2.1 Media and reagents
Recombinant mouse AOAH (rAOAH) was purchased from Cloud-Clone Corp® (Suzhou, China. Lot: P20210203211) and dissolved in phosphate-buffered saline (PBS). α-Minimum essential medium (αMEM), penicillin/streptomycin, and fetal bovine serum (FBS) were obtained from Gibco BRL (Grand Island, NY). Mouse Recombinant mouse macrophage-colony stimulating factor (M-CSF) and receptor activator for nuclear factor-κB ligand (RANKL) were purchased from PeproTech (London, UK). Cell Counting Kit-8 (CCK-8), alkaline phosphatase (ALP), alizarin red, and lipopolysaccharide (LPS) were supplied by Beyotime Biotechnology (Shanghai, China). Specific antibodies against p65, p-p65, p-ERK, ERK, JNK, p-JNK, IκBα, AKT, p-AKT, p38, p-p38, NFATC1, c-Fos, and β-actin were obtained from Cell Signaling Technology (Cambridge, MA) and Absin Bioscience (Shanghai, China). TRAP staining kit was obtained from Sigma-Aldrich (St. Louis, MO). The auric acid and Paclitexal were supplied by Bozhi Biotechnology (Suzhou, China). ELISA kits were purchased from Sinobestbio (Shanghai, China).
2.2 Animals and tissues
All animal experiments followed the guidance of the Institutional Animal Ethics Committee of Taizhou Hospital. The C57BL/6− Aoahtm1cyagen mice were obtained from Cyagen Research Laboratories (Suzhou. China. Lot: KOAIP180601JN1). We generated Aoah−/− offspring and their WT littermates by crossing two heterozygous C57BL/6-Aoahtm1cyagen mice. In the experiment of mouse model of LPS-Induced bone loss, 10 WT male mice (8-week-old) and 10 Aoah−/− male mice (8-week-old) were sacrificed. All mice were housed in SPF animal room with a controlled environment of 22–24°C, 50–60% humidity, 12h light/dark cycles, and free access to sterile rodent chow (Jiangsu Syung Pharmaceutical Bio-engineering Corporation) and sterile water.
WT mice and Aoah−/− mice were randomly divided into 3 groups (5 mice in each group): 4, 8 and 12-week-old mice. After the mice were euthanized, blood was collected through retro-orbital blood collection and centrifuged to obtain serum. The bone marrow from the mouse femur and tibia was flushed with 0.5 ml phosphate buffed saline (PBS) using an 18-gauge needle, via a hole formed at the joint end of each bone, and bone marrow was ground for further analysis. The feces were collected when the first excrement was defecated from mice with a hand-caught. The feces were weighed out 1g of the feces, then put into grinding machine with the 500µl PBS. A 1 cm long intestine was removed from the abdominal cavity of the mouse. The intestine is first washed with sterile saline. Then the intestine was soaked in 75% alcohol for 3 minutes, was weighed out 1 g, add 500 µl PBS, and was crushed in a grinding machine. The biological samples were sent for ELISA measurement. Femurs and tibias of mice were also collected and ground for mRNA and protein extraction.
2.3 LPS-induced calvaria bone loss mouse model
8-week-old WT male mice and Aoah−/− male mice were randomly divided into 2 groups (5 in each group): sham operation (only PBS injection) and LPS injection (5 mg/kg body weight) group. Under mild general anesthesia, mice received subcutaneous injection over the sagittal midline suture of the calvaria. The PBS and LPS were injected every other day for 14 days. At the end of the experimental period, all mice were sacrificed and their calvaria were excised, fixed in 4% paraformaldehyde, and subjected to micro-CT scanning. All mice showed normal behaviors during the experiment and no adverse effects or death were observed.
2.4 Cell viability determination
The CCK-8 assay kit (Beyotime Biotechnology, Shanghai, China) was used to test cell viability/proliferation. In brief, BMMs or BMSC were culture in complete α-MEM medium containing macrophage colony stimulating factor (M-CSF) (30 ng/ml) for 48, 72, 96, or 120 h. CCK-8 reagent (10 µL) was added to the petri dish. After 3 h of incubation, the optical density of the petri dish was measured at 450 nm with a Multiskan FC microplate photometer (Thermo Fisher Scientific, Waltham, MA).
2.5 Osteoclastogenesis test
BMMs were flushed from the femur and tibia of Aoah−/− and WT mice (8 weeks old) and cultured in complete α-MEM medium containing 10% fetal bovine serum (FBS) and M-CSF (30 ng/ml) 4 d or until 90% confluence. The adherent cells were considered to be bone marrow monocytes/macrophages (BMMs). The BMMs were then incubated with complete α-MEM medium containing M-CSF (30 ng/ml) and RANKL (50 ng/ml) for another 5 d. The medium containing RANKL and M-CSF was changed every 2 d. Next, the BMMs were stained by TRAP activity kit (Sigma-Aldrich, St. Louis, MO) and observed using an inverted optical microscope. ImageJ software (NIH, Bethesda, MD) was used to quantify the number and area (percentage of holes) of TRAP-positive osteoclasts (≥ 3 nuclei).
2.6 Osteoblast differentiation assay
Bone marrow cells were washed from the femur and tibia of Aoah−/− and WT mice (8 weeks old) and cultured in complete α-MEM medium containing 10% FBS for 6 d or until 90% confluence. For the osteoblast differentiation assay, BMSCs were seeded on a 12-well plate at a density of 5×104 cells/well and placed in osteogenic medium (DMEM supplemented with 10 mM β-glycerophosphate, 50 µM ascorbic acid, and 100 nM dexamethasone). The medium was changed every other day for 7 d and alkaline phosphatase (ALP) activity was stained, or for 21 d and mineralized bone nodules was stained with Alizarin Red staining solution.
2.7 Micro-CT scan
All bone samples were scanned by Scanco Medical micro-CT 100 high-resolution scanner (Brüttisellen, Switzerland). The scanning and image acquisition parameters were as follows: 70kV voltage, 200µA current and 20 µm isometric resolution. The images were reconstructed by software and the images in three dimensions were processed for morphometric analysis. After the three-dimensional (3D) image was reconstructed, a square region of interest (ROI) is then selected around the middle suture of the skull. Bone volume/tissue volume (BV/TV) and the total porosity ratio of each sample were tested using scanco µ100 evaluation (Switzerland).
2.8 Western blot
Cells were lysed with RIPA Lysis Buffer (Beyotime Biotechnology, Shanghai, China) containing the protease inhibitor phenylmethanesulfonyl fluoride (PMSF). Cell lysates were collected and centrifuged. The BCA assay (Beyotime Biotechnology, Shanghai, China) was used to quantify the protein concentration of the supernatant containing the post-nuclear extract. The protein sample was mixed with SDS-PAGE loading buffer, boiled at 100°C for denaturation, and loaded in a 10% SDS-PAGE gel for electrophoresis. The separated proteins were transferred to a nitrocellulose membrane, and the membrane was blocked with QuickBlock blocking solution (Beyotime Biotechnology, Shanghai, China) for 10 min, and then incubated with the specific primary antibody (diluted 1:1000, v/v) at 4°C overnight. The membrane was then treated with an appropriate HRP-conjugated secondary antibody (1:10,000 dilution, v/v; Beyotime Biotechnology, Shanghai, China) for 1 h. The protein-antibody complexes were detected after exposure to Luminata enhanced chemiluminescence reagent (Millipore Sigma, Billerica, MA) and imaged on the ImageQuant LAS-500 imaging system (GE Life Sciences, Chicago, IL). ImageJ software was used to measure the density of protein bands.
2.9 RNA isolation and qPCR
RNAiso Plus Total RNA Extraction Reagent (Takara Bio, Otsu, Japan) was used to extract total RNA from cultured cells or bone tissue ground by small steel balls from each well according to the manufacturer's protocol. Primer Script RT cDNA Synthesis kit (Takara Bio) was used and 1µg of the extracted RNA template was used to synthesize first-strand cDNA. Then, SYBR Green-based real-time PCR master mix (Takara Bio) was used and the following cycle parameters for qPCR were set: 40 cycles at 94°C for 20 sec; 60°C for 20 sec; and 72°C for 30 sec. The results were normalized to β-actin, an internal housekeeping gene. The primer set used includes: Actb (forward: 5ʹ-AGC CAT GTA CGT AGC CAT CC-3ʹ, reverse: 5ʹ-CTC TCA GCA GTG GTG GTG AA-3ʹ); Acp5 (forward: 5ʹ-TCC TGG CTC AAA AAG CAG TT-3ʹ, reverse: 5ʹ-ACA TAG CCC ACA CCG TTC TC-3ʹ); Ctsk (forward: 5ʹ-CTT CCA ATA CGT GCA GCA GA-3ʹ, reverse: 5ʹ-TCT TCA GGG CTT TCT CGT TC-3ʹ); Fos (forward: 5ʹ-CCA GTC AAG AGC ATC AGC AA-3ʹ, reverse: 5ʹ-AAG TAG TGC AGC CCG GAG TA-3ʹ); Nfatc1 (forward: 5ʹ-CAG CTG C CG TCG CAC TCT GGT C-3ʹ, reverse: 5ʹ-CCC GGC TGC CTT CCG TCT CAT A-3ʹ); Dcstamp (forward: 5ʹ-CTT GCA ACC TAA GGG CAA AG-3ʹ, reverse: 5ʹ-TCA ACA GCT CTG TCG TGA CC-3ʹ); and Atp6v0d2 (forward: 5ʹ-AAG CCT TTG TTT GAC GCT GT-3ʹ, reverse: 5ʹ-TTC GAT GCC TCT GTG AGA TG-3ʹ).
2.10 Calcein-alizarin red staining
On day 0 and day 4, mice were injected intraperitoneally with 20 mg/kg calcein (1 mg/ml in 2% NaHCO3 solution) and 40 mg/kg alizarin red (2 mg/ml, in PBS). On the 7th day, the mice were sacrificed and the bones were fixed, dehydrated and embedded with Embed-812 (Electron Microscopy Sciences). A hard tissue cutter was used to slice the sample into 5 µm. Serial sections were stained with silver nitrate to measure bone morphology. Then, a confocal microscope was used to take the image.
2.11 Enzyme-linked immunosorbent assay (ELISA)
The concentrations of inflammatory cytokines IL-17, RANKL, IFN-γ, TNF-α, and LPS in the supernatant were determined using commercial mouse ELISA kits (Sinobestbio, Shanghai, China) according to the manufacturer's protocol. A standard curve with the concentration of the standard substance as the x-axis and the OD value on the y-axis was made. The concentration of target protein was calculated by multiplying the concentration calculated from the standard curve with dilution ratio.
2.12 Bone resorption measurement
In order to determine the bone resorption capacity, BMMs from 8-week-old Aoah−/− and WT mice were seeded into 100 µm bovine bone slices at a density of 1 × 104 cells/well (Beijing Rongzhi Haida Biotechnology Co., Ltd., China) in α-MEM supplemented with 100 ng/ml RANKL. In order to reveal the reverse effect of rAOAH, 8-week-old Aoah−/− mouse BMMs were seeded into bovine bone slices inα-MEM containing 100 ng/ml RANKL and 0, 50 or 100 ng/ml rAOAH. The medium was changed every 2 d and cultured for 14 d. The cultured cells were removed by mechanical agitation and sonication, and bone resorption pits were examined under a scanning electron microscope (Field Environmental Instruments Inc, Hillsboro, OR). The area of the reabsorption pits was measured by Image-Pro Plus software.
2.13 Statistical analysis
The results were expressed as the mean ± standard deviation (SD), and a value of p < 0.05 was measured statistically significant. The statistical significance of the differences between experimental groups was measured using a two-tailed Student’s t-test or one-way analysis of variance (ANOVA) following the Tukey post hoc test. Statistical analyses were performed using the SPSS software package (Version 22.0 for Windows; SPSS Inc., USA). All experiments were repeated at least three times using independent samples.