RA modeling, grouping, and treatment
Type II collagen-induced rat models have several similarities to human RA and have been widely used in RA studies in vivo. To build this model, type II collagen (Sigma, USA) and Freund's incomplete adjuvant (1:1) (Sigma) were mixed and emulsified on ice. In the RA group (n = 6), 200 µL of the above mixture was injected subcutaneously into the tail of SD rats (5 weeks old, 200 ± 20 g) (Vital River Laboratory Animal Technology Co., Ltd., Beijing, China). On day 21, a booster injection of the above mixture (200 µL) was conducted by intradermal injection at another site. The control group rats received equal doses of PBS injections in the same manner (n = 6). Rats were allowed free access to food and water, and their growth conditions included a temperature of 22–26°C, humidity of 40–60%, and a light/dark cycle of 12 h. All the protocols were approved by the Research Ethics Committee of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China (Ethical code: 2021B009). The animal experiments were performed following the guidelines for the Care and Use of Laboratory Animal Experience of the Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences.
For Ad-shGZMK or Ad-shCCL5 treatment, Ad-NC, Ad-shGZMK, or Ad-shCCL5 (108 pfu) (GenePharma, Shanghai, China) were administered via intraarticular injection into their left hind ankle joints at 15, 20 and 25 days after the first immunization (n = 6/group). All rats were sacrificed with intraperitoneal sodium pentobarbital (100 mg/kg, Sigma) on the 36th day to harvest synovial or ankle joint tissues for further experimental analysis. The left posterior ankle joints were X-rayed to observe the joint alterations using an MRAD-D50S RADREX-I system (Toshiba Medical Manufacturing Co., Ltd., Japan). Images were read and assessed by a radiological pathologist.
Assessment of the severity of arthritis
From the first immunization, the rats were forced to undergo clinical examinations to assess severity every other day. The rat's paw scores were as follows [15]: no macroscopic evidence of erythema and swelling was observed (0 points); Erythema and mild soft-tissue swelling were observed localized to the tarsal/ankle joint (1 point); Erythema and mild soft-tissue swelling from the ankle to the tarsal bone were observed (2 points); erythema and moderate soft-tissue swelling extending from the ankle to the tarsal bone (3 points) were observed; significant erythema and severe swelling (including ankles, plantar surfaces, and toes) or stiffness of the extremities (score 4) were observed. Ankle joint staining of rats was conducted by Hematoxylin and eosin (HE) (Beyotime, Beijing, China) and safranin O/fast green staining (Beyotime.) following the manufacturer's scheme. A digital pathology slide scanner (3DHISTECH; The Digital Pathology Company, Budapest, Hungary) was used to scan all sections.
Gene expression profiling
Synovial samples were submitted to Majorbio Biopharm Technology Co., Ltd. (Shanghai, China) for total RNA extraction, purification, library preparation, and sequencing. Total RNA was isolated from synovial samples using TRIzol reagent (Invitrogen, Waltham, MA, USA). RNA purity and concentrations were analyzed by a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Wilmington, DE, USA). RNA quality was determined by a 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Ribosomal RNA digestion was done using the Ribo-Zero Gold kit (Illumina, San Diego, CA, USA). The libraries were then constructed and sequenced on an Illumina HiSeq 2000 platform [16]. Differential expressions of mRNAs were analyzed using DEGseq. The two groups of differentially expressed indicators were presented using fold change (FC), with statistical differences evaluated by t-test. A P value < 0.05 indicates differential expression.
Data retrieval
Microarray analysis data on the adjuvant-induced arthritis model were downloaded from the GSE115662 dataset in the GEO database, and DEGs were screened. The heatmap and volcano plot were drawn to present the differential expression of DEGs. DEGs with P < 0.05 and |log2 FC| > 1 were considered statistically significant.
Synovial fibroblast isolation and cell culture
The synovia of rats in RA and control groups were used for the isolation of SFs, named RA-SFs and control-SFs. Small pieces of fresh ankle joints synovial tissues (1 mm3) cut with scissors under sterile conditions were gently pipetted using a Pasteur pipette attached to the cell culture flask wall and cultured in DMEM/high-glucose medium (Hyclone, Logan, UT, USA) containing 20% (v/v) fetal bovine serum (FBS) (Invitrogen), streptomycin (100 mg/mL) (Beyotime), and penicillin (100 U/mL) (Beyotime). The culture flask was placed upright for 6 h in an incubator at 37°C with 5% CO2 to allow tissue adhesion. After fibroblast-like cell colonies developed, the adherent cells were digested. All experiments were conducted using SFs from the 4th to 7th generations.
Reverse transcription-quantitative polymerase chain reaction
Synovial tissues or SFs were used for total RNA extraction following the manufacturer's scheme by TRIzol reagent (Invitrogen). RNA purity and concentrations were determined by a NanoDrop ND-1000 spectrophotometer. Reverse transcription was conducted using the PrimeScriptTM RT reagent kit with a gDNA eraser (Takara, Tokyo, Japan). Quantitative PCR was executed on the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA) with the SYBR® Premix Ex TaqTM II Kit (Takara). Relative mRNA levels were determined by the 2−ΔΔCtmethod [17]. The primer sequences are exhibited in Supplementary Table 1.
Coimmunoprecipitation (Co-IP)
The co-IP Kit (P2179S, Beyotime) was used to verify the GZMK-CCL5 interaction. SFs (107cells/mL) were lysed in a pre-cooled lysis buffer with a protease inhibitor cocktail and incubated with antibodies against GZMK (MAB209Mu27, Wuhan Yunclone Technology Co., Ltd., China) and CCL5 (#D163610, Sangon Biotech, Shanghai, China) at 4°C overnight. After incubation with protein A Sepharose (Sigma), the immunoprecipitates were washed, and the co-IP results were analyzed by western blotting.
Western blotting
Total protein from synovial tissues or SFs was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime) containing protease inhibitors (Beyotime) and quantified by a BCA kit (Beyotime, China). Protein samples were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies. The membranes were then incubated with goat anti-rabbit IgG (HRP) (1:2000, Abcam, ab6721) for 2 h. Protein bands were detected using an enhanced chemiluminescence detection system (Invitrogen). The antibodies used were as follows: GZMK (MAB209Mu27, Wuhan Yunclone Technology Co., Ltd), CCL5 (#D163610, Sangon Biotech), p-ERK (#sc-7383, Santa Cruz Biotechnology, Santa Cruz, California, USA), ERK (#sc-514302, Santa Cruz Biotechnology), and GAPDH (#sc-365062, Santa Cruz Biotechnology).
Cell transfection and treatment
Small interference RNAs targeting GZMK (si-GZMK-1 and si-GZMK-2) and its control si-NC, as well as CCL5 and GZMK overexpression plasmids and pcDNA control vectors (Ctrl) were obtained from GenePharma. Cell transfection was performed using Lipofectamine 2000 (Invitrogen, USA) following the manufacturer's instructions. For ERK activation, ERK phosphorylation agonist Ro 67-7476 (MCE, South Brunswick Township, NJ) (10 µM) was added.
Cell counting kit-8 (CCK-8)
A diluted suspension of SFs (100 µL, 1 × 106 cells/mL) was added to 96-well plates and cultured for 24, 48, 72, or 96 h. Each well was added with 10 µL of CCK-8 reagent (Dojindo, Tokyo, Japan). After 2 h of incubation, the absorbance was read at 450 nm with a microplate spectrophotometer (Envision, Perkin Elmer, Waltham, MA).
5-Ethynyl-2’-deoxyuridine (EdU) assay
Cell proliferation was evaluated by an EdU detection kit (RIBBIO, Guangzhou, China). The SFs were then incubated for 24 h. The EdU labeling cell procedure was performed following the instructions. The staining results were observed and photographed under a fluorescence microscope (Olympus BX51, Japan).
Cell cycle analysis
SFs (1 × 105 cells/well) in the logarithmic phase were plated in 6-well plates for 48 h. The cells (1 × 105) were fixed with 70% ethanol (1 mL) at 4 ℃ overnight and then incubated with 100 µg/mL RNase (BD Bioscience, NJ, USA) at 37 ℃ for 30 min. The cell cycle was detected using a BD FACSCalibur flow cytometer after staining with 50 µg/mL PI (BD Bioscience) at 4 ℃ for 30 min. The data were analyzed using FlowJo 10 software (FlowJo).
Cell apoptosis
An Annexin V/PI Apoptosis Detection kit (Solarbio, Beijing, China) was used for flow cytometry. SFs (200 µL, 3 × 105 cells/mL) were resuspended in binding buffer and stained using Annexin V/FITC (5 µL) at room temperature for 5 min and PI (5 µL) at room temperature for 5 min. Cell apoptosis was assessed using a fluorescence-activated cell sorting cytometer (BD Biosciences).
Transwell migration and invasion assays
The migration of SFs was performed using a transwell migration assay with a filter (6.5 mm diameter, 8.0 µm pore size) (Corning, NY, USA). Briefly, SFs (1 × 105 cells/mL) suspended in serum-free DMEM were seeded to the chambers' upper compartments. A medium containing 10% FBS as a chemoattractant was added to the lower compartments. After 24 h incubation, the migrated SFs were fixed in methanol for 15 min and stained with 0.1% crystal violet (Solarbio) for 15 min. The number of the migrated SFs was analyzed with a microscope (Olympus BX51). We performed a similar experiment in chambers coated with BD Matrigel basement membrane matrix (BD Biosciences) to measure cell invasion.
Reactive oxygen species assay
ROS production in SFs was evaluated using a DCFH-DA probe (Beyotime, Shanghai, China). The cells were added at a concentration of 10 µM/mL DCFH-DA medium and incubated at 37°C for about 20 min. After removing the remaining probes, cell precipitates were collected and resuspended, and samples were analyzed by flow cytometry.
Detection of ferrous iron (Fe2+), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD)
An iron assay kit (MAK025, Sigma, USA) was used to analyze the levels of Fe 2+ in the transected SFs. The MDA content and the activities of GSH and SOD in the transected SFs were detected by the MDA (S0131, Beyotime, China), GSH (S0053, Beyotime), and SOD (S0101S, Beyotime) assay kits. These experiments were conducted based on the manufacturer's protocols.
FerroOrange fluorescence probe for analyzing intracellular Fe2+levels
For intracellular Fe2+ detection, a FerroOrange fluorescent probe was used following the manufacturer's protocol. SFs were incubated with 250 µM FerroOrange for 30 min. After washing, cells were observed under an inverted fluorescence microscope (Olympus BX51). The fluorescence intensity of Fe2+ was quantified with ImageJ software.
Immunofluorescence (IF)
SFs were seeded in 6-well plates with a coverslip at the same density as described above. When the cells reached 80% confluence, they were pre-treated with 5 µM H2O2 for 12 h. Briefly, 4% paraformaldehyde (1 mL) was added to each well and incubated for 30 min at room temperature. The cells were incubated with 0.5% Triton X-100 for 20 min. The wells were blocked and then incubated with primary antibodies α-sma (ab124964, Abcam), Ki67 (ab279653, Abcam), caspase-3 (sc-7272, Santa Cruz Biotechnology), mmp-9 (sc-21733, Santa Cruz Biotechnology), and GPX4 (sc-166570, Santa Cruz Biotechnology) at 4°C overnight. The cells were rinsed thrice with PBS before adding a goat secondary antibody to mouse IgG (Alexa Fluor® 488) (ab150117, Abcam) at 2 µg/mL (shown in green) and a goat secondary antibody to rabbit IgG (Alexa Fluor® 594) (ab150080, Abcam) at 2 µg/mL (shown in red) for 1 h. DAPI (Beyotime) was then added and incubated for 10 min in the dark. The cells were photographed under a fluorescence microscope (Olympus BX51).
Statistical analysis
Data are presented as the mean ± standard deviation. Unpaired Student's t-test or one-way analysis was performed for statistical analysis using GraphPad Prism 7 (GraphPad Software, Inc.). All data were obtained from at least three independent experiments. P < 0.05 was considered to indicate a statistically significant difference.