Human MSC culturing
The protocol for the use of human cells was approved by the committee of the Second Hospital of Shandong University, and bone marrow cells were harvested from bone fragments of patients from the Second Hospital of Shandong University, with written consent forms acquired from all patients. All bone marrow aspirates were diluted with low-glucose Dulbecco’s modified Eagle’s medium (DMEM, Thermo, Waltham, MA, USA), and then subjected to Ficoll gradient centrifugation (1200×g for 30 min at room temperature). The cells from the interface were harvested, followed by two washes in phosphate-buffered saline (PBS). Mononuclear cells re-suspended in complete DMEM were counted with a hemocytometer and seeded in 10 cm2 tissue culture dishes at a density of 5 × 106 cells/10 mL. After two days, floating cells were discarded, and the adherent cells were kept for culture at 37 °C with 5% humidified CO2. After reaching a confluence of 75–85%, cells were detached with 0.05% trypsin/1 mM EDTA and re-plated, and expanded cells with < 9 passages were used in the experiments.
Colony formation assay
A total of 1 × 105 MSCs were plated into a 10-cm petri dish and continuously cultured for up to 21 days in the presence of 0, 1, 5 and 10 μM of hesperidin, respectively. Crystal violet (0.5%, SIGMA, MO, USA) was used to stain the formed colonies for 15 mins followed by counting under light microscope. Colonies larger than 2 mm in diameter were counted, which typically ranged from 50 to 200 per dish (calculated from 10 randomly chosen fields in each dish).
Proliferation assay
The proliferation of cells was assessed by commercial CCK-8 kit (Dojindo, Kumamoto, Japan). In brief, 1 × 105 MSCs were plated into each well of 6-well plate and continuously cultured for up to 7 days in the presence of 0, 1, 5 and 10 μM of hesperidin, respectively. 10 ul CCK-8 solution was then added into each well and the chromogenic reaction was carried out at 37 °C for 15 mins. Microplate reader (Molecular Devices, Sunnyvale, CA, USA) was used to record the absorption at 450 nm and relative cell viability was calculated.
Chondrogenesis assays
MSCs were cultured in chondrogenic induction medium (DMEM, 0.2 mM ascorbate-2-phosphate, 20% FBS, and 10 mM glycerol-2-phosphate) for 14 days in the absence or presence of 5 μM hesperidin, with fresh medium exchanged every 2 days. Chondrogenesis was evaluated using Alcian Blue (Millipore, Billerica, MA, USA) staining.
mRNA extraction and real-time PCR
Total mRNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA), and reverse transcribed to complementary cDNAs with Superscript II following manufacturer’s instructions (Biorad, Hercules, CA, USA). Triplicate PCR reactions were conducted using cyber green-based system (Applied Biosystems, Waltham, MA, USA) with the following conditions: 15 s at 95 °C, 1 min at 60 °C for 40 times. The relative expression levels were calculated using GAPDH as the internal control. Primers used in this study were: Sox9 forward 5′-GTA CCC GCA CTT GCA CAA-3′, reverse 5’-TCT CGC TCT CGT TCA GAA GTC-3′; p65 forward 5’-ACA TCC ATG CGG AGA ACG AGG AG-3′, reverse 5′-AGT GCT GCG AGT GAG TCA AGA GG-3′; GAPDH forward 5’-CTG ACT TCA ACA GCG ACA CC-3′, reverse 5′-TAG CCA AAT TCG TTG TCA TAC-3′.
Western blot
Cell resuspension was prepared in the lysis buffer containing 150 mM NaCl, 50 mM Tris-HCl, 10 mM HEPES, 0.1% NP-40 alternative, 0.5 mM NaF, 0.25% Na-deoxycholate, 1 mM Na3VO4, pH 7.4 (Protease Inhibitor Cocktail, Roche, 1 tablet/10 ml). Cell lysates were quantitated using BCA protein assays, and 30 μg total protein was then run on SDS-PAGE followed by transfer to PVDF membranes. The membranes were subsequently blocked with 1% BSA (bovine serum albumin, Sigma, USA), and incubated with primary antibodies at 4 °C overnight. Primary antibodies for p65 and GAPDH were both purchased from Abcam. HRP conjugated secondary antibodies were utilized to visualize bands in an ECL-based imaging system.
p65 overexpression and knockdown
Stable overexpression and knockdown of p65 were established using p65 lentiviral particle (LPP-F0160-Lv105) and p65 shRNA particle (HSH016213-CH1), both of which purchased from GeneCopoeia (Rockville, MD, USA). Cells were first transduced by respective lentiviral particles for 24 h, followed by selection with puromycin for 2 weeks, according to vendor’s instructions.
Enzyme-linked immunosorbent assay (ELISA)
The MSCs were treated in the absence or presence of 5 μM hesperidin for 2 days. Cells were then completely removed by centrifugation and clear medium was collected for ELISA analysis. The levels of IFN-γ, IL-2, IL-4 and IL-10 were measured with the commercially available ELISA kits (Abcam, MA, USA) following the manufacturer’s instructions.
Statistical analysis
All data were analyzed using SPSS 22.0 system (IBM, Armonk, NY, USA), and presented as mean ± standard deviation (SD) from at least three independent experiments. The differences between groups were determined by Student’s T tests and single factor variance analysis (ANOVA). P values less than 0.05 were considered statistical significant.