-blind. Numbers, instead of group names, are marked on test tubes or slices containing samples to ensure the examiner blinded. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of Third Military Medical University, and the mice were euthanized according to the AVMA Guidelines for the Euthanasia of Animals.
Apoptotic body extraction and identification
After the formation of osteoclasts, the cell supernatant was removed, 5 μM STS in DMEM medium was added, and the cells were further cultured at 37 °C for 4 h to induce osteoclast apoptosis. The culture medium produced after osteoclast apoptosis was collected, centrifuged at 300 × g for 5 min to precipitate cells and debris and removed, and the supernatant was centrifuged at 3000 × g for 30 min to precipitate apoptotic bodies. Apoptotic bodies were resuspended and washed in PBS, and re-pelleted by centrifugation at 3000 × g for 30 min. All centrifugation operations were under 4 °C and performed by using Optima XE-90 (Beckman Coulter). For identification, apoptotic body suspensions were incubated with FITC-Annexin V antibody for 15 min in the dark, after which they were dropped into confocal dishes and visualized using confocal microscopy (Zeiss LSM-800, Germany). The purity of apoptotic body suspensions supplemented with standard size polyethylene microspheres (#M122073& #M122077, Aladdin, Shanghai, China) was examined by flow cytometry (CytoFLEX, Beckman, USA). Western blot was used to detect apoptotic body markers C1QC, C3B, H3 and H2B.
H&E and TRAP staining
The extracted hindlimbs of mice were fixed in 4% paraformaldehyde for 72 h and then soaked in EDTA decalcification solution for 4 weeks. The decalcified tissue was graded dehydrated and then embedded in paraffin. Subsequently, 5 μm thick sections were made for staining by using Leica microtome (RM2235, Leica Biosystems, Germany). The sections were immersed in hematoxylin staining solution for 1 min, rinsed with water for 30 s, and differentiated with 1% hydrochloric acid alcohol for 10 s. Sections were immersed in eosin staining solution again for 3 min and rinsed with water for 30 s. The slides were sealed with neutral resin after gradient dehydration and observed under microscope. For TRAP staining, bone tissue sections were stained with TRAP staining kit (CS0740, Sigma, Shanghai, China), and the method steps were referred to the reagent manufacturer’s instructions. TRAP staining of osteoclast was also used the above-mentioned kit. The images were acquired and analyzed by the ZEISS Axio Scan-Z1 Fully Automatic Digital Slide Scanning System (Carl Zeiss, Germany).
Osteocalcin staining
Sections were gradient dewaxed and blocked in 3% H2O2 for 15 min, then the sections were immersed in sodium citrate antigen retrieval solution at 95 °C for 10 min. After 15 min of incubation in immunohistochemical blocking solution, sections were incubated with diluted rabbit anti-OCN (1:200, diluted in blocking solution) for 2 h at room temperature. Further, the sections were washed 3 times with PBS and incubated with horseradish peroxidase-conjugated secondary antibody for 2 h, and then DAB chromogenic reagent was added dropwise for observation under a microscope. The images were acquired by the ZEISS Axio Scan-Z1 Fully Automatic Digital Slide Scanning System and analyzed by ImageJ v.1.8.0 software.
Osteogenic induction and alizarin red staining
We extracted the hind limbs of eight-week-old female BALB/c mice and collected bone marrow cell suspensions by repeated marrow cavity flushing. Subsequently, the bone marrow cells were cultured in a-MEM medium containing 10% FBS for 8 h to obtained adherent bone marrow mesenchymal stem cells (BMSCs). 1 × 105 BMSCs were seeded into 24-well plates and cultured with basal medium (a-MEM supplemented with 10% FBS) for proliferation. When the cell confluence reaches more than 80%, basal medium was changed to osteogenic induction medium (100 mL a-MEM medium supplemented with 10% FBS, 10 μL 1 mM Dexamethasone, 500 μL 10 mM Vitamin C and 1 mL 1 M β-Glycerophosphate) for continue culture in an incubator at 37 °C with 5% CO2. For the observation of calcium salt deposition, cells were fixed with 4% paraformaldehyde for 20 min and washed 3 times with PBS. Cells were covered with alizarin red staining solution (ALIR-10001, OriCell®, Suzhou, China) and protected from light, incubated at room temperature for 30 min and washed with PBS three times. The images were acquired by the Leica DMIL microscopic imaging system (Carl Zeiss, Germany) and the mineralized area was analyzed by ImageJ v.1.8.0 software.
Micro-CT
Images of the femur were acquired using a Bruker MicroCT Skyscan 1272 system (Kontich, Belgium) with a resolution of 8.0 μm voxel size and data reconstruction was performed using Nrecon (Kontich, Belgium). Select regions of interest (ROI) and acquire 2D images in DataViewer 1.5.6.2 (Bruker, Kontich, Belgium). The bone volume fraction and other data of the ROI were obtained by CTAn 1.20.8.0 (Bruker, Kontich, Belgium) software.
Real-time polymerase chain reaction (PCR)
Total RNA was isolated from the cultures using the RNA-Quick Purification Kit (YiShanBiotech, Shanghai, China) according to the manufacturer’s instructions. The cDNA was synthesized using SuperScript III (Life Technologies). The real-time PCR was performed using SYBR Green Supermix (Toyobo) and gene-specific primers. The primers included Alp forward 5′-AACCCAGACACAAGCATTCC-3′; reverse 5′-GAGACATTTTCCCGTTCACC-3′. Sp7 forward 5′- ATGGCGTCCTCTCTGCTTG-3′; reverse 5′- TGAAAGGTCAGCGTATGGCTT-3′. Col1a1 forward 5′-GCTCCTCTTAGGGGCCACT-3′; reverse 5′-ATTGGGGACCCTTAGGCCAT-3′. Runx2 forward 5′-ATGCTTCATTCGCCTCACAAA-3′; reverse 5′-GCACTCACTGACTCGGTTGG-3′. GAPDH forward 5′-TGGATTTGGACGCATTGGTC-3′; 5′-TTTGCACTGGTACGTGTTGAT-3′. GAPDH was used as an endogenous control for osteoclasts. Real-time PCR was detected on a CFX96™ Real-Time PCR System (Bio-Rad).
Western blot analysis
Cells or ABs were lysed in RIPA lysis buffer (CWBIO, Beijing, China) mixing with protease phosphatase inhibitor (Beyotime Biotechnology, Shanghai, China). The lysis system was centrifuged at 12,000 × g for 15 min to obtain the supernatant after incubated on ice for 30 min. The BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China) was used to detect the concentration of proteins. 50 μg protein samples were diluted in loading buffer (Beyotime Biotechnology, Jiangsu, China) and electrophoresed, followed by transferred onto polyvinylidene fluoride membranes (ImmobilonTM-PSQ Membranes, Sigma-Aldrich, China) and blocked in 5% bovine serum albumin (BSA). Protein on membranes were incubated with anti AKT, anti p-AKT, anti PI3K, anti p-PI3K, anti S6K, anti p-S6K and anti β-actin (1:1000, diluted in 5% BSA) antibodies for 12 h at 4 °C. After washing in Tris Buffered Saline Tween buffer three times, secondary antibodies linked HRP (1:10,000, diluted in 5% BSA) against primary antibodies were used to incubate membranes. Super ECL Plus (Biosharp, Anhui, China) and the ChemiDoc XRS + gel imaging system (BioRad) were used to detect chemiluminescent signals.
Statistics
All experiments were repeated at least three times carried out with at least three biological replicates. Comparisons between two groups were analyzed by using independent unpaired two-tailed Student’s t-tests. One-way ANOVA analysis was used to compare multiple sets of data. GraphPad Prism 8.0 software was used for statistical analysis. P values < 0.05 and P values < 0.01 were used to represent the significance of the difference. The error bars in the figures represent the standard deviation (SD).