Animals
All animal procedure, perations and experimental were approved and performed and experimental protocols were approved by the guidelines of the Animal Care Committee of the Fourth Military Medical University, Xi’an, Shaanxi, China. Two-month-old female C57BL/6J mice were purchased from the Animal Experimental Center of Fourth Military Medical University. Sixteen-month-old female C57BL/6J mice were purchased from Changzhou Kaiwensi Laboratory Animal Center, Jiangsu, China. All mice were housed under specific pathogen-free conditions (22°C, 50%-55% humidity) on a 12 h light/12 h dark cycle with food and water easily accessible.
Micro-computed tomography analysis
The mouse femora at mid-diaphysis were scanned with the GE micro-CT system (GE, USA). X-ray source was set at 80 kV, and 80 μA microfocus. Three-dimensional images were reconstructed and data analysis was performed with GEHC MicroView analysis software. The relevant bone morphometric parameters, including trabecular bone mineral density (BMD), trabecular volume relative to total volume (BV/TV), and cortical bone thickness(Ct.Th)were measured.
Isolation and culture of C57B/L BMMSCs
Bone marrow-derived mesenchymal stem cells were harvested from femora and tibiae of 2m-old young C57BL/6J and 16m-old aged C57BL/6J mice. Mice were sacrificed with cervical dislocation and sterilized with 75% alcohol. Femora and tibiae were separated and attached muscle was stripped. After epiphyses were amputated and bone marrow was exposed, primary BMMSCs were drawn out, cultured with basal medium containing α-MEM medium (Gibco, Grand Island, NY, USA), 20% FBS- (Sijiqing, Hangzhou, China), 2 mM L-glutamine (Invitrogen, Carlsbad, CA), 100 U/mL penicillin and 100 U/mL streptomycin (North China pharmaceuticals company, Shijiazhuang, China), and incubated at 5% CO2 at 37°C. The medium was changed every 3 day. Cells were digested with 0.25% trypsin when confluence reached 90%. BMMSCs used for the majority of experiments was at passage one in this study.
Isolation and culture of C57B/L myoblasts
Muscles were harvested from the hind limbs of 2-month-old young C57BL/6J and 16-month-old aged C57BL/6J mice. Firstly, the excessive connective tissues and fat were separated from muscle in cold sterile PBS. Then, muscles were cut into small pieces and enzymatically digested with 400 IU/ml collagenase II (Worthington) at 37 °C for 1 h. The digested slurry was sequentially passed through a 70-μm and then 30-μm cell strainer (BD Falcon). The filtrate was centrifuged at 1000g, and the pellets were suspended in myoblast growth medium (Ham’s F-10 medium with 10% FBS) and incubated at 5% CO2 at 37°C. Briefly, the cell suspension was seeded for 15-30min to allow quick adherence of fibroblasts, thus leaving a purer population of myoblasts in the supernatant, which was then transferred to another dish for subculturing. Myoblasts were digested with 0.25% trypsin when confluence reached 80%.
Senescence-associated β-galactosidase staining
The femora were fixed in 4% paraformaldehyde, decalcified with 17% EDTA (pH7.0), dehydrated with 30% sucrose and embedded in OCT. 15 μm-thick longitudinal sections were prepared and collected on slide for SAbetaGal staining. The β-galactosidase activity was assessed with a SAbetaGal staining kit (Cell signaling). Briefly, the slices were washed twice with PBS, and fixed with Fixative Solution of the SAbetaGal staining kit at room temperature for 10-15 min. Then, the samples were washed twice with PBS, covered with SAbetaGal staining solution and incubate at 37°C overnight. On the next day, the slides were washed with PBS three times and 80% glycerin was mounted on the samples. Then, coverslips were inverted onto slide and excess glycerin were removed. The SAbetaGal positive cells were observed under a microscope.
Osteogenic and adipogenic differentiation assays
Young and aged BMMSCs were seeded at the density of 4.2×104 cells per cm2 on 6-well or 12-well plastic plates and cultured with basal medium. When cell confluence reached 80%,cells were induced with osteogenic differentiation medium (100 nmol/L dexamethasone, 50 μg/mL ascorbic acid, and 5mmol/L β-glycerophosphate) up to one week for western blot assay or two weeks for Alizarin Red staining assay, with the medium changed every 3 days. To assess osteogenic differentiation, cells were fixed with 4% paraformaldehyde and stained with 1% alizarin red. The expressions of Runx2 and ALP were detected by western blot.
Young and aged BMMSCs were cultured the same method described above. When cell confluence reached 85%, cells were induced with adipogenic differentiation medium (0.5 mol/L 3-lisobutyl-1-methyxanthine, 200 μmol/L indomethacin, 1μmol/L hydrocortisone and 10 μg/ml insulin) up to 5 days for western blot or 7 days for Oil Red O staining , with the medium changed every 3 days. Lipid droplet formation in cells was detected by staining with Oil Red O solution. The expression of PPAR-γ was detected by western blot.
Oil Red O staining
For cells Oil Red O staining, the BMMSCs were fixed with 4% paraformaldehyde for 15 minutes at room temperature, and then stained with Oil Red O staining for 15 minutes at room temperature. The stained cells were washed twice with PBS and then observed under microscope. The images were analyzed with Image pro plus software.
Alizarin Red staining
Alizarin Red staining was performed to determine mineralization after 14 days of osteogenic induction. BMMSCs were fixed with 60% isopropanol for 90 seconds, and washed once with ddH2O. Then the cells were stained with 1% Alizarin Red (Sigma Aldrich) for 3-5 minutes and washed twice with ddH2O. Quantitative parameters of the mineralized area were analyzed with Image J software.
Alcian blue staining
Alcian blue staining was carried out to detect the chondrocytes in growth plates of femora. Firstly, the samples of decalcified femurs were cut into 10μm-thick and mounted on slides. Then, samples were stained with 0.1 % Alcian blue staining solution containing acetic acid for 20 minutes at room temperature. Then slides were washed twice with ddH2O and the positive areas were observed under microscope.
Immunofluorescent staining
For cells immunofluorescence staining, cells were seeded on 3.5 cm confocal dish at the density of 3×104. Cells were treated with or without the autophagy-flux inhibitor chloroquine (CQ)(50 μM) 3-5 hours before staining. In sequence,cells were fixed with 4% paraformaldehyde at 4°C for 10-15 minutes, washed with PBS, incubated with 0.5% triton-100 at room temperature for 10 minutes, and blocked with PBS containing 1% BSA at room temperature for 40 minutes. Next, the samples were incubated with primary antibodies to LC3 (Cell Signaling Technology, 1274, 1:100), aggrecan (GeneTex, GTX54920, 1:100), collagen Ⅱ (Abcam, ab34712, 1:100 ), OCN (Santa Cruz Biotechnology, sc-390877, 1:100), PPAR-γ (Abcam, 2435, 1:50), TRAP (Santa Cruz Biotechnology, sc-30833, 1:100) overnight at 4°C and subsequently incubated with fluorescent secondary antibodies respectively. The positive cells were examined under a laser scanning confocal microscope (Olympus FluoViem FV 1000, Tokyo, Japan). Quantitative histomorphometric analysis was conducted with Image pro plus software.
Transmission Electron Microscopy (TEM) analysis
Cells were harvested and fixed in 4% glutaraldehyde in 0.1 M PB (pH 7.4) for 24 hours, followed by 1% osmic acid for 2h. After fixation, cells underwent osmosis by acetone and 812 resins. Then, samples were embedded with Epon 812, and kept in a thermostatic drying oven for 4 hours at 36℃, 6 hours at 45 ℃ and 12hours at 60℃. Afterwards, embedding blocks were successively cut into semithin sections (1-2 μm) and ultrathin sections(50-100 nm). Then, samples were stained with uranyl acetate and lead citrate. Finally, images were captured with a transmission electron microscope (FEI, USA) at an accelerating voltage of 80–120 kV.
qRT-PCR analysis
Total RNA was extracted from BMMSCs, myoblasts, bone marrow and muscle by RNAiso plus (TaKaRa, Japan) according to the manufacture’s instruction. Then, the mRNA was reversely transcribed into cDNA by PrimescriptTM RT master mix(TaKaRa,RR036A). Real-time PCR was performed with SYBR Premix Ex TaqTMⅡ (TaKaRa), and detected by CFX96 Trademark Real-time PCR detection system (Bio-rad, USA). The primers used in Real-time PCR were listed in the supplementary table 1.
Western blotting analysis
Total proteins were harvested from BMMSCs, bone marrow and other organs with RIPA lysis buffer (Beyotime, China) and quantified by BCA assay. Next, the proteins were separated on sodium dodecyl sulfonate-polyacrylamide gels (SDS-PAGE), transferred to PVDF membranes (Millipore, Billerica, MA, USA), blocked in TBST containing 5% BSA, and incubated in first antibodies with Beclin1 (Cell signaling, 3738, 1:1000), ATG7 (Cell signaling technology, 8558, 1:1000), LC3 (Cell signaling technology, 1274, 1:1000), p62 (Proteintech, 18420-1-AP, 1:1000), HDAC9 (Abcam, ab59718, 1:1000), H3K9ac (Abcam, ab10812, 1:1000), H3K18ac (Cell signaling technology, 9675, 1:1000), H4K16ac (Abcam, 13534, 1:1000), H3 (Cell signaling technology, 9715, 1:1000), p53 (Cell signaling technology, 2524, 1:1000), phospho-p53 Antibody (R&D systems, AF1043, 1:2500), Runx2 (Cell signaling technology, 2435, 1:1000), ALP (R&D systems, AF2910, 1:500), PPAR-γ (Abcam, 2435, 1:300), GAPDH (Cwbiotech, CW0100, 1:4000), respectively. Then, the membranes incubated in secondary antibodies which coupled to peroxidase (Cwbiotech, China). Finally, the signals were detected by an enhanced chemiluminescence kit (7seapharmtech, China).
HDACs inhibitor TSA and NaB treatment
BMMSCs were cultured in the medium with Trichostatin A (TSA, sigma) at 50, 100 and 200 (n mol/L) or sodium butyrate (NaB, sigma) at 50, 100, 200 and 400 (μ mol/L). To assess the effects of TSA or NaB on inhibition of HDACs in cells, HDAC1-11 was detected by qRT-PCR. The expressions of HDAC9 and acetylated H3k9 were detected by western blotting. To examine the effects of HDACs on BMMSCs differentiation, the cells were treated with or without NaB or TSA at their effective concentrations selected above.
siRNAs interference
Small interfering RNA (siRNA) targeting mice BECN1(Ribobio, China), or HDAC9 (Santa Cruz, USA)were transfected into cells at a final concentration of 50 nM using riboFECT™ CP(Ribobio, China). siNC(Ribobio, China)was used as negative controls. All steps were performed according to the instruction in the riboFECT™ CP kit. The following experiments were performed according to the experimental designed. In detail, siRNA silencing HDAC9 was transfected into BMMSCs to investigate the effects of HDAC9 on osteogenic and adipogenic differentiation of BMMSCs and binding to the promoter of autophagy-related genes. In addition, aged BMMSCs were cotransfected with siHDAC9 and siBECN1 to investigate the hypothetical HDAC9-autophagy axis which may regulate BMMSCs lineage differentiation.
Chromatin immunoprecipitation (ChIP)
To confirm the interaction between HDAC9 and targeting genes, chromatin immunoprecipitation assays were performed according to the manufacturer’s protocol (Millipore, LSKMAGG02, USA). Antibodies against HDAC9 (Abcam, ab59718) and polyclonal anti-Histone H3 (acetyl K9) (Abcam, ab10812) were incubated with randomly interrupted genome DNA samples. Normal rabbit IgG (Merck Millipore) was used as negative control. Then, antibodies-DNA complexes were captured by protein A/G magnetic beads. Finally, the precipitated DNA samples were detected by qRT-PCR, and the results were normalized to the input value. The primers designed according to the promoters of Atg7, BECN1, LC3a and LC3b (Sangon biotech, China) are listed in Supplementary Table 2.
shHDAC9 virus injection in vivo
The shRNA sequences for targeting HDAC9 were, forward sequence:5'-CCGGCTGGGCACAAAATTCTGAACACTCGAGTGTTCAGAATTTTGTGCCCAGTTTTTG-3', reverse sequence: 5'-AATTCAAAAACTGGGCACAAAATTCTGAACACTCGAGTGTTCAGAATTTTGTGCCCAG-3. The shHDAC9 lentivirus was packaged by cotransfecting shHDAC9 lentiviral vector with two packaging systems (pMD2.G and psPAX2) in 293T cells, and the medium containing virus was collected and concentrated 48 hours later. Detailly, the medium was centrifuged at 800rpm and 4°C for 10 mins, and the supernatant was filtered by a filter with 0.45μm. Then, the filtrate was mixed with Lenti-X (TaKaRa, 631231) and kept at 4°C overnight. Next day, the mixture was centrifuged for 40 minutes at 1500g and the precipitate was resuspended with PBS whose volume is 1/100 of collected medium. The final virus titer was more than 1×108/ml. To evaluate the effect of HDAC9 on bone mass/skeletal metabolism in senescence induced bone loss, aged mice (n=21) were randomly divided into three groups (7/each group), named the control group, the shScr group and the shHDAC9 group, respectively. Aged mice anesthesized through intraperitoneal injection with 1% pentobarbital sodium and were administrated with 20 μl of lentivirus containing shHDAC9 or empty vector every two weeks for 1 month through intra-bone marrow injection in the distal femora. After an one-month or a two- months treatment, the mice from above three groups were sacrificed by cervical dislocation and femora from each group were collected for micro-CT scanning, Oil Red O staining and SAbetaGal staining. BMMSCs were harvested and cultured from femora for lineage differentiation analysis and autophagy analysis as above methods
Statistics
The data were presented as the mean ± s.d. Comparisons were made using t- test and one-way ANOVA for experiments with more than two groups. All of the experiments were repeated more than three times, with representative experiments shown. The P values of less than 0.05 were considered significant.