Reagents and antibody

GSL were supplied by Holistol International Ltd. (Hong Kong, China). Docetaxel, cisplatin, 5-fluorouracil, dexamethasone, ascorbic acid, β-glycerol phosphate, indomethacin, and rosiglitazone were obtained from Sigma–Aldrich (St. Louis, MO, USA). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), and 0.25% trypsin/0.02% EDTA were purchased from Gibco-BRL Life Technologies (New York, NY, USA). Antibodies against CD73 (1:200, ab175396), CD90 (1:200, ab3105), CD105 (1:200, ab221675), pro-caspase-3 (1:10000, ab32499) and cleaved caspase-3 (1:1000, ab13847), β-actin (1:1000, ab8226) were purchased from Abcam (Cambridge, UK).

Isolation and culture of BMSCs

C57BL/6 mice were euthanized by asphyxiation with CO2. Bone marrow cells were collected by flushing the femurs and tibias and cultured in DMEM supplemented with 10% (vol/vol) FBS and antibiotics (100 U/mL penicillin G and 100 µg/mL streptomycin). Then, the cells were seeded into normal tissue culture flasks under the condition of 37℃ and 5% CO2. The culture medium was changed on day 3 and non-adherent cells were carefully removed. Subsequently, the medium was replaced after 4 days and the BMSCs were used within two or three passages.

BMSC treatment

Two passages of BMSCs were seeded in 6-well plate at a density of 3000 cells/cm2 and were divided into four groups: control, TPF, GSL pre-treated, and GSL co-treated. The GSL was dissolved in dimethyl sulfoxide (DMSO). The control group was treated with 1% DMSO. The TPF group was treated with docetaxel (1.5 ng/mL), cisplatin (2 µg/mL), and 5-fluorouracil (1 µg/mL) in a total volume of 80 µg. The GSL pre-treated group was treated with 0.1% GSL for 7 days followed by 2 days of 80 µg TPF at the above-defined doses. The GSL co-treated group was simultaneously treated with 0.1% GSL and 80 µg TPF at the above-defined doses for 2 days.

Cell viability assay

Cells (1 mL) were seeded at a density of 3,000/mL in 24-well plates with 10% FBS. The medium was replaced with fresh medium. The cell number was determined by counting each sample in duplicate using a hemocytometer under an inverted microscope (Olympus, Tokyo, Japan) every 48 h for 8d.

Flow cytometry analysis

The surface markers CD73, CD90, and CD105 in BMSC were detected using flow cytometry. BMSCs were incubated at 4℃ with primary monoclonal anti-CD73, -CD90, and -CD105 for 30 min. After washing the cells with phosphate buffered saline (PBS) supplemented with 5% fetal calf serum and 0.4% NAN3 (FACS buffer), they were incubated with the indicated primary antibodies for 15 min. After washing the FACS buffer, flow cytometry was performed using a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA) and analyzed using CellQuest version 5.1 (BD Biosciences). The experiments were repeated in triplicate.

BMSC apoptosis analysis

BMSC apoptosis was analyzed using an Annexin V-FITC/propidium iodide (PI) apoptosis detection kit (BD Biosciences). BMSCs in each group were detached using trypsin/EDTA at the end of treatment. After washing with PBS, the cells were stained using the Annexin V-FITC Apoptosis Detection Kit. Apoptotic BMSCs were detected using FACS Calibur and CellQuest (BD Biosciences).

BMSC senescence analysis

Staining of senescence-associated β-galactosidase (SA-β-GAL) was performed as previously described [22]. BMSCs in each group were fixed and then stained with β-galactosidase staining kits according to the manufacturer’s instructions (Cell Signaling Technologies, Boston, MA, USA). Images of labeled cells were captured using a microscope (Olympus) and staining intensity was quantified using ImageJ.

ROS detection

The levels of intracellular reactive oxygen species (ROS) were measured using an ROS assay kit (Beyotime Biotechnology, S0033, Shanghai, China) according to the manufacturer’s instructions. After treatment, BMSCs in each group were incubated with serum-free medium containing 10 µM dichlorodihydrofluorescein diacetate (DCF-DA) at 37 ℃ for 30 min and washed with PBS three times. Fluorescence was observed using an inverted fluorescence microscope (Olympus). Fluorescence intensity was measured using ImageJ.

BMSC differentiation assay

After treatment, the normal medium of each BMSC group was replaced with the differentiation medium. Osteogenic differentiation medium consisted of DMEM containing 10% FBS, 10 mM b-glycerophosphate, 1 µM dexamethasone, and 0.2 mM ascorbic acid. The medium was replaced every 3 days until day 21. Adipogenic differentiation was induced by DMEM containing 10% FBS, 2 mM L-glutamine, 1 µM dexamethasone, 500 µM 1-methyl-3-isobutylxanthine, 10 µg/mL insulin, and 100 U/mL penicillin/streptomycin; BMSCs were incubated in adipogenic induction medium for 7 days. When morphological signs of BMSC differentiation were visible, cells were fixed with polyformaldehyde for 20 min. Chondrogenic differentiation was performed using a STEMPRO Chondrogenesis Differentiation Kit (Gibco Life Technologies, Frankfurt, Germany) according to the manufacturer’s protocol. Osteogenesis, adipogenesis, and chondrogenesis were examined using Alizarin Red S, Oil Red O, and Alcian blue staining, respectively. The cells were observed under an inverted phase microscope.

Real-time polymerase chain reaction

Total RNA from MSCs after differentiation induction was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA samples were converted into complementary DNA using the Superscript III First-Strand Synthesis System for Reverse Transcription-Polymerase Chain Reaction (RT-PCR) (Invitrogen) according to the manufacturer’s instructions. Gene transcripts were quantified on a 7,500 Real-time PCR System using Power SYBR Green dye (Applied Biosystems). PCR conditions were one cycle of 50 ℃ for 5 min and 95 ℃ for 10 min, followed by 50 cycles of 94 ℃ for 10 s and 60 ℃ for 1 min. The primer sequences used for RT-PCR were: glyceraldehyde-3-phosphate dehydrogenase (GAPDH) forward, 5’-tgtgatgggtgtgaaccacg-3’; GAPDH reverse, 5’-cagtgagcttcccgttcacc-3’; osteocalcin forward, 5’-tacacgtgcaggtcaatccc-3’; osteocalcin reverse, 5’-gggcagcacaggtcctaaat-3’; parathyroid hormone receptor (PTHr) forward, 5’-aagcacgaagtgggagtagc-3’; PTHr reverse, 5’-ggagccattaaggaagccgt-3’; peroxisome proliferation-activated receptor-γ (PPARγ) forward, 5’-aagcacgaagtgggagtagc-3’; PPARγ reverse, 5’-ggagccattaaggaagccgt-3’; lipoprotein lipase (LPL) forward, 5’-ccagctgggcctaactttga-3’; LPL reverse, 5’-aactcaggcagagccctttc-3’.

Western blotting

BMSCs from each group were washed with ice-cold PBS and lysed in Laemmli sample buffer containing protease and phosphatase inhibitors. After centrifugation at 12,000 rpm for 20 min, the protein concentration of the supernatant was determined using a bicinchoninic acid assay. Equal amounts of proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto methanol-activated polyvinylidene difluoride membranes (Millipore, Massachusetts, USA). The membranes were blocked in Tween supplemented with 5% non-fat skim milk at room temperature for 2 h, incubated with primary antibody against caspase 3 at 4 ℃ overnight, and blotted with appropriate secondary antibody conjugated to horseradish peroxidase. Electrochemiluminescence was performed using a chemiluminescence system (Alpha, California, USA) according to the manufacturer’s instructions.

Animal model

Forty male C57BL/6 mice were provided by the Animal Facility of Shenzhen University, Shenzhen, China. All mice were 7 weeks old and weighed 36 g. Mice were fed under standard laboratory conditions (25 ± 2 ℃, 60% ± 10% relative humidity, and 12 h light–dark cycle). All experimental procedures were approved by and conducted in accordance with the guidelines of the Institutional Animal Ethics Committee of Shenzhen University Medical School.

The mice were randomly allocated into four groups: control group (n = 10), TPF group (n = 10), GSL pre-treated group (n = 10), and GSL co-treated group (n = 10). Mice in the TPF group received TPF via intraperitoneal injection (docetaxel 20 mg/kg, cisplatin 6 mg/kg, and 5-fluorouracil 17 mg/kg). Mice in the pretreated group received 1 mg/kg GSL daily for 7 days and were intraperitoneally injected with TPF for 2 days. Mice in the co-treated group simultaneously received 1 mg/kg GSL and TPF for 2 days. The control group received equal quantities of saline by oral gavage.

Routine blood analysis

At the end of treatment, blood samples (0.5 mL) from the posterior vena cava of CO2 anesthetized mice were collected into tubes containing ethylenediaminetetraacetic acid. Routine blood analysis was performed using an automatic hematology analyzer (VetAutoread, USA) for white blood cell (WBCs) and peripheral platelet (PLTs) counts.

Hematopoietic colony-forming cell assay

Bone marrow cells were collected and cultured in Iscove’s Modified Dulbecco’s Medium supplemented with FBS, bovine serum albumin, β-mercaptoethanol, and methylcellulose. For colony-forming cell assay, the bone marrow cells (2 × 104 cell per dish) were seeded into 35 mm dishes with the following growth factors: 100 ng/mL recombinant mouse SCF, 10 ng/mL recombinant mouse interleukin 3, 10 ng/mL recombinant mouse interleukin 6, 10 ng/mL mTPO, 3 U/mL recombinant human erythropoietin, 10 ng/mL recombinant human granulocyte colony-stimulating factor. After 10 days of incubation, the dishes were observed under a microscope, and the granulocyte–macrophage colony-forming units (CFU-GM), erythroid colony-forming units (CFU-E), and erythroid burst-forming units (BFU-E) were counted.

Statistical analysis

Data are expressed as mean ± standard deviation (SD). Statistical differences were analyzed using one-way analysis of variance (ANOVA) and Tukey’s test. Statistical significance was set at p < 0.05. All experiments were repeated at least three times.