Patients’ serum for enzyme linked immunosorbent assay (ELISA)
We enrolled 49 RA and 25 OA patients with comprehensive medical records from the Rheumatology department, Integrated Traditional Chinese and Western Medicine hospital, Southern Medical University, China between Oct. 2017 to Dec. 2018. Exclusion criteria included other autoimmune diseases, acute inflammation, fever, thyroid disease, diabetes, liver and kidney diseases. Patients’ sera were collected for ELISA assays of AIM2 (R&D Systems, USA). The ELx808TM absorbance microplate reader was used to measure the absorbance values at 450 nm. Concentration of proteins in the samples were calculated using a standard curve for each protein. General information of patients recruited are given in Table 1.
Table 1
General information of patients recruited for ELISA in this study
General information | RA(n = 49) | OA(n = 25) |
Gender (male/female) | 11/38 | 7/18 |
Age(y/o) | 50.87 ± 9.35 | 58.48 ± 9.69** |
disease course (years) | 7.27 ± 5.11 | 6.41 ± 8.88 |
ESR (mm/h) | 56.12 ± 40.36 | 34.7 ± 24.88* |
CRP (mg/L) | 12.16 ± 20.36 | 2.21 ± 2.76* |
RA, rheumatoid arthritis; OA, osteoarthritis; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein. *, p < 0.05; **, p < 0.01. |
Patients’ synovium for immunohistochemical (IHC) staining
Arthroscopic surgery was applied to 41 RA and 26 OA patients for therapeutics purpose (patients’ general information in Table 2). Regular streptavidin biotin-based immunoperoxidase staining for Aim2, Aim2, ASC, caspase-1, and IL-1β was performed to formalin fixed, paraffin embedded pathology keen synovium specimens. H score was applied to quantify the staining intensity10
Table 2
General information of patients recruited for IHC in this study
General information | RA (n = 41) | OA (n = 26) |
Gender (male/female) | 6/35 | 6/20 |
Age (y/o) | 51.6 ± 2.408 | 60.62 ± 2.296 |
Disease course (years) | 6.538 ± 0.8864 | 6.417 ± 1.428 |
ESR (mm/h) | 79.68 ± 6.896 | 38.38 ± 6.636*** |
CRP (mg/L) | 30.26 ± 5.437 | 7.015 ± 4.216** |
RA, rheumatoid arthritis; OA, osteoarthritis; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein. **, p < 0.01; ***, p < 0.001. |
Patients’ synovium for quantitative real-time polymerase chain reaction (qPCR)
Synovium specimens from 10 RA patients and 9 OA patients obtained through knee arthroscopy (patients’ general information in Table 3). Specimens were soaked in TRIzol® Reagent (Thermo Scientific, USA) after remove adipose tissue under sterilized environment, then stored under − 20℃ refrigerator for qPCR on mRNA of Aim2, ASC, caspase-1, and IL-1β. qPCR was also applied to evaluate relative expression of mRNA AIM2, ASC, caspase-1, and IL-1β in FLS after transferred with AIM2 siRNA.
Table 3
General information of patients recruited for RT-qPCR in this study
General information | RA (n = 10) | OA (n = 9) |
Male: female | 2:8 | 5:4 |
Age (years) | 54.2 ± 8.65 | 66.6 ± 8.61** |
ESR (mm/h) | 86.5 ± 50.5 | 37.8 ± 29.3** |
CRP (mg/L) | 19.1 ± 13.63 | 5.13 ± 7.19* |
RA, rheumatoid arthritis; OA, osteoarthritis; ESR, erythrocyte sedimentation rate; CRP, C-reactive protein. *, p < 0.05; **, p < 0.01. |
The primers for amplifying the target mRNAs are listed in Table 4.
Table 4
qPCR primers used in this study
Primer name | Sense primer/sequence | Antisense primer |
AIM2 | AGCAAGATATTATCGGCACAGTG | GTTCAGCGGGACATTAACCTT |
ASC | CCTACTGTTCTTTCTGTGGGAAG | CGAGGTCGTCAGCCATCAC |
CASPASE-1 | TTTCCGCAAGGTTCGATTTTCA | GGCATCTGCGCTCTACCATC |
IL-1β | TTCGACACATGGGATAACGAGG | TTTTTGCTGTGAGTCCCGGAG |
GAPDH | ACAACTTTGGTATCGTGGAAGG | GCCATCACGCCACAGTTTC |
Immunofluorescent staining for AIM2
For immunofluorescent staining, both sections of RA and OA patients were blocked with normal goat serum in 0.01 M phosphate-buffered saline for 1 hour. The primary rabbit anti-rat AIM2 antibody (1:200) was incubated overnight at 4°C together with either mouse anti-human vimentin ( 1:400); The following day, the sections were incubated for 60 minutes at 37°C with FITC-conjugated goat and anti-mouse (1:1000) and anti-rabbit (1:1000). The nuclei of cells were stained with DAPI. The results were examined under a fluorescence microscopy.
AIM2 siRNA preparation
AIM2 siRNA were produced by Ribobio Company, China. The following siRNA sequences were used: AIM2 siRNA 1 (5′-GAGCTCTTCACCACTTTCA-3′), AIM2 siRNA 2 (5′-GGAGCGGGTGTATTTACAT-3′), AIM2 siRNA 3 (5′-CGTCGAGTCTTTGTCAGAA-3′).
Isolation and culture of fibroblast-like synoviocytes (FLS)
FLS were derived from synovial tissue specimens harvested from patients by needle arthroscopy. FLS were isolated by enzyme digestion and subsequently cultured in Dulbecco’s modified Essential medium (DMEM) containing 10% fetal bovine serum (FBS, Invitrogen) containing antibiotics (penicillin and streptomycin) at 37°C with 5% CO2. Cells cultured between passages 4 and 9 were used for this study. Cells were frozen with cell freezing medium and stored in -80℃ freezer until used.
Methyl thiazolyl terazolium (MTT) assay
MTT (methyl thiazolyl terazolium) assay was used to ascertain the effects of AIM2 siRNA on FLS viability at different concentrations. FLS samples were digested using 0.25% pancreatin were transferred to two 96-well plates with 3-5×103 cells/well. After 24 h of culture, the FLS were transferred with AIM2 siRNA (20 nM). Six replicates were used at each time point. At different time intervals (0, 24, 48 and 72 h), MTT solution (5 mg/ml, Sigma-Aldrich, St. Louis, USA) was added, followed by 4 h incubation period. Then the culture medium was aspirated and 150 µl of dimethyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals. Optical density (OD) was measured at 490 nm using Universal Microplate Reader (Bio-Tek instruments, Winooski, USA). Data curves were plotted with OD values on the y-axis and time intervals on the x-axis. Each experiment was done in triplicates.
Flow cytometry for apoptosis
Flow cytometry was performed to evaluate the effects of SiRNA Aim2 on FLS apoptosis. 48 hours after FLS treated by SiRNA aim2 and NC in each groups in 6-well plates, the cells were collected (around 3×105/well), washed two times with PBS, and resuspended in 500µl 1×binding buffer, mixed with 5µl of Annexin-V-fluorescein isothiocyanate (FITC) and 5µl of propidium iodide (PI), and eventually detected by flow cytometer (BD LSRFortessaTM, USA). The scatter diagram was distributed as follows: Q3: healthy cells (FITC-/PI-); Q2: apoptotic cells at an advanced stage (FITC+/PI+); Q4: apoptotic cells at an early stage (FITC+/PI-). The apoptosis rate = ratio of apoptotic cells to the total cells in Q4 + ratio of apoptotic cells to the total cells in Q2. Each experiment was conducted three times.
Transwell test
For the matrigel invasion assay, cells at the logarithmic growth phase were digested, collected, re-suspended and diluted into a concentration of 3×104/mL in serum-free medium. The cell suspension (200 μl) was added to the upper chamber coated with Matrigel (BD Bioscience) that were diluted with DMEM media (1:3), while 500 μl DMEM media containing 10% FBS was added in the lower chamber. After incubation for 12 h at 37 °C, the cells in the upper membrane were discarded and cells on the lower membrane were fixed using 4% Paraformaldehyde for 25 min and stained with 0.1% crystal violet (Beyotime, USA) for 10 min. Next, five random fields were counted.
Automated electrophoresis western blot analysis
FLS were seeded at 1-2×105 cells per well in 6-well plates and incubated for adherence. Medium was replaced in the wells with fresh medium containing metformin (5 mM) or saline for 48h. After aspiration of the medium, cell monolayers were rinsed with 1 ml ice-cold PBS and lysed in 80 μl of lysis buffer (20 mM Tris–HCl pH 7.5, 150 mM NaCl, 1mM EDTA,1mM EGTA, 1%(v/v) TritonX100, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate) supplemented with fresh 1 mM Na3VO4, 1mM dithiothreitol containing 1 X protease inhibitor cocktail (Roche Molecular Biochemicals, Basel, Switzerland). Lysates were pre-cleared by centrifugation at 18000 g for 15 min at 4℃. Supernatants were collected, protein concentrations were measured using Bradford assay (Thermofisher, Massachusetts, USA) and lysates were adjusted to 5 mg/ml protein concentration. Capillary electrophoresis western blot analysis was carried out using manufacturer’s reagents provide in the the user manual (ProteinSimple WES, San Francisco, USA). Briefly, 5.6 μl of the cell lysate was mixed with 1.4 μl of fluorescent master mix and heated at 95℃ for 5 min. The samples, blocking reagent, wash buffer, antibody of tublin, AIM2, and TNF-α (1:100 R&D Systems), secondary antibody and chemiluminescent substrate were dispensed into the microplates. The electrophoretic separation and immunodetection was performed automatically using default settings. The data was analyzed using in-built Compass software for SW 4.0. The truncated and full-length AIM2 and TNF-α intensities (area under the curve) were normalized to that of the tubulin peak (control). In most of the figures, electropherograms are represented as pseudo-blots, generated using Compass software.
Statistical analysis
Statistical analysis was conducted with GraphPad Prism 7.0 software. All the data were denoted as mean ± SD. Differences between two groups were evaluated for statistical significance using Student’s t-test. Kruskal-Wallis and Dunn’s multiple comparison post-hoc test was used to evaluate the differences among three or more groups. Correlations were evaluated using Spearman’s Rank correlation test. P ≤ 0.05 was considered as statistically significant.