Patient samples and ethical approval
Synovial tissue and synovial fluid were obtained from RA (n = 9) and osteoarthritis (OA) (n = 9) patients who underwent arthroscopic surgery at Shanghai East Hospital of Tongji University. All patients fulfilled the diagnostic criteria of the American College of Rheumatology (ACR) for RA and OA. None of the patients received preoperative therapy. Each patient provided their informed consent. This study was approved by the Ethics Committee of Shanghai East Hospital (No. 2020tjdx) and was conducted in accordance with the guidelines of the Declaration of Helsinki.
Primary cell culture
Intraarticular synovial tissue obtained during arthroscopic surgery was immediately cut into 1 mm3 pieces under aseptic conditions. The tissue fragments were placed in a 25-cm2 culture flask, and a small amount of RPMI 1640 complete medium (containing 10% FBS, 1% penicillin, and streptomycin) was added. The culture flask was upside-down in a 5% CO2 incubator at 37 °C for 4–6 h. After the fibroblasts emerged from the tissue mass, a proper amount of complete medium was added for culture. When the primary cells grew to a fusion rate of approximately 80%, passaging was performed. The cells used in this study were cultured between 3 and 8 passages. FLSs were cultured under normoxia condition (21% O2, 5% CO2) and/or hypoxia condition (3% O2, 5% CO2) at 37 °C in BioSpherix oxygen control system. RPMI 1640 and FBS were purchased from ThermoFisher Scientific, MA, USA.
Detection of amino acids by LC–MS/MS
Amino acid concentrations in the synovial fluid of RA and OA and L-arginine concentrations in the culture supernatant of RA FLSs were determined using LC–MS/MS. Briefly, RA FLSs transfected with CAT-1-siRNA (GenePharma, Shanghai, China) were plated at 5 × 106 cells/well in 6-well plates and cultured under normoxic or hypoxic conditions for 24 h. The supernatant was collected and injected on an Agilent HILIC Plus RRHD column (Agilent, CA, USA) at 5 μl. The flow rate was held constant at 400 μl/min, and amino acids were detected.
Cytometric bead array
A cytometric bead array (CBA)(BD Bioscience, NJ, USA) was used to detect cytokines: 2 ml of blood was intravenously collected, and the serum was separated. Fifty microliters of serum, 50 µl of a mixture of 6 cytokines, and 50 µl of PE fluorescent dye were fully mixed and reacted at room temperature for 3 h. Then, 1 mL of solution was added to each tube and centrifuged for 5 min (2000 r/min), and the supernatant was discarded. Then, another 120 µl of solution was added, shaken thoroughly, and detected 3–5 min later. A standard curve was created for each batch of kits, a dilution of the standard product was prepared according to the kit instructions, a standard tube and negative control were set during the experiment, and CBA software from American BD Company was used for analysis.
CAT-1 knockdown in RA FLSs
To knock down CAT-1 expression in RA FLSs, we used two siRNA targeted to knockdown CAT-1 (si-CAT1-833: sense 5′-GGACACACAUGACUCUGAATT-3′, antisense 5′-UUCAGAGUCAUGUGUGUCCTT-3′; si-CAT1-1704:sense 5′-CCGUCUCUGUUUGAACAAUTT-3′, antisense 5′-AUUGUUCAAACAGAGACGGTT-3′), and a nonsilencing-siRNA (GenePharma, Shanghai, China) as a control. CAT1-siRNA or Ctrl-siRNA was transfected into RA FLSs using Lipofectamine 2000 Reagent (ThermoFisher Scientific, MA, USA) according to the manufacturer’s instructions. Forty-eight hours after transfection, RA FLSs were used to determine their function.
Cell cycle and apoptosis assessment
RA FLSs in the logarithmic growth phase were plated at 2 × 105 cells/well in 6-well plates and cultured under normoxic or hypoxic conditions for 24 h. D-Arginine (Sigma-Aldrich, MO, USA) at different concentrations (2 mM, 4 mM, 8 mM, 16 mM, and 32 mM) was added, and the control was set at the same time. After culturing for 48 h, the cells were collected and washed twice with PBS. Each sample was stained with 5 μL Annexin V and 5 μL PI solution (BD Bioscience, NJ, USA) at 4℃ for 30 min. The apoptosis rate of FLSs was detected using a BD Aria II flow cytometer and analyzed using Diva software.
Cells were collected using the same method described above, and 1 × 105 FLSs were fixed in 1 mL 70% ethanol solution at 4℃ overnight. Next, the samples were centrifuged at 1000 rpm for 5 min, and the fixation solution was removed. After washing with precooled PBS 3 times, 100 μL of 100 μg/mL RNase (BD Bioscience, NJ, USA) was added to each tube. The samples were incubated with RNase at 37℃ for 30 min to degrade RNA, and then 100 μL of 50 μg/mL PI (BD Bioscience, NJ, USA) staining solution was added and incubated at 4℃ for 30 min. The cell cycle was detected using a BD FACSCalibur flow cytometer, and results were analyzed using ModFit software.
One hundred micrograms of synovial tissue from RA or OA patients and cultured RA FLSs transfected with siRNA or under hypoxic conditions for 48 h were added with 100 µL of RIPA cell lysate (Beyotime, Inc., Shanghai, China) to extract total cellular protein. Total protein concentration was determined using a BCA kit (Beyotime, Inc., Shanghai, China). Equal protein samples were separated on 10% polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes. After the membranes were blocked in 5% skim milk for 1 h while shaking at room temperature, the primary antibody (CAT-1, 1:1000, Sigma-Aldrich, MO, USA; HIF-1α, 1:1000, Cell Signaling Technology, WWLP, USA) was added and incubated overnight at 4℃. We used β-actin (1:1000, Santa Cruz, TX, USA) as the loading control. The next day, the secondary antibody labeled with horseradish peroxidase was added to membranes and incubated for 1 h. The protein expression levels of CAT-1, HIF-1α, and β-actin were detected using the Odyssey Infrared Imaging System. Densitometry of the corresponding proteins was quantitatively analyzed using ImageJ software.
RA FLSs in the logarithmic growth stage were plated into 96-well plates at a concentration of 5 × 103 cells/mL, with 3 repeat wells in each group. Next, 200 µl cell suspension was added to each well. The 96-well plate was incubated at 37℃ for normal oxygen and 3% hypoxia culture, and an MTT test was performed on the first, second, third, and fourth days of culture as follows: 5 mg/mL MTT reagent (Promega, WI, USA) was added to each well (20 µl), and culture was continued for 4 h. The supernatant was subsequently removed, 150 µl DMSO (Sigma-Aldrich, MO, USA) was added to each well, and the plate was shaken for 15 min. Then, the OD value of each well was measured using a microplate reader (wavelength 570 nm).
RNA extraction and real-time PCR
Total RNA of RA and OA synovial tissues was extracted using a TRIzol™ kit (ThermoFisher Scientific, MA, USA) and reverse transcribed into cDNA according to the instructions of the first-strand cDNA synthesis kit (TaKaRa, Kyoto, Japan). Real-time polymerase chain reaction (real-time PCR) was performed using SYBR Premix Ex Taq II PCR (TaKaRa, Kyoto, Japan) on ABI Q6. The reaction conditions were as follows: predenaturation at 95 °C for 30 s, denaturation at 95 °C for 5 s, and annealing at 60 °C for 30 s, for a total of 40 cycles. Dissolution curve analysis was performed, and each sample was run in triplicate. The reaction volume was 20 µL, including 2 µL cDNA, 0.8 µL primer, 10 µL SYBR Premix and 0.4 µL ROX, and ultrapure water was added to 20 µL. The 2−ΔΔCt method was used for data analysis. The sequences of the primers are shown in Supplementary Table 1.
Partial specimens of RA and OA synovial tissues were fixed in 10% formaldehyde solution (Beyotime, Inc., Shanghai, China), embedded in paraffin, sliced at a thickness of 4 µm, baked in an oven at 80 °C, dehydrated with ethanol, inactivated for endogenous peroxidase, and hydrated with citric acid buffer solution at high temperature and high pressure. Samples were then rinsed with PBS 3 times, and the primary antibody (CAT-1, Sigma-Aldrich, MO, USA) was added at 4℃ overnight. Subsequently, samples were washed with PBS 3 times, and the secondary antibody (rabbit anti-mouse IgG-HRP, Santa Cruz, TX, USA) was added. At room temperature, diaminobenzidine (DAB) (Beyotime, Inc., Shanghai, China) was added for color rendering, hematoxylin was used for contrast staining, and 1% hydrochloric acid ethanol was added for 5 min. Samples were then washed with water for 5 min before undergoing gradient ethanol dehydration (80%, 85%, 90%, 95%, 100%, 100%) and xylene transparency treatment, drying, and finally sealing with gum. The negative control was PBS instead of primary antibody.
Cell migration assay
According to Corning® BioCoat™ Matrigel® Invasion assay kit (Corning, NY, USA), 1 × 105 cells/well RA FLSs transfected with siRNA or treated with D-arginine were seeded into the upper chambers in FBS-free media, while the bottom chambers were supplied with RPMI 1640 media containing 10% FBS. After cultured under normoxic or hypoxic conditions for 24 h, non-invaded FLSs in the upper chambers were removed and invaded cells on the bottom surface were fixed with methanol, stained with crystal violet, and counted in five randomly chosen visual fields.
Collagen-induced arthritis (CIA)
Female DBA/1 mice (n = 18), aged 8 weeks, were randomly divided into three groups: L-arginine-deprived feeding group, treatment group, and control group. Mice in the L-arginine-deprived feeding group were fed an arginine-free diet 2 weeks before model induction, while mice in the treatment and control groups were fed a normal diet. The treatment group was given water containing 2 mg/ml D-arginine 7 days prior to bovine type II collagen injection. CIA induction was performed based on reported methods : bovine type II collagen (Sigma-Aldrich, MO, USA) was dissolved in 0.1 mol/L acetic acid to a final concentration of 2 g/L, mixed and emulsified with complete Freund’s adjuvant (V:V = 1:1) (Sigma-Aldrich, MO, USA) to prepare a type II collagen emulsion. The emulsion was intradermally injected into the tails of mice at primary and secondary immunization on days 0 and 21, respectively.
Arthritis index (AI) evaluation
The incidence and severity of multiple arthritis lesions were recorded by AI score every 3 days beginning on day 1 of primary immunization. AI was divided into 5 levels (scored from 0 to 4): 0 = no redness or swelling; 1 = redness and swelling of the little toe joint; 2 = all joints and toes were red and swollen; 3 = redness and swelling appeared below the ankle joint; 4 = all areas were red and swollen, including the ankle joint. Arthritis induction was considered successful if at least one foot was red and swollen, including the ankle joint.
All data mentioned above are presented as the mean ± SD of at least 3 independent experiments. The results were analyzed using the statistical software SPSS 17.0 and GraphPad Prism 8 software. The quantitative data of two groups were analyzed using two-tailed Student’s t-tests, and comparisons among three or more groups were conducted using one-way ANOVA. A correlation analysis was performed between amino acids and cytokines using Pearson’s correlation method. A p-value less than 0.05 was considered statistically significant.