BMSC cell culture
Primary cultures of rat BMSCs were prepared from Sprague Dawley rats aged 3 to 4 weeks weighing 100 ± 10 g. Male rats maintained in specific-pathogen-free (SPF) conditions were provided by the Animal Experimental Center of the Second Affiliated Hospital of Harbin Medical University, Harbin, China. For the experiments involving in vitro osteogenic differentiation of BMSCs, rats were obtained from the Animal Experimental Center and sacrificed by cervical dislocation after being euthanized with CO2 inhalation. This study was approved by the Institutional Animal Care and Use Committee of Harbin Medical University.
After CO2 euthanasia, rats were sacrificed by cervical dislocation and soaked in 75% ethanol for 10 min. The bilateral femur and tibia of rats were removed under aseptic conditions. The periosteum and muscle tissue were dissected, and the femur and tibia were washed with phosphate buffered saline (PBS) three times. Next, the medullary cavity was cut and washed repeatedly with DMEM/F12 medium (Hyclone; Logan City, Utah, USA) containing 10% fetal bovine serum (Biological Industries; Beit Haemek, Israel). The bone marrow cells were inoculated into cell culture flasks and cultured in a humidified atmosphere with 5% CO2 at 37˚C. When reaching 80% confluence, the adherent cells were passaged after trypsin digestion, and third generation BMSCs (P3 cells) at the logarithmic growth phase were used for subsequent experiments.
To induce commitment toward an osteogenic lineage, P3 cells were cultured in osteogenic differentiation induction (OS) medium, which was comprised of DMEM/F12 culture medium supplemented with fetal bovine serum (10%), 50 μg/ml vitamin C, 10 mmol/L β-glycerophosphate, and 0.1 μmol/L dexamethasone (Solarbio; Beijing, China), as previously reported[27].
Immunofluorescence analysis of BMSCs
BMSCs at the third passage were fixed with 4% paraformaldehyde for 15 min and blocked with 5% bovine serum albumin (BSA) in TBST (Tris-buffered saline, 0.1% Tween 20). Cells were probed with primary antibodies (1:100 dilution) against CD44, CD90, CD31, and CD34 (Absin, Shanghai, China) overnight at 4°C. Bound antibodies were detected with Cy3-conjugated goat anti-rabbit IgG (1:400 dilution; Absin, Shanghai, China) and FITC-conjugated goat anti-mouse IgG (1:100 dilution, Zhongsu Jinqiao, Beijing, China), followed by a nuclear staining with 4’,6-diamidino-2-phenylindole (DAPI). Cells stained with secondary antibody only were considered negative controls. Fluorescent signals were detected using a fluorescence microscope (Nikon Eclipse Ti, Nikon, Japan).
Cell treatment and grouping
The P3 cells cultured in OS medium were treated with H2O2 (50 μmol/L to 200 μmol/L) or 0.5 μmol/L Wortmannin (phosphatidyl inositol-3 kinase (PI3K) inhibitor; Solarbio; Beijing, China), as indicated in each experimental design. H2O2 (100 μmol/L) was selected to test the impacts of Moringa leaf-containing serum (10%). The P3 cells were divided into the following groups: osteogenic induction in regular OS medium (Control group), osteogenic induction in medium supplemented with 10% Moringa leaf-containing serum (MO group), osteogenic induction in OS medium supplemented with 100 μmol/L H2O2 (OS+H2O2 group), osteogenic induction in medium supplemented with 10% Moringa leaf extract-containing serum and 100 μmol/L H2O2 (MO+H2O2 group), osteogenic induction in medium supplemented with 10% Moringa leaf-containing serum, 100 μmol/L H2O2, and 0.5 μmol/L Wortmannin (MO+H2O2+Wor group), and osteogenic induction in medium supplemented with 10% Moringa leaf-containing serum, 100 μmol/L H2O2, and DMSO (MO+H2O2+DMSO group; DMSO is solvent of Wortmannin, and its dosage was the same as the MO + H2O2 + Wor group). H2O2 treatment lasted 24 h starting at 0 h, and the cells in the MO+H2O2+Wor group were treated with 0.5 μmol/L Wortmannin for 24 h starting from 0 h. The OS medium or the medium supplemented with 10% Moringa leaf extract-containing serum was changed every 3 days. Cell cycle, oxidative stress, and protein expression were all assayed at 48 h after initiation of osteogenic differentiation. After 7 days of induction, the expression levels of genes for osteogenic markers were determined. ALP staining was examined on day 14 and osteogenesis determination was examined on day 21 after osteogenic differentiation.
Preparation and analysis of drug-containing serum
Male rats weighing 250 ± 20 g were administered Moringa leaf solution through oral dosing (0.5 g/kg/day) at the frequency of twice per day for 3 consecutive days. Rats in the control group were given normal saline. At 1 h after the last dosing, blood was extracted from the heart after the euthanasia. Blood was collected and centrifuged at 300 rpm for 20 min at room temperature. The serum was sterilized by suction filtration and stored at -20 °C. This study was approved by the Institutional Animal Care and Use Committee of Harbin Medical University.
The drug-containing serum was analyzed using Non-target High Performance Liquid Chromatography (HPLC). LC-MS/MS analyses were performed using an UHPLC system (1290, Agilent Technologies; Santa Clara, CA, USA) with a UPLC HSS T3 column (2.1 mm × 100 mm, 1.8 μm) coupled to Q Exactive (Orbitrap MS, Thermo Fisher Scientific; Waltham, MA, USA). The mobile phase A was 0.1% formic acid in water for positive, and 5 mmol/L ammonium acetate in water for negative, and the mobile phase B was acetonitrile. The elution gradient was set as follows: 0 min, 1% B; 1 min, 1% B; 8 min, 99% B; 10 min, 99% B; 10.1 min, 1% B; and 12 min, 1% B. The flow rate was 0.5 mL/min. The injection volume was 3 μL. The QE mass spectrometer was used because of its ability to acquire MS/MS spectra on an information-dependent basis (IDA) during an LC/MS experiment. In this mode, the acquisition software (Xcalibur 4.0.27, Thermo Fisher Scientific) continuously evaluates the full scan survey MS data as it collects and triggers the acquisition of MS/MS spectra depending on preselected criteria. ESI source conditions were set as follows: Sheath gas flow rate as 45 Arb, Aux gas flow rate as 15 Arb, Capillary temperature 400°C, Full ms resolution as 70,000, MS/MS resolution as 17,500, Collision energy as 20/40/60 eV in NCE model, and Spray Voltage as 4.0 kV (positive) or -3.6 kV (negative), respectively.
The raw data were converted to the mzXML format using ProteoWizard and processed using MAPS software (Version 1.0). The preprocessing results generated a data matrix that consisted of the retention time (RT), massto-charge ratio (m/z) values, and peak intensity. In-house MS2 database was applied for metabolite identification.
MTT assay
At 72 h after osteogenic differentiation under different treatment conditions, 20 μL MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)] solution (5 g/L) was added to each well of the 96-well plates, and cells were incubated for another 4 h in the incubator (37°C and 5% CO2). The medium in each well was aspirated and 100 μl of DMSO was added. Cell viability was quantified by measuring absorbance at 490 nm on a microplate reader. The optical density (OD) values of samples in experimental groups were normalized to that of the control groups without H2O2 or drug treatments at the same time point.
Measurement of intracellular peroxide markers
The reactive oxygen species (ROS) test kit was purchased from Beyotime Biotechnology (Shanghai, China). The SOD (superoxide dismutase), MDA (malondialdehyde), and GSH-PX (glutathione peroxidase) test kits were obtained from Nanjing Institute of Bioengineering Research Institute (Nanjing, China). Assays to measure intracellular ROS and MDA levels, as well as SOD and GSH-PX activity of BMSCs were conducted following the manufacturer's protocols. Briefly, after removing the medium, 10 μmol/L DFCH-DA diluted in serum-free medium was added to each group of cells and incubated at 37°C for 20 min in the dark. Then the cells were washed three times with serum-free cell culture medium, and the fluorescence intensity of each well was detected using a fluorescence microplate reader at an excitation wavelength of 488 nm to calculate intracellular ROS levels. To determine SOD activity, total protein was extracted and the protein concentration was determined using the BCA method. The SOD inhibition rate was calculated according to the formula provided by the manufacturer. To determine MDA levels, the homogenate of treated cells was prepared, and the MDA value was calculated using a colorimetric measurement of each tube at 532 nm after the addition of the reagents. To detect GSH-PX activity, absorbance of the samples was determined at 412 nm at the end of the reaction using a microplate reader. All results were calculated according to the manuals provided with the kits.
Cell cycleanalysis
Cells were fixed with 70% ethanol at 4˚C overnight. After centrifugation at 1,500 rpm for 5 min and two washes with PBS, cells were incubated with 0.1 mg/mL RNase A (Thermo Fisher Scientific) at 37˚C for 30 min and then 0.05 mg/ml propidium iodide (PI) at 4˚C for another 30 min. The DNA content was determined using a flow cytometer (FACSCalibur, BD Biosciences; Franklin Lakes, NJ, USA), and 2 - 3 million cells were collected. Data were analyzed using the Modfit LT software (Verity Software House).
Osteogenesis-related gene expression
Total RNA was extracted using the Extreme RNA extraction kit (HaiGene; Harbin, China) following the manufacturer’s protocols. The eluted RNA was proportionally added to 2× RNA Loading Buffer and denatured at 65°C for 10 min. The concentration of RNA was measured using an ND5000 ultra-micro UV spectrophotometer with the ratio of OD 260/280. Synthesis of cDNA was performed using Golden 1st cDNA Synthesis Kit (HaiGene). Quantitative real-time PCR (RT-qPCR) was performed on a Fluorescence quantitative PCR system using SYBR Green Fluorescence Quantification PCR Kit (HaiGene). The reaction conditions were 15 min at 95°C, 40 cycles of 10 sec at 95°C, and 30 sec at 60°C. The relative expression levels of each gene were analyzed using the 2-ΔΔCt method and normalized to GAPDH expression. The sequences of primers for each gene were as follows:
ALP, 5’-CGGCGGATGATAAGGAGGAC-3’ (forward),
5’-GGCGTGTAACAGATGGAAACC-3’ (reverse);
Collagen I, 5’-AAGGGGTGGGGTGGGAAG-3’ (forward),
5’-GAGAGCAGGCTGGAGTTGG-3’ (reverse);
Osteopontin (OPN), 5’-GCTTGGCTTACGGACTGAGG-3’ (forward),
5’-CTGGGCAACTGGGATGACC-3’ (reverse);
Runx2, 5’-GCGGAACAACAACAACAACAAC-3’ (forward),
5’-GAAAGCAAATCTTGGGCAATAGC-3’ (reverse);
GAPDH, 5’-AACTCCCATTCTTCCACCTTT-3’ (forward),
5’-CTCTTGCTCTCAGTATCCTTG-3’ (reverse).
Alkaline phosphatase(ALP) activity assays
ALP activity was detected using the ALP activity kit (Solarbio; Beijing, China) according to the manufacturer’s protocol. Briefly, BMSCs were fixed with the ALP fixative for 3 min. Then, the ALP incubation solution was added, and cells were incubated in the wet box for 20 min in dark. After washing with distilled water, the stained cells were visualized under a microscope and photographed.
Quantifying matrix mineralization via alizarin red staining
Matrix mineralization was evaluated using alizarin red S staining. At 21 days after BMSC osteogenic differentiation under the indicated conditions, cells were washed twice with PBS and fixed with 4% paraformaldehyde for 30 min at 25℃. Then the cells were stained with 1% Alizalin Red (pH 4.2; Solarbio; Beijing, China) for 30 min and visualized under a microscope and photographed as previously described[28].
Western blot analysis
Total protein was extracted from the treated cells with RIPA (radioimmunoprecipitation assay) lysis buffer and the total protein concentration was determined using a BCA assay kit (Beyotime; Shanghai, China). Protein samples in loading buffer were heated at 100°C for 5 min, and an equal amount of protein for each sample was loaded onto 12% polyacrylamide gels (Beyotime Biotechnology). After electrophoresis, proteins were transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% BSA and incubated with the primary antibodies overnight a 4°C and the secondary antibody for 1 h at room temperature. The primary antibodies were as follows: anti-caspase-3 (1:1,000 dilution; Cell Signaling Technology; Danvers, MA, USA), anti-Akt (1:1,000 dilution; Cell Signaling Technology), anti-phosphorylated (p)-Akt (1:2,000 dilution; Cell Signaling Technology), and anti-Foxo1 (1:1,000 dilution; Cell Signaling Technology). Secondary antibodies including goat anti-mouse (H+L)/HRP (horseradish peroxidase) (1:5,000 dilution) and goat anti-mouse (H+L)/HRP (1:5,000 dilution) were purchased from Zhongshan Golden Bridge Biotechnology Co., Ltd. (Beijing, China). Proteins of interest were visualized using the Supersensitive ECL (enhanced chemiluminescence) Primer kit purchased from HaiGene (Harbin, China). Signals were detected using an Imaging System (Clinx; Shanghai, China) and data are expressed as normalized ratios to GAPDH.
Statistical analysis
The experimental results are expressed as mean ± standard deviation (mean ± SD), and statistical analysis was performed using the SPSS 24.0 software (IBM, Armonk, NY, USA). One-way analysis of variance (One-Way ANOVA) was used to compare multiple-sample groups. Differences between groups were analyzed by Tukey's post hoc test (Figure 2, Figure 3, Figure 4A, 4C, 4D, and Figure 5) or Dunnett’s T3 test (Figure 4B). Differences were statistically significant at P < 0.05.