Construction of ovariectomized osteoporosis rat model
A total of 32 female Sprague‑Dawley rats (3 months old) were purchased from the Experimental Animal Center of Sun Yat‑Sen University. All rats were numbered and randomly divided into ovariectomy group(24) and sham operation group(8). The ovariectomy group were performed bilateral oophorectomy, The sham operation group preserved the complete structure and function of ovary and fallopian tube, and excised the same volume of adipose tissue around the ovary. Penicillin 20000 U/kg was injected intramuscularly for 3 days. Three months after operation, the rats in each group were sacrificed by cervical dislocation in accordance with the guidelines of ARRIVE, lumbar spine bone mineral density (BMD) was measured by Micro-CT. The lumbar bone tissue and adipose tissue stored in liquid nitrogen for further experimentation. The present study was approved by the Animal Care Committee of Sun Yat‑Sen University [no. (2019)196] and was performed in accordance with the guidelines for the use of laboratory animals.
Immunohistochemistry
The decalcification of bone tissue and adipose tissue was used for Immunohistochemistry. First, Copy: paraffin section, 37 ℃ oven overnight, Bake in 60 ℃ oven for 2h before dewaxing. Dewaxing and hydration: put the slices into xylene I and II for 10min respectively, Ethanol gradient (100%, 95%, 80%, 70%) for 5min, Wash with water for 5min, TBS 3min×3, then filter paper to dry around the tissue and nail polish. Antigen repair: hyaluronidase+pepsin repair, hyaluronidase 37 ℃, 30min, TBS 3min×3, Pepsin 37 ℃, 30min, TBS 3min×3. Sealing: 5% BSA sealing solution at 37 ℃ for 30min, dry without washing. Incubate the primary antibody: dilute the primary antibody with TBS or PBS and wet box at 4 ℃ overnight. The next day, incubate the secondary antibody: TBS for 5min×5. Add 3% H2O2 dropwise on the slices, keep away from light at room temperature for 10min, add polymerized HRP labeled secondary antibody dropwise, 37 ℃, 30min, TBS for 5min×5. Counterstaining: counterstaining with hematoxylin, blue returning with saturated sodium monohydrogen phosphate solution. Seal: dehydration: 70%, 80%, 95% and 100% ethanol for 3min respectively. Transparent: xylene I and II for 5min respectively. Neutral gum seal. Photographing: low magnification first and then high magnification, determine the exposure and contrast satisfactory for dyeing, make a unified ruler, and take photos from different visual fields for preservation.
Isolation and culturing of BMSCs
A total of 10 female Sprague‑Dawley rats (4-6 weeks old; 100-120 g) were purchased from the Experimental Animal Center of Sun Yat‑Sen University for use in the present study. The rats were allowed to adapt to the housing conditions for 7 days, during which the rats were maintained under a 12-h light/dark cycle at 22˚C with free access to food and water (23,24). Afterward, the animals were sacrificed by cervical dislocation in accordance with the guidelines of ARRIVE, BMSCs were harvested from the femoral and tibial medullary cavities by flushing with a 5 ml syringe and suspended in Dulbecco's modified Eagle medium F‑12 (DMEM/F12) growth medium (GM) with 10% FBS(Gibco, cat.no.10099141C,Australia). The cells were cultured in a humidified incubator at 37˚C under an atmosphere with 5% CO2 and the third generation of BMSCs were used in the experiments. The present study was approved by the Animal Care Committee of Sun Yat‑Sen University [no. (2019)196] and was performed in accordance with the guidelines for the use of laboratory animals.
Flow cytometry
BMSCs were isolated from the bone marrow, and sorted by conducting CytoFLEX fow cytometry (Beckman Coulter, USA). In details, the obtained cells were then re-suspended in PBS supplemented with 0.1% FBS and 0.5% NaN3, centrifuged, and rinsed. After re-suspension, the cells were incubated with antibodies specifc to CD29, CD34, CD45, and CD105 (Abcam, UK) at room temperature in conditions devoid of light for 30 min. The supernatant was discarded and the precipitate was rinsed in PBS. After that, the precipitate was centrifuged at 1000 rpm for 5 min and the supernatant was removed after centrifugation. The centrifugation was repeated for three times. The harvested precipitate was re-suspended in 300 μl PBS. Before testing, the instrument was sterilized under 30 min ultraviolet irradiation, while the fow tubes were sterilized by hypochlorous acid. Sorting parameters were set based on the forward scatter (FSC), side scatter (SSC), and size of the cells. The separator was installed where the cells were sorted. Cells with the largest volume and quantity and most intra-cellular granules were selected as target cells for further experimentation.
Osteogenic differentiation
When BMSCs fusion reached 60%-70%, add 2 ml osteogenic induction medium (Glutamine, ascorbic acid β-Sodium glycerophosphate, dexamethasone,Cyagen,USA) into the culture dish. Fresh osteogenic induction medium is used every 3 days. After 2-4 weeks of induction, calcium nodules were stained with Alizarin Red S dye.
Adipogenic differentiation
When BMSCs fusion reached 100% or over fusion, add 2 ml of adipogenic induction medium solution A (Glutamine, insulin, 3-isobutyl-1-methylxanthine, rosiglitazone, dexamethasone,Cyagen,USA) to the culture dish. After 3 days of induction, suck out solution A and add 2 ml of adipogenic differentiation medium solution B (insulin,Cyagen,USA), after 24h, suck out liquid B and replace it with liquid A for induction. After the alternating of solution A and solution B for 3-5 times (12-20 days), continue to maintain the cells with solution B for 4-7 days until the lipid droplets become large and round enough.
Lentivirus-Phactr1 transfection
Phactr1 lentivirus were procured from Shanghai GeneChem Co. 1ml transfection enhancer Polybrene fresh culture medium(8μg/ml) was added to the well plate, and then the diluted and blown Phactr1 lentivirus and positive control virus suspension were 100μl respectively add to the well plate with gently mixing. After 48 hours, the medium containing virus was replaced with fresh medium (no Polybrene). On the third day, the virus with fluorescent label began to express the fluorescent signal observed by fluorescence microscope. On the third day after BMSCs transfection, G418 culture with screening concentration was added for one week, the remaining cell population was digested with 0.25% enzyme and divided into two groups. One was screened and cultured in normal whole medium, Another cell was subcultured. The transfection efficiency was determined by western blot analysis of the control group and Lenti-Phactr1 group.
siRNA-Phactr1 Interference
siRNA Phactr1 was procured from Shanghai GeneChem Co. The day before interference, 3×105 cells/cm2, 2.5ml growth medium without antibiotics was added to each well. Remove the growth medium from the cells, Add 2.5 ml of fresh serum-free growth medium. Sterile ddH2O configuration concentration is 20 μmol siRNA mother liquor, sub packed, frozen at -20℃ for standby. Mix an selected amount of interference chain with transfection reagent, gently blowed at room temperature for 15-20 minutes. Add the mixture to the cell culture dish containing serum-free growth medium and shake it gently. After 5-6 hours, The medium containing serum was added for 48 hours. The interference efficiency was determined by western blot analysis of the control group and siRNA-Phactr1 group.
Western blot analysis
Western blot analysis was performed using standard techniques. Following 0, 3 ,7 days of osteogenic differentiation in control, Lenti-Phactr1 and KD025 groups, Following 0, 3 ,7 days of adipogenic differentiation in control, siRNA-Phactr1 and KD025 groups, BMSCs were lysed in 50 µl of protein extraction reagent (M-PER) (BestBio) and protease inhibitor cocktail. The protein samples were harvested following centrifugation for 15 min (12,000×g,4˚C) and boiled for 5 min, total protein concentration was determined using a NanoDrop 2000 spectrophotometer. Equal volumes (20 µl) of the samples were separated via 10% SDS-PAGE and subsequently transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were blocked with 5% non-fat dry milk and then incubated overnight at 4˚C with primary antibodies specific for rabbit anti-Phosphatase and Actin Regulator 1 (Phactr1,1:1000, cat. no. 23446-1-AP, Cell Signaling Technology, Inc.), Mouse ranti-Runt-related transcription factor 2 (Runx2,1:500,ab76956), rabbit anti-CCAAT/Enhancer-Binding Protein (C/EBPα,1:500,ab140479), rabbit anti-Rho-associated coiled-coil-containing protein kinase 2 (ROCK2,1:500,ab66320),and anti-β-tublin (1:1000, cat. no. 2128, Cell Signaling Technology, Inc.). The samples were rinsed with tris-bufered saline tween 20 (TBST) three times (10 min each time). After that incubation with a alkaline phosphatase-conjugated goat anti-IgG (1:2,000, cat. no. 7054; Cell Signaling Technology, Inc.) for 1 h at room temperature. The immunoreactive proteins were visualized using a chemiluminescence kit (EMD Millipore), and The gray value of target band was quantifed by ImageJ software.
Alizarin red staining
After osteogenic differentiation, use 1×PBS flushing1-2 times. Add 2 ml of 4% neutral formaldehyde solution to each well and fix for 30 min. Absorb neutral formaldehyde solution and use 1×PBS flushing twice. Add 1 ml alizarin red dye solution to each well for 3-5 min. Absorb alizarin red dye and use 1 × PBS flushing 2-3 times. The culture plate was placed under the microscope to observe the calcium nodules.
Oil red O staining
After adipogenic differentiation, used 1×PBS flushing 1-2 times. Add 2 ml of 4% neutral formaldehyde solution to each well and fix for 30 min. Absorb neutral formaldehyde solution and use 1×PBS flushing twice. Add 1 ml of oil red O dye working solution into each well for 30min (working solution preparation method: oil red O storage solution: distilled water = 3:2, mix well and filter with neutral filter paper). Absorb the oil red O dye solution and use 1×PBS flushing 2-3 times. The culture plate was placed under the microscope to observe the effect of lipid droplets staining.
Co-Immunoprecipitation
Generally, The proteins of BMSCs on the 7th day of osteogenic and adipogenic differentiation were extracted respectively, add specific antibody 2 μg, gently incubate at 4 °C for 12h on a shaking bed, add protein A/G agarose beads 40 μg, continue to incubate at 4 °C for 6h, centrifuge at 2500 rpm for 15min, remove the supernatant, wash with PBS buffer for 3 times (2500 rpm, 5min each time), and finally lyse the agarose beads and immunoprecipitation complex with lysate, Collect the lysate, The rest steps(Electrophoresis and Transfer, Immunoblotting and Detection) are the same as Western blot analysis.
Active RhoA detection
Active RhoA detection was carried out using a kit from Cell Biolabs, Inc. (San Diego, USA). In brief, the cells were collected and washed with cold PBS, and lyzed in a Lysis/Binding/Wash Buffer. The cell lysates were incubated with glutathione resin and GST-Rhotekin-RBD to allow GTP-bound GTPase binding to the glutathione resin through GST-linked binding protein. The bound proteins were eluted with SDS buffer containing DTT, then run on SDS-PAGE gel and analyzed by Western blotting to detect the presence of active RhoA using a rabbit anti-RhoA antibody. HRP-linked anti-rabbit IgG was used as the secondary antibody.
Statistical analysis
Statistical analyses were performed using SPSS 22.0 software (SPSS, Inc.). The data are presented as the means ± standard deviation. Differences between the quantitative values among multiple groups were analyzed with one-way analysis of variance (ANOVA) with the Tukey's test. P<0.05 was considered to indicate a statistically significant difference.