Autophagy induction contributes to the resistance to methotrexate treatment in rheumatoid arthritis fibroblast-like synovial cells through high mobility group box chromosomal protein 1

Synovial tissue specimens were obtained from seven patients with OA and seven patients with RA during joint replacement surgery (Department of Joint Surgery, Hong Hui Hospital, Xi’an, China). This study was approved by the human research ethics committee of the Xi’an Hong Hui Hospital, and all patients fulfilled the American College of Rheumatology criteria for classification of RA. All patients provided informed consent. Clinical data, laboratory examination results, and patient medications are summarized in Table 1.

MTX and antibodies against microtubule-associated protein 1 light chain 3 (anti-LC3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Anti-β-actin antibodies were purchased from Biosen (Beijing, China). High mobility group box chromosomal protein 1 (HMGB1), anti-poly(ADP-ribose) polymerase (PARP), anti-phosphorylated mammalian target of rapamycin (anti-p-mTOR), and anti-mTOR antibodies were purchased from Abcam (Cambridge, UK). Anti-Beclin-1, anti-Akt, and anti-phosphorylated Akt (anti-phospho-Akt; Ser473) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Synovial fibroblasts were isolated from synovial tissue specimens obtained from patients with RA and patients with OA. Cells were cultured as described elsewhere [18] and used between passages 3 and 8 for all experiments. The concentrations of MTX used in the different experiments ranged from 0.01 μM to 1 μM, and the culture periods ranged from 24 h to 96 h of continuous exposure to MTX. On the basis of pharmacokinetic analysis, the ingestion of a 20-mg tablet of MTX yields plasma MTX concentrations of approximately 0.5 μM after 1 h and of approximately 0.1 μM after 10 h [19]. Controls were treated with matched amounts of dimethyl sulfoxide (DMSO); 0.1 μM of bafilomycin A1 (Sigma-Aldrich), which inhibits the fusion of autophagosomes with lysosomes, was added to cell cultures for the last 4 h of treatment.

Cell viability was measured using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The cells were seeded at 5 × 10⁴ cells/well in 96-well plates, incubated overnight, and then exposed to the indicated concentrations of MTX for the indicated times. Thereafter, 20 μl of MTT solution (5 mg/ml) was added to each well, and the cells were incubated for another 4 h at 37 °C. After removal of the culture medium, the cells were lysed in 200 μl of DMSO, and the optical density (OD) was measured at 570 nm with a microplate reader (Thermo Fisher Scientific, Waltham, MA, USA). The following formula was used: cell viability = (OD of the experimental sample/OD of the control group) × 100 %.

After treatment, cells were detached with trypsin, washed twice with 1× phosphate-buffered saline (PBS), and resuspended in annexin V binding buffer (7SeaPharmTech, Shanghai, China) at a concentration of 3 × 10⁵ cells/ml. Next, the cells were incubated with fluorescein isothiocyanate–annexin V (7SeaPharmTech) for 15 minutes at room temperature in the dark and with propidium iodide (7SeaPharmTech) for 5 minutes at 4 °C in the dark, and then the cells were analyzed by flow cytometry (guava easyCyte HT; EMD Millipore, Billerica, MA, USA).

Synovial fibroblasts and tissue specimens were washed twice with ice-cold PBS and solubilized in Triton X-100 lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 0.2 mM ethylenediaminetetraacetic acid, 1 % Triton X-100, 1 % sodium deoxycholate, 0.1 % sodium dodecyl sulfate [SDS]) and protease inhibitor cocktail (Beyotime, Shanghai, China) on ice, then quantified using the Lowry method. Cell lysate proteins (40 μg) were separated by SDS-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose membranes (Immobilon-P; EMD Millipore). The membranes were blocked in 5 % skim milk in Tris-buffered saline with Tween 20 (TBST) at room temperature for 1 h and incubated overnight at 4 °C with the indicated primary antibodies. After a washing step with TBST buffer, the membranes were reacted with the appropriate horseradish peroxidase–conjugated secondary antibodies for 1 h at room temperature. After incubation with the secondary antibody, the membranes were washed three times with TBST and developed via electrochemiluminescence (Thermo Fisher Scientific) and using a Western blotting detection system (GeneGnome 5; Syngene, Cambridge, UK).

After treatment, cells were detached with trypsin, washed twice with PBS, and fixed in ice-cold 2 % glutaraldehyde/0.1 M phosphate buffer (pH 7.2), postfixed in 1 % osmium tetroxide, washed, dehydrated with a graded ethanol series (30 %, 50 %, 70 %, 90 %, and 100 %), and embedded in 1:1 propylene oxide/embedding resin. The resin blocks were cut with a LKB V ultramicrotome (LKB, Bromma, Sweden). Thin (60-nm) sections were picked up on 200-mesh copper grids and stained with uranyl acetate and lead citrate. The sections were examined with an H-7650 transmission electron microscope (HITACHI, Ibaraki, Japan).

Both small interfering RNA (siRNA) targeting Beclin-1 complementary DNA (cDNA) sequence (5′-CAGTTTGGCACAATCAATATT-3′) and HMGB1 cDNA sequence (5′-CCCGTTATGAAAGAGAAATTT-3′) and a control siRNA (5′-UUCUCCGAACGUGUCACGUTT-3′) were obtained from Shanghai GenePharma (Shanghai, China). Cells were transfected with either Beclin-1 siRNA or control siRNA at 75 nmol/L using X-tremeGENE siRNA transfection reagent (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s guidelines. Twenty-four hours after transfection, the cells were treated as indicated and then harvested for Western blot analysis or flow cytometry.

Mean ± SD values were calculated. According to whether data are normally distributed, either the Mann–Whitney U or Student’s t test was used for statistical evaluation of the data (GraphPad Prism 5.0 software; GraphPad Software, La Jolla, CA, USA). p Values less than 0.05 were considered significant.