Mice and reagents
All animal protocols were approved by Institutional Review Board of Longhua hospital, Shanghai University of Traditional Chinese Medicine (China). C57BL/6J wild-type mice, OPG knockout (KO) mice and OPG Wild-type (WT) mice were purchased from Shanghai Biomodel Organism Science & Technology Development Co.,Ltd(China). Osthole, with 98% purity, was purchased from the Shanghai Institute for Drug and Quarantine Bureau. Recombinant human M-CSF and the receptor activator of nuclear factor-kappa B ligand (RANKL) proteins were purchase from R&D system (Minneapolis, MN, USA). Dimethyl sulfoxide (DMSO) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Fetal bovine serum, DMEM dehydrated medium, and penicillin streptomycin combination were purchased from Invitrogen Corporation (Carlsbad, CA). RNeasy Mini RNA kit was purchased from Qiagen Corporation (Valencia, CA). iScriptcDNA synthesis kit and PVDF membrane were purchased from Bio-Rad Laboratories, Inc (Hercules, CA). Absolute QPCR SYBR Green Master Mix and E-PER protein extraction reagents were purchased from Thermo Scientific (Waltham, MA). Tartrate-resistant acid phosphatase staining (Trap) kit was purchased from Sigma-Aldrich (St. Louis, MO). Mouse bone gla-protein (BGP) ELISA kit was purchased from GENTAUR (SanJose, CA). Rabbit anti-OPG monoclonal antibody, Rabbit anti-β-catenin monoclonal antibody and mouse anti-β-Actin monoclonal antibody were purchased from Cell Signaling Technology, Inc (Beverly, MA). Adenovirus-GFP and Adenovirus -Cre were purchased from Baylor College of Medicine (Houston, TX, USA).
Animal study
Twelve12-month-old male mice were randomized into two groups, treatment group and control group, respectively. 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 sacrificed, the lumbar vertebrae were harvested for evaluation.
Micro-computed tomography (μCT) analysis
The fifth lumbar vertebrae (L5) were 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 one-hundred slices. The 3D images were reconstructed and analyzed with the evaluation software of the μCT system.
Histological and histomorphometrical assays
The third lumbar vertebrae (L3) were fixed in 4% paraformaldehyde, decalcified, dehydrated, cleared with dimethylbenzene, and then embedded in paraffin. At least 3 consecutive 7-μm sectionswere obtained from the coronal planes, and performed TRAPstaining for identifying osteoclasts. Histomorphometrical assay was performed to determine the number of osteoclasts andthe percentage of osteoclast surface by using an imageauto-analysis system (Olympus BX50; Japan).
Immunohistochemical staining
The paraffin sections of L3were deparaffinized by immersing the tissue in xylene, fixing it with4% paraformaldehyde for 15 minutes, and treating it with0.5% Triton for 15 minutes, followed by fixation with 4%paraformaldehyde for another 5 minutes.The sections were then incubated with rabbit anti-OPG monoclonal antibody(1:50dilution) and rabbit anti-β-catenin monoclonal antibody(1:50dilution), at 4 °C over night. Afterthorough wash, the slides were then incubated with a horseradish peroxidase (HRP)–conjugated secondary antibody for 30 minutes. After being mounted, slides wereexamined by using an Image Analysis System (OlympusBX50, Japan).
Bone marrow stemcells (BMSCs) culture and treatment
Primary bone marrow stem cells, extracted from 3-month-old C57BL/6J mice, or 3-month-old OPG-/- mice and its littermates of OPG+/+ mice, were cultured with incubation of M-CSF and RANKL for one week, and then treated with various doses (0.5-100 μM) of Osthole for 48 hours.
TRAP staining
Primary BMCswere seeded in 96-well plate at a density of 3x105/ml. The cells were treated with M-CSF (30 ng/ml) and RANKL (30 ng/ml), at the present or absence of Osthole (100 µM).The medium was changed every 3 days. After 7-day-incubation, the cells was fixed and performed TRAP staining to calculate the number of multi-nuclear (>=3 nucleus) osteoclasts.
Real-time qPCR analysis
Primary BMSCs extracted from 3-month-old C57BL/6J mices, were seeded in 6-well plates at a density of 1x106 cells/well. After 2-day culture, Cells were treated with variation doses of Osthole (1-100 μM) or Vehicle for 48 hours. Total cellular mRNA was isolated respectively using RNeasy Mini Kit. One microgram of total RNA was reverse-transcribed separately into cDNA using the iScriptcDNA synthesis kit. Quantitative polymerase chain reaction (qPCR) analysis was carried out using Absolute QPCR SYBR Green Master Mix in a total volume of 20 μl of buffered solution containing 1μl 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 is internal reference gene. Conditions were 15-min polymerase activation at 95 °C followed by 45 cycles, 95 °C for 20 s, 58 °C for 20 s and 72 °C for 30 s. All reactions were performed in triplicate independently.
Western-blotting analysis
Primary BMSCs isolated from 3-month-old OPG-/- homozygous mice and its littermates of OPG+/+ mice, were seeded in 6-well plates at a density of 1x106 cells/well. Cells were treated with Osthole (100 μM) or Vehicle for 48 hours and cells lysates were, respectively, extracted with E-PER protein extraction reagents (Thermo Scientific, Waltham, MA). Proteins were transferred onto a PVDF membrane (Bio-Rad, Hercules, CA) and the membrane was blocked with 5% non-fat milk in PBST solution for 1 h at room temperature (RT). After incubation with the primary antibody overnight at 4 °C and the HRP-conjugated secondary antibodies (Thermo Scientific, Waltham, MA) for 1 h at RT, the protein expression was detected using a SuperSignal West Femto Maximum Sensitivity Substrate Kit (Thermo Scientific, Waltham, MA). Rat anti-OPG monoclonal antibody and Rabbit anti-β-catenin monoclonal antibody were used as primary antibodies. Mouse anti-β-Actin monoclonal antibody was used as secondary antibody.
In vitro deletion of the 𝛽-catenin gene
In vitro deletion of the β-catenin gene was performed as previouslydescribed[24]. BMSCs isolated from 3-month-old β-cateninfx/fx mice were seeded in 6-well culture plates at a density of 1 × 106 cells per well and cultured for6 days. Cells were infected with Ad-GFP or Ad-Cre (Titer:4 × 108 pfu/mL; Baylor College of Medicine, Houston, TX,USA) for 72 hours. Ad-GFP was used as a control and tomonitor infection efficiency. After recovery for 48 hours, cellswere treated with or without Osthole (100 uM) for 48 hours. Real-time qPCR assay was performed to examine the expression of β-catenin and OPG. All reactions were performed in triplicate independently.
Statistical analysis
Based on all experiments conducted independently at least three times, the date was expressed as mean ± SD and analyzed using SPSS 24.0 software and GraphPad Prism 8. We analyzed the statistically significant differences using Student’s t test and one-way analysis of variance. ImageJ software was employed to measure the grayscale analyses. In figures,*P < 0.05 or lower was considered statistically significant.