Mice and reagents
All animal protocols were approved by the Institutional Review Board of Longhua Hospital, Shanghai University of Traditional Chinese Medicine (China). C57BL/6J wild-type mice, Osteoprotegerin (OPG) knockout (KO) mice, and OPG wild-type (WT) mice were purchased from the Shanghai Bio model Organism Science and Technology Development Co., Ltd (China). Osthole, with 98% purity, was purchased from the Shanghai Institute for Drug and Quarantine Bureau. The reagents used in the experiment are listed in Table S1.
Six-month-old C57BL/6 mice, specific pathogen-free (SPF), were purchased from the Institute of Zoology, Chinese Academy of Sciences. Four months later, C57BL/6 mice of 1-month-old were purchased again, and the experiment was performed when the mice were 3- and 12-months-old. Next, the 12-month-old male mice were randomized equally (n=6) into two groups: treatment and control group. The treatment group was intervened with osthole (5 mg/kg/day) by intraperitoneal injection once a day for 4 weeks, and the control group was intervened with vehicle (corn oil) by intraperitoneal injection once a day for 4 weeks. After sacrifice, the lumbar vertebrae were harvested for evaluation.
Microcomputed tomography (μCT) analysis
The fourth lumbar vertebra (L4) was scanned at 18-μm voxel size using the μCT scanner (μCT80, Scanco Medical AG, Bassersdorf, Switzerland). The trabecular bone under the growth plate was segmented using a contouring tool, and the contours were morphed automatically to segment the trabecular bone on all the 100 slices. The three-dimensional (3D) images were reconstructed and analyzed using the software of the μCT system.
Histological and histomorphometric assays
The fifth lumbar vertebra (L5) was fixed in 4% paraformaldehyde, decalcified, dehydrated, cleared with dimethyl benzene, and embedded in paraffin. At least three consecutive 7-mm sections were obtained from the coronal planes and subjected to tartrate resistant acid phosphatase(TRAP) staining to identify osteoclasts. The histomorphometric assay was performed to determine the number of osteoclasts and the proportion of osteoclast surface using an image auto-analysis system (Olympus BX50; Japan).
The paraffin sections of L3 were deparaffinized by immersing the tissue in xylene, fixing with 4% paraformaldehyde for 15 min, and permeabilizing with 0.5% Triton X-100 for 15 min, followed by fixation with 4% paraformaldehyde for an additional 5 min. Then, the sections were incubated with rabbit anti-OPG monoclonal antibody (1:50) and rabbit anti-β-catenin monoclonal antibody (1:50) at 4 °C overnight and then with horseradish peroxidase (HRP)-conjugated secondary antibody for 30 min. Finally, the slides were mounted and examined using an Image Analysis System (Olympus BX50).
Cell culture and treatment
Primary BMSCs, extracted from 3-month-old OPG-/- mice and the littermates of OPG+/+ mice, were cultured by incubation with macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor (NF)-κB-ligand(RANKL for 1 week, and then treated with various doses (0.5–100 μM) of osthole for 48 h. Transgenic mice were presented by Professor Chen, Department of Orthopedics, University of Rochester. Primary calvaria osteoblasts, extracted from 3-day-old C57BL/6J mice or 3-day-old OPG-/- mice and the littermates of OPG+/+ mice
Primary BMSCs were seeded in a 96-well plate at a density of 3x105/mL and treated with M-CSF (44 ng/mL) and RANKL (100 ng/mL) in the presence or absence of osthole (100 µM). The medium was changed every 3 days. After 7-day incubation, the cells were fixed and subjected to TRAP staining to calculate the number of multinuclear (≥3 nuclei) osteoclasts.
Real time-quantitative polymerase chain reaction (qPCR) analysis
Primary calvaria osteoblasts, extracted from 3-day-old C57BL/6J mice, were seeded in 6-well plates at a density of 1×x106 cells/well. After 2-day culture, the cells were treated with various doses of osthole (1–100 μM) or vehicle for 48 h. Total cellular mRNA was isolated using the RNeasy Mini Kit(Qiagen Corporation, Valencia, CA) . An equivalent of 1 mg of total RNA was reverse-transcribed into cDNA using the iScript cDNA synthesis kit(Bio-Rad Laboratories, Inc, Hercules, CA). qPCR analysis was carried out using Absolute QPCR SYBR Green Master Mix in a total volume of 20 μL reaction containing 1 μL of the diluted (1:5) reverse transcription product in the presence of sense and antisense primers of target genes listed as Table 1. β-actin served as the internal reference gene. The reaction was as follows: polymerase activation at 95 °C for 15 min, followed by 45 cycles of 95 °C for 20 s, 58 °C for 20 s, and 72 °C for 30 s. All reactions were performed in triplicate.
Western blotting analysis
Primary calvaria osteoblasts, isolated from 3-day-old OPG-/- homozygous mice and the littermates of OPG+/+ mice, were seeded in 6-well plates at a density of 1×106 cells/well. Cells were treated with osthole (100 μM) or vehicle for 48 h and cell lysates were extracted using protein extraction reagents(PER) (Thermo Scientific, Waltham, MA, USA). The protein concentration is 10ug, and by Sodium Dodecyl Sulfonate-Polyacrylamide gel electrophoresis(SDS-PAGE) and the 10% of gel. Proteins were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA, USA). Subsequently, the membrane was blocked with 5% non-fat milk in Phosphate Buffered Saline and Tween-20(PBST) for 1 h at room temperature and probed with primary antibodies overnight at 4 °C, followed by HRP-conjugated secondary antibodies (Thermo Scientific) for 1 h at room temperature. The intensities of the immunoreactive bands were detected using a SuperSignal West Femto Maximum Sensitivity Substrate Kit (Thermo Scientific). Rat anti-OPG and rabbit anti-β-catenin monoclonal antibodies were the primary antibodies, and mouse anti-β-actin monoclonal antibody was used as an internal reference.
In vitro deletion of the 𝛽-catenin gene
The in vitro deletion of the β-catenin gene was performed as described previously . calvaria osteoblasts isolated from 3-day-old β-cateninfx/fx mice were seeded in 6-well culture plates at a density of 1×106/well and cultured for 6 days. Then, the cells were infected with Ad-GFP or Ad-Cre (Titer: 4×108 pfu/mL; Baylor College of Medicine, Houston, TX, USA) for 72 h, and Ad-GFP was used as a control to monitor infection efficiency. After recovery for 48 h, cells were treated with or without osthole (100 mM) for 48 h. Real-time qPCR assay was performed to examine the expression of β-catenin and OPG. All reactions were performed in triplicate.
All experiments were conducted independently at least three times. The data were expressed as mean±standard deviation (SD) and analyzed using SPSS 24.0 software and GraphPad Prism 8. The statistically significant differences were analyzed using Student’s t-test of variance. We compared 3-month-old mice vs. 12-month-old mice and Osthole vs. vehicle. ImageJ software was employed to quantitate the grayscale intensities.,*P<0.05 indicated statistically significant difference.