2.1. Reagents and Antibodies
The anti-COL2A1 and anti-aggrecan antibodies from Santa Cruz Biotechnology (sc-52658, sc-166951, Santa Cruz, CA, USA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Millipore (MAB374, Bedford, MA, USA), and horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG from Sigma-Aldrich from SeraCare Life Sciences, Inc. (5220 − 0341, Milford, MA, USA) were used in the study. AGE3 was prepared as described in the previously described(Yamazaki et al. 2021; Nishinaka et al. 2022), in short BSA (FUJIFILM Wako, Osaka, Japan) was incubated with glycolaldehyde dimer (Sigma-Aldrich) in 0.2 M phosphate buffer (pH 7.4) at 37°C for 7 d under sterile conditions. Then, AGEs were dialyzed for 2 d against phosphate-buffered saline (PBS) at 4°C. RAGE antagonist, FPS-ZM1 was purchased from Millipore.
2.2 Cell Culture and Treatment
We used a OUMS-27 cells in our study because chondrocytes become unstable and lose properties in vitro(Lafont 2010), and many previous studies have used OUMS-27 instead of primary chondrocytes(Yaykasli et al. 2009; Gun Bilgic et al. 2020). OUMS-27 cells were cultured in Dulbecco’s modified Eagle medium (DMEM, Nissui Pharmaceutical, Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS), and maintained at 37°C in a humidified chamber with 5% CO2 and 20% O2. OUMS-27 cells (2.0 x 105) were seeded per well in six-well tissue culture plates, followed by culture in the presence or absence of AGE3 for 7 d.
2.3 Cell Viability Assay
OUMS-27 cell viability was assessed using the CCK-8 kit (Dojindo, Tokyo, Japan). Briefly, OUMS-27 cells were seeded in 96-well plates (5×104 cells) and exposed to AGE3. The cells were then incubated for 1–8 d at 37°C under 5% CO2 and the optical density was measured at 450 nm using a Bio-Rad model 680 microplate reader (Bio-Rad Laboratories, Hercules, CA). Cell viability was determined as a percentage of proliferation compared with that in control cells.
2.4 RNA Extraction and Quantitative Real-Time Polymerase Chain Reaction (qPCR)
TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was used to extract and quantify total RNA from cell samples according to the manufacturer's guidelines. The RNA concentration was measured at 260 nm using a NanoDrop One spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The extracted total RNA was then subjected to reverse transcription using ReverTra Ace (Toyobo, Osaka, Japan). The reaction was carried out at 25°C for 10 min, 42°C for 60 min, and 97°C for 5 min with random primers (Toyobo).
qPCR was performed on a StepOnePlus system (Applied Biosystems, Foster City, CA, USA) using SYBR Green PCR Master Mix (Applied Biosystems). The PCR reaction mixture consisted of 5 µL of 2X SYBR Green mix, 0.5 µL (10 uM) of each target gene primer, and GAPDH as an endogenous internal control gene. The reaction was performed under the following conditions 10 min at 95°C, followed by 40 cycles of one-step thermal cycling, consisting of 3 s at 95°C and 30 s at 60°C, in a 96-well reaction plate. The mRNA expression levels of the target genes were determined by the comparative Ct (ΔΔCT) method and normalized to GAPDH.
2.5 Western Blot Analysis
2.5 Western Blot Analysis
OUMS-27 cells were cultured at a density of 1×106 cells/well in six-well plates using DMEM supplemented with 10% FBS at 37°C and 5% CO2. Following AGE3 stimulation, the cells were washed with cold PBS and lysed in radio-immunoprecipitation assay (RIPA) buffer containing protease inhibitors and protein extracted as described previously(Hatipoglu et al. 2023). The inhibitors used were antipain, leupeptin, and aprotinin, with a concentration of 2.0 µg/mL each, from the Peptide Institute Inc. (Osaka, Japan) and Nacalai Tesque (Kyoto, Japan). After centrifugation, the total protein concentration was determined using a Bradford protein assay kit from Bio-Rad Laboratories. The cell lysates were then mixed with 6X reducing sample buffer, denatured at 95°C for 5 min and separated on 8% SDS-PAGE gels. The separated proteins were then transferred to polyvinylidene difluoride membranes (Merck Millipore Ltd). The primary antibodies used in the experiment were anti-COL2A1, anti-aggrecan, and anti-β-actin, with dilutions of 1:1,000 and 1:10,000. Secondary antibody, anti-mouse IgG, was used at dilutions of 1:2,000 and 1:10,000. To obtain accurate results, the membranes were blocked with 5% skim milk in TBS-0.05% Tween 20 (TBS-T) for 1 h at room temperature. The membranes were then incubated with primary antibodies overnight at 4°C. After three washes with 1X TBS-T buffer, the membranes were incubated with secondary antibody for 1 h at room temperature. Signals were visualized by chemiluminescence using Amersham™ ECL™ Prime (Cytiva, WA, USA), then imaged using an Amersham Imager 600 and quantified by densitometry using ImageJ software (NIH, Bethesda, MD, USA).
2.6 Statistical Analysis
Data are shown as the mean ± standard deviation of the mean. Differences between groups were analyzed by unpaired Student's t-test or by ANOVA followed by Dunnett's post hoc comparison. All experiments were repeated at least three times. In all analyses, p < 0.05 was considered statistically significant.