The study population included patients with OA and RA with effusion in at least one knee joint. All RA patients fulfilled the ACR/EULAR classification criteria for RA (Aletaha et al. 2010). Patients were recruited from the outpatient department of Rheumatology and clinical immunology at the Institute of post graduate medical education and research (IPGME&R), Kolkata, who have already been administrated with DMARDs with or without NSAIDs. Patients with any other rheumatological disease, DM, HTN, hypothyroidism, BMI≥ 25, pregnancy, renal and/or hepatic impairment, receiving steroids or any antioxidants were excluded. This study was approved by the Institutional Ethics Committee (No: Inst/IEC/24.02.2014), and informed consent was obtained from all participants. Baseline demographic and clinical data were collected from all the patients. Approximately 10 ml of Synovial fluid was aspirated from the knee joint of the participant maintaining strict aseptic measures.
Cell culture and reagents:
Isolated cells were cultured in DMEM (Dulbecco's Modified Eagle Medium, D5648Sigma, St Louis, MO, USA) supplemented with 10% fetal bovine serum/FBS (Invitrogen, Rockford, IL, USA), 1% penicillin-streptomycin (10,000 U/ml), [Cat no-15140122] from Invitrogen at 37 °C with 5% CO2 in a humidified incubator. Theaflavin 3, 3’digallate/TF3 [Cat no-92223-1MG] and Methotrexate/MTX [Cat no-M8407] were purchased from (Sigma, St Louis, MO, USA). For protein estimation, Bradford reagents were obtained from bio rad (Cat no- 5000006). Primary antibodies for human [HIF-1α(ab51608),GRP78(#3183), CHOP(#2895), HSP-70(#4876), Bcl-2(#4223), Bax (#2772), Beclin-1(#3738) p62 (#5114), α-tubulin (#2144), 10x cell lysis buffer Cat no-#9803 and Protease Inhibitor Cocktail, PKC (100x) Cat no-5871] were obtained from Cell-Signaling Technologies Inc. (Danvers, MA, USA). Nitrocellulose membrane was procured from MF-Millipore [Cat no- HAWP04700] 0.45 µm pore size. Caspase-9 and Caspase-3 were obtained from Biolegend (Cat No: 621902 and 634101 respectively). Anti-LC3b antibody (ab51520) was procured from Abcam. The concentration of VEGF-A and ANG-1 ELISA Kits [(ELH-VEGF-1) and (IQH-Angiopoietin1-1)] were procured from (Ray Biotech, Norcross, GA). For detection of apoptosis FITC Annexin V Apoptosis Detection Kit, I (Cat no-556547) was used from BD, Biosciences.
Culture of synovial fluid-derived fibroblasts:
SF from patients with RA and OA was centrifuged (10X3000rpm), the cell pellet was re-suspended in (%) DMEM supplemented with 15% FBS, 2mM L-glutamine, 50 U/ml penicillin, 50µg/ml streptomycin, and plated in 25cm2 flasks. After 24h, non-adherent cells were discarded. Spindle-shaped cells were observed after the third passage. The presence of fd-FLS was confirmed by phenotypic characterization by flow cytometry using FITC conjugated CD90 (Stebulis et al. 2005).
Extracellular cytokines measurement by ELISA:
Extra-cellular pro-inflammatory cytokines (TNF-α, IL-6) along with angiogenic markers (VEGF-A and ANG-1) as well as inflammatory marker CRP were measured from cell-free supernatant of cultured fd-FLS.
Cell viability assay:
The pro-apoptotic activity of TF3 alone and in combination with MTX was evaluated in fd-FLS obtained from RA patients by MTT assay (Sen et al. 2007). Briefly, 5x104 cells/ml were seeded in 96 well plates in DMEM medium (supplemented with 10% FBS and antibiotics) and incubated with TF3 (0-50 µM) respectively for 48h.For combinatorial treatment, one dose of MTX (125nM) was fixed with the variable dose of TF3 (5, 10, 15, 20, 30µM) for 24h. After treatment incubation, fd-FLS viability was measured using 3-(4, 5 dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide, and the optical density (OD) was measured at 570nM in a microplate reader (Byonoy absorbance 96 plate reader). The results were expressed at IC50 values i.e. the concentration that inhibited 50% of cell growth, enumerated by graphic extrapolation using GraphPad PRISM software (version 5).
Identification of CD90+ cell by flow cytometry and analysis of apoptosis:
Isolated cells were treated for 24h with both MTX and TF3, followed by washing twice with 1xPBS. Cells (1x106 cells/ml) were seeded in 1x PBS. Cells were then centrifuged for 10min, 3000 rpm, the cell pellet was resuspendedin100 µl of (1x) annexin V binding buffer and kept for 10 min, dark, RT. FITC conjugated annexin V and propidium iodide (PI) was added, kept for 15 min and 5 min respectively, dark, RT(Room temperature). Cells were acquired and analyzed in FACS VerseTM (BD Bioscience) after the addition of 400µl of binding buffer. Each experiment was performed in triplicate.
RNA isolation and cDNA synthesis:
Total RNA was extracted from synovial fluid-derived synovial fibroblast cells using 300µl TRIzolTM (Invitrogen, Carlsbad, CA, USA) at a ratio of (1:1). Cells were slightly flush and stored overnight at -20°C. On the next day, the mixture was centrifuged at 10,000 rpm for 5 min at 4°C. The clear supernatants were transferred to a fresh microcentrifuge tube and allowed to stand for 5 min at room temperature. Next, 200µl of chloroform was added and shaken vigorously. Then the mixture was allowed to stand at room temperature for 2-5 min and was again centrifuged at 10,000 rpm for 10 min. The exact upper aqueous phase was then transferred to a fresh tube followed by the addition of 0.5 ml of isopropanol and was again subjected to vigorous shaking. The mixture was then kept at room temperature for 5-10 min. Centrifugation was carried out at 10,000 rpm at 4°C to settle down the pellet from the mixture. The RNA pellet was collected, washed with 75% ethanol followed by centrifugation at 10,000 rpm, and allowed to air dry at room temperature. Finally, the dried RNA pellet was re-suspended in DEPC water and quantified in a nano-drop spectrophotometer (Thermo Scientific, USA). Approximately, 1μg of cDNA was prepared from isolated RNA with a total volume of 11.5μl (RNA+DEPC water). Next, oligo dT (1μl) was added and incubated at 70°C for 5 min inside the PCR machine (Applied Biosystems, USA). The mixture was taken out and placed in chilled-ice for 5 min followed by the addition of PCR master mix. The mixture was given a short vortex to mix well followed by incubation at 37°C for 5 min. Then, 1μl of reverse transcriptase was added and incubated at 42°C for 60 min inside the PCR machine. Finally, the mixture was again incubated at 70°C for 10 min after that the mixture was taken out and kept on ice for 5-10 min and stored at -20°C for further processing.
Reverse transcriptase (RT)-PCR:
The gene expressions of the prepared cDNA samples were analyzed by real-time quantitative PCR analyses. The reactions were carried in 0.2ml strip tubes/96well plates (Applied Biosystems, USA) having a final reaction volume of 10μl. The reaction mixture containing 10ng cDNA, 2ng of both forward and reverse primers along with 1X SYBR-Green PCR master mixture (Power SYBR-Green PCR Master Mix; Applied Biosystems, USA) was prepared. The quantitative real-time PCR was carried out using RT-PCR system (Applied Biosystems, USA). All the reactions were performed in triplicates and the expression of enzyme GAPDH was used as a housekeeping gene for the following experimental studies. Primer sequences for spliced XBP-1 mRNA was forward 5’-CCCTCCAGAACATCTCCCCAT-3’ and reverse 5’-ACATGACTGGGTCCAAGTTGT-3’ and unspliced sequences was forward 5’-CAGACTACGTGCACCTCTGC-3’ and reverse 5’-AGTTGTCCAGAATGCCCAACA-3’. Primer sequences for IRE1-α were forward 5’-CCGAACGTGATCCGCTACTTCT-3’ and reverse 5’-CGCAAAGTCCTTCTGCTCCACA-3’ (IDT Inc., Singapore). The reaction mixtures were incubated at 95oC for 2 min, followed by 40 cycles of 5 sec at 95oC and 30 sec at 60oC respectively (Ahmadiany et al. 2019). Relative expression of transcript levels of each sample was demonstrated according to the 2 ΔΔCT methods.
After washing twice with 1X PBS, cultured fd-FLS were lysed in cell lysis buffer (1X) contains PKC (Protein Kinase C). Quantification of total protein was done using Bradford reagent. Cell lysate proteins (50µg) were separated by SDS-polyacrylamide gel electrophoresis, transferred into nitrocellulose membrane, and (3% BSA dissolved in Tris buffer saline or TBS) was used for blocking for 1 hr, RT and incubated overnight at 4oC with desired 10 antibodies (Dilution- 1:10000). After washing twice with TBST (Tris Buffer solution with Tween), the membrane was incubated with HRP conjugated 20antibody (Dilution- 1:500) for 2hrs, RT. The membrane was washed four times with TBST followed by development via chemiluminescence using a gel documentation system (Bio-Rad-Chemidoc, XRS+), quantified by Image J, (Image J, 1.40, USA), and normalized by α-tubulin for further analysis.
Caspase 3 assay:
Caspase-3 activity in synovial fibroblast cells was detected by using a caspase-3 assay kit [(BioVision, Sandiego, USA) (cat no: K106)]. Cells were seeded in a 6 well plate at 1X104 cells/well, incubated overnight with a combination of MTX (125nM) and TF3 (10µM) for 24 h. After completing the treatment, cells were lysed using cell lysis buffer (cell signaling) and 2X reaction buffer was added to each sample with 5µl of DVD-pNA substrate, and cells were incubated at 37oC for 1-2 h, followed by measuring by spectrophotometer at 400-405nM.
Immunofluorescence staining and confocal microscopy:
Synovial fibroblast cells were seeded (1X106 cells/ml) in a confocal dish (SPL Life Sciences) and incubated overnight. After treatment with [either rapamycin or MTX+TF3 and rapamycin (200nM)], cells were fixed with 4% v/v paraformaldehyde (in 1x PBS) at RT, washed twice, permeabilized with 0.2% triton X-100 in 1x PBS for 20 mins at RT. Cells were then blocked with 2% BSA, washed, and incubated overnight with primary antibody LC3B (ab51520) (1:100 dilution) at 4oC. Cells were treated with fluorophore conjugated secondary antibody (Invitrogen) and Hoechst (for nuclear staining, Invitrogen) diluted in 1% BSA –1x PBS and viewed under Olympus FV10i-LIV confocal laser-scanning microscope (magnification: 60*4X/1.35).
Data are expressed as mean ± SD or median (inter quartile range, IQR). Student t-test has been done for parametric data, for non-parametric data paired t-test (Mann Whitney U test) were done. Repeated measures ANOVAs was done for more than two data set. The correlation was done using Pearson’s correlation coefficient test for parametric data and Spearman rank correlation coefficient for nonparametric data using Graph Pad Prism software, version 5.0, (Graph Pad Software Inc, La Jolla, CA, USA). p<0.05 was considered as significant for all statistical analysis.