Experimental verification
Extraction, culture and identification of rat primary BMSCs
BMSCs were extracted from rats by whole bone marrow cell culture, and cell isolation, culture and identification were performed as previously described [
9]. Briefly, the rats were euthanized and sterilized in 75% ethanol for 30 min, and the long bone shaft of the rats was isolated and collected. BMSCs were flushed out by an injection of Dulbecco’s modified Eagle’s medium (DMEM, C11885500BT, Gibco, USA) using a 20-mL syringe under sterile conditions. After centrifugation, the BMSCs were cultured in OriCell® rat bone marrow mesenchymal stem cell complete medium (batch No.: RAXMX-90011, Saiye (Suzhou) Biotechnology Co., Ltd., Suzhou, China). All of the abovementioned cells were cultured at 37 °C in an incubator containing 5% CO
2.
Cell proliferation assay
BMSC viability was detected using the Cell Counting Kit-8 (CCK-8, C0038, Beyotime, China) assay following the manufacturer’s instructions. Specifically, BMSCs were plated at a density of 2500 cells per well in a 96-well plate. After 24 h of incubation, different concentrations (0.1 μM, 0.5 μM, 1 μM, 5 μM, 10 μM, and 20 μM) of fisetin (S2298, Selleck, USA) were added to the cells and incubated for 24, 48, and 72 h, respectively. Dimethyl sulfoxide (DMSO, D2650, Sigma, USA) was used as the control group. At the end of fisetin treatment, each well was subjected to 10 μL CCK-8 and incubated at 37 °C for 2 h. The absorbance of the optical density at 450 nm was determined using a microplate reader (M1000 Pro, Tecan, Switzerland).
Osteogenic differentiation determination and mineralization assessment
The surface of the 6-well plate for osteogenesis induction was coated with gelatin, and the BMSCs were plated at a density of 1 × 105 cells per well. Osteogenically induced differentiation of the cells was performed using the Mesenchymal Stem Cell Osteogenic Differentiation Medium Kit (batch No.: RASMX-90021, Saiye (Suzhou) Biotechnology Co., Ltd., Suzhou, China) after reaching 70–80% confluence. For treatment, fisetin (10 μM) and/or ICG-001 (10 μM) were added to the osteogenic differentiation medium. The DMSO wells served as the control group. Mineralization of the calcium nodules was detected at day 30 of differentiation using alizarin red S (ARS) solution. Images were taken using an inverted microscope (ECLIPSE Ti2-E, Nikon, Japan). After that, 10% cetylpyridinium chloride was used to elute ARS staining, and the absorbance was detected at 562 nm using a microplate reader.
Alkaline phosphatase (ALP) activity assay
BMSCs were plated at a density of 1 × 105 cells per well in a 6-well plate. The medium was replaced with osteogenic differentiation medium after 24 h. For treatment, fisetin at various concentrations (DMSO, 1 μM, 5 μM, and 10 μM) was added to the medium. The DMSO wells served as the control group. The ALP activity of cells was determined at day 10 of differentiation using an ALP Staining Kit (batch No.: P0321S, Beyotime Biotechnology, China). The absorbance at 405 nm for ALP was detected using a microplate reader.
Reverse transcription and real-time PCR (RT-PCR)
After rat BMSCs were treated with the different drugs, total RNA was extracted using TRIzol® Plus RNA Purification Kit (12183018A, Invitrogen, USA), and 1 μg of RNA from each sample was then reverse-transcribed to cDNA using the High Capacity cDNA Reverse Transcription Kit (4368814, Applied Biosystems, USA)) according to the manufacturer's protocol. For real-time PCR, ABI PrismTM 7500 (Thermo Life, USA) was performed using Fast SYBR@GREEN Master Mix (4385612, Applied Biosystems, USA) with the following primers. The primers used for ALP, Collagen I, RUNX2, Osteopontin, Wnt3, CTNNB1, and GAPDH amplifications are listed as follows: 5′-GGCGTCCATGAGCAGAACTACATC-3′ (ALP-Forward), and 5′-CAGGCACAGTGGTCAAGGTTGG-3′ (ALP-Reverse); 5′-TGTTGGTCCTGCTGGCAAGAATG-3′ (Collagen I-Forward), and 5′-GTCACCTTGTTCGCCTGTCTCAC-3′ (Collagen I-Reverse); 5′-CTTCGTCAGCGTCCTATCAGTTCC-3′ (RUNX2-Forward), and 5′-TCCATCAGCGTCAACACCATCATTC-3′ (RUNX2-Reverse); 5′-GACGATGATGACGACGACGATGAC-3′ (Osteopontin-Forward), and 5′-GTGTGCTGGCAGTGAAGGACTC-3′ (Osteopontin-Reverse); 5′-CAGCCTGACTTCCGAGCCATTG-3′ (Wnt3-Forward), and 5′-ACTCCCGATGCTTCTCCACCAC-3′ (Wnt3-Reverse); 5′-ACAAGCCACAGGACTACAAGAAACG-3′ (CTNNB1-Forward), and 5′-TCAGCAGTCTCATTCCAAGCCATTG-3′ (CTNNB1-Reverse); 5′-TCTCTGCTCCTCCCTGTTCT-3′ (GAPDH-Forward), and 5′-GTTCACACCGACCTTCACCA-3′ (GAPDH-Reverse). Relative gene expression was normalized to GAPDH as an internal standard, and the expression levels of target mRNAs were calculated by the 2−△△CT method. Each assay was conducted in triplicate.
Western blotting (WB)
After BMSCs were treated with the different drugs, cell lysis buffer for WB and IP (P0013, Beyotime Biotechnology, China) was used to collect the total protein from the cells. The expression of relative proteins was detected by WB according to the protocol we described[1]. The primary protein antibodies were as follows: anti-RUNX2 (1:1000, 12556, CST), anti-Osteopontin (1:1000, ab63856, Abcam), anti-collagen I (1:1000, ab260043, Abcam), anti-β-actin (1:2000, 3700, CST), anti-β-catenin (1:1000, 8480, CST), and anti-Wnt3 (1:1000, ab32249, Abcam). HRP-conjugated goat anti-rabbit or anti-mouse secondary antibodies were used (7074, 7076, CST, USA).
Statistical analysis
The data are expressed as the mean ± standard deviation. T test was used for comparison between the two groups, and one-way ANOVA was used for multiple groups. p < 0.05 was considered to indicate statistical significance.