Sixty-week-old male apoE−/−mice with a C57Bl/6J background were purchased from Nanjing Biomedical Research Institute (Nanjing University, Nanjing, China) and fed with regular rodent chow (containing 20.5% protein, 4.68% fat, 1.23% calcium, and 0.91% phosphorus). Twenty-four mice were randomly allocated to two groups. Twelve mice (Atorvastatin group) were orally gavaged with 10 mg kg−1 day−1 atorvastatin (Pfizer, New York, USA) dissolved in DMSO (dimethyl sulfoxide) for 12 weeks. The other mice served as the control group and received an equivalent amount of DMSO as placebo. The mice were maintained in groups under a strict 12 h light/dark cycle in a temperature-controlled environment (25°C) with free access to food and water. The procedures involving animals and their care were conducted in conformity with the guidelines of the department of laboratory animal science of Fudan University. All animal experiments were conducted with the approval of the Animal Resources Committee of Huadong Hospital, Fudan University (2012-03-FYS-GJJ-01).
Microcomputed Tomography (Micro-CT) Analysis
The femurs collected from apoE–/– mice at 72 weeks of age were harvested, fixed, and scanned by the SkyScan-1176 micro-CT instrument (Bruker, Kontich, Belgium). For cortical bone, measurements were performed on an 0.5 mm region of the mid-diaphysis of the femur. For trabecular bone, micro-CT evaluation was performed on a 1 mm region of the metaphyseal spongiosa on the distal femur. The regions were located 0.5 mm above the growth plate. NR Econ software, version 1.6 was used for the three-dimensional (3D) reconstruction and viewing of images .
Biochemical and Bone Markers Assays of Serum
Serums from two groups’ mice were analyzed for OCN by enzyme-linked immunosorbent assays (ELISAs) using a Mouse Osteocalcin EIA Kit (Immutopics International, San Clemente, CA, USA) and for TRAP5b using MouseTRAPTM [TRAcP5b] ELISA(Immunodiagnostic Systems Limited, London, England) Serum total cholesterol (TC) was measured by a Hitachi 917 autoanalyzer (Roche, Meylan, France), and ox-LDL was measured by a mouse ox-LDL ELISA kit (Uscn Life Science & Technology, Wuhan, China) .
BMSCs were obtained from the femurs and tibias of apoE–/– mice (male, 18 weeks old). The second generation of cells was seeded at 1 × 105 cells/well in a six-well culture plate and cultured in L-DMEM containing 10% fetal bovine serum.
(1) BMSCs were cultured with atorvastatin at different concentrations (0, 10, 100, 1000 nmol/L) at 6 h for qRT-PCR and at 3 days for Western blot. (2) BMSCs were cultured with EX-527 (Sigma, USA) at a concentration of 10 μM for 2 h; then, the medium was changed to atorvastatin (100 nmol/L) at 6 h for qRT-PCR and at 3 days for Western blot.
Quantitative Real-Time Reverse Transcription Polymerase Chain Reaction (qRT-PCR)
Total RNA was extracted from tissues and cultured BMSCs. Real time PCR was conducted as previously described . The primers were obtained from SBSgene (www.sbsgene.com) with the following primer sequences: Sirt1, 5′-GCA GGT TGC AGG AAT CCA A-3′ and 5′-GGC AAG ATG CTG TTG CAA A-3′ (63 bp, XM_006514342.1); Runt-related transcription factor 2 (Runx2), 5′-TGT TCT CTG ATC GCC TCA GTG-3′ and 5′-CCT GGG ATC TGT AAT CTG ACT CT-3′ (146 bp, XM_006523545.1); ALP, 5′-CAC GCG ATG CAA CAC CAC TCA GG-3′ and 5′-GCA TGT CCC CGG GCT CAA AGA-3′ (479 bp, XM_ 006538500.1); osteocalcin (OCN), 5′-ACC CTG GCT GCG CTC TGT CTC T-3′ and 5′- GAT GCG TTT GTA GGC GGT CTT CA-3′ (240 bp, NM_007541.3); and glyceroldehyde -3-phosphate dehydrogenase (GAPDH), 5′-AGC CTC GTC CCG TAG ACA-3′ and 5′-CTC GCT CCT GGA AGA TGG-3′ (255 bp, NM_008084.3).
Western Blot Analysis
All samples were collected, and the Western blot was conducted as previously described . The primary antibodies used were rabbit anti-Sirt1 (Cat.No:13161-1-AP;1:2000; Proteintech, Rosemont,USA), mouse anti-Runx2 (Cat.No:ab54868;1:1000; Abcam, CA, USA), and mouse anti-GAPDH (Cat.No:KC-5G4;1:10,000; Kangcheng, Shanghai, China). The protein bands were quantitatively analyzed using an image analysis system (QuantityOne software; Bio-Rad, Hercules, CA, USA).
All data are presented as the mean ± standard deviation. We used two-tailed t-tests to determine the significance between two groups. We analyzed the results of multiple groups by two-way ANOVA with Bonferroni post-hoc tests using SPSS version 17 software. For all statistical tests, we considered a P value < 0.05 statistically significant.