- Chemicals and Reagents
Anti-actin antibody, 4HR, TRAP staining kit, and other reagents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti TRAP, NFAT c1 and Cathepsin K antibodies were from Proteintech Group (Wuhan, China). Phosphorylated-IKKβ (pIKKβ) and total IKKβ antibodies were from Cell Signaling Technology (Beverly, MA). Recombinant soluble M-CSF and RANKL were obtained from PeproTech (Rocky Hill, NJ). Cell counting kit‐8 (CCK‐8) kit was purchased from Wuhan Boster Biological Technology Company (Wuhan, China). Serum cross-linked C-telopeptide of type I collagen (P1NP) and C-terminal telopeptide of type1 collagen (CTX1) were from Cloud-Clone (Katy, TX, USA).
- Animals and OVX surgery
All animal work was approved by Tongji Medical College, Huazhong University of Science and Technology. C57BL/6J mice used in the study were purchased from Experimental Animal Center of Tongji Medical College. All mice were kept under standard laboratory conditions with 12 h light and 12 h dark cycle, water and food ad libitum. All animal experiments were performed according to the guidelines of the Care and Use of Laboratory Animals by the National Institute of Health, China.
For OVX surgery and drug treatment, 4 months old female C57BL/6J were used, mice were anaesthetized by 2% isoflurane, and the ovaries were bilaterally removed according to established protocol except the sham-operated mice. OVX mice received equal volume of DMSO intraperitoneally injection daily for 60 days after surgery. Another OVX group received 1mg/kg 4HR intraperitoneally injection daily for 60 days after OVX surgery (OVX+4HR). Mice were sacrificed after the final administration, the tibias of the all the mice were collected and scanned using microCT.
- Bone specimen collection and microCT scanning
The tibias were dissected and fixed in neutral buffered formalin for 48 hrs. After fixation, the tibias were scanned using Viva CT 40 (Scanco Medical, Switzerland) at 15µm resolution and 70kVP and 112µA x-ray energy. The parameters of BV/TV (Bone volume per tissue volume), Tb.N (Trabecular Number), Tb. Th（Trabecular Thickness）, Tb. Sp (Trabecular Separation) were collected and analyzed from each sample.
After microCT scanning, the tibias were decalcified in 10% EDTA for 4 weeks. Before sectioning, the tissues were paraffin embedded. Sections were cut at 5 µm thickness and then stained with hematoxylin and eosin (H&E). TRAP staining was performed using 387A kit (Sigma-Aldrich, St. Louis, MO, USA) following a standard protocol.
- In vitro osteoclastogenesis
The tibia and femur were dissected from 6-8 week C57BL/6J mice and BMMs were flushed from the tibia and femur (N=3 each group, male mice were used). Then the cells were centrifuged and plated onto 100 mm tissue culture dish containing 10% FBS and 10 ng/ml M-CSF. The next day, non-adherent bone marrow progenitor cells (monocyte-macrophage) were collected. The BMMs were cultured in α-MEM plus 10%FBS supplemented with 10 ng/ml M-CSF. For osteoclastogenesis, BMMs were cultured in α-MEM containing 10% FBS in the presence of 10 ng/ml M-CSF and 100 ng/ml RANKL for 5 days. Mature osteoclasts number and area in each well were quantified using imageJ. RNA was extracted using Trizol reagent from 3 days of osteoclastogenesis BMMs. The total RNA was reverse transcribed to cDNA for RT-qPCR. The primers were designed as below, NFATc1, forward primer, 5’-TCTTCCG AGTTCACATCCC-3’ and reverse primer, 5’-GACAGCACCATCTTCTTCC-3’; TRAP, forward primer,5’-cagcagccaaggaggactac-3’, reverse primer, 5’-acatagcccacaccgttctc-3’; Cathepsin K forward primer, 5’-ccagtgggagctatggaaga-3’, reverse primer, 5’-tggttcatggccagttcata-3’, and those genes mRNA levels were normalized by mouse β-actin, forward primer, 5’-tgttaccaactgggacgaca-3’, reverse primer, 5’-ggggtgttgaaggtctcaaa-3’. The relative mRNA levels of target genes were calculated by the comparative CT method  .
- Western-blot analysis
Protein extracts were prepared in radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor and phosphatase inhibitors. The proteins were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis 10% gel and then transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA). Membranes were blocked with 5% BSA in Tris‐buffered saline Tween 20 (TBST) for 60 min and then incubated with the indicated primary antibodies overnight. Then the membranes were incubated with the appropriate horseradish peroxidase‐conjugated secondary antibodies. Finally, the membranes were visualized using electrochemiluminescence reagents and the band densities were quantified using Image Lab 5.1 software (Bio‐Rad, Hercules, CA) and normalized to actin.
- Luciferase assay
HEK293T cells were transfected with NF-κB -luciferase reporter plasmid together with control vector (Promega, USA) as reference controls using Lipofectamine 3000 (Invitrogen, USA). 48 hours later, cells were subject to different treatments. The luciferase activity measurement was performed as described in Dual luciferase reporter assay kit (Promega, USA).
- Analysis of serum biomarkers
Serum P1NP, an indicator of bone formation, and CTXI level, an indicator of bone resorption, were measured using an ELISA kit (Cloud-Clone, Katy, TX, USA) followed the instructed methods .
- Statistical analysis
Data were shown as mean ±SD and were analyzed using SPSS software (SPSS Inc, Chicago, IL, USA). One-way ANOVA followed by LSD’s post hoc tests were used to test the differences among groups. Student t test was applied to compare difference between two groups. The levels of significance are presented as *p<0.05, **p<0.01, and ***p<0.001.
All data supporting the conclusions are provided in the manuscript.