Induction and treatment of collagen-induced arthritis (CIA) in rats

The animal study was approved by the Animal Ethics Committee of the Institute of Clinical Pharmacology, Anhui Medical University. Six- to eight-week-old male Wistar rats (purchased from Shanghai SLAC Laboratory Animal Co., Ltd, Shanghai, China) were housed in a pathogen-free laboratory at the Institute of Clinical Pharmacology, Anhui Medical University. An emulsion of chicken type II collagen (Catalogue #20011, Chondrex, Woodinville, WA, dissolved in 0.1 mol/L acetic acid) and Complete Freund’s adjuvant (CFA; 4 mg/ml, Catalogue #7001, Chondrex, Woodinville, WA) was applied for intradermal injection of rats on Day 0 and Day 7 to establish the rat CIA model. Six normal rats and 6 CIA rats were compared. In the treatment study, when the joints exhibited swelling on Day 14, 15 CIA rats were randomly divided into 3 groups based on the arthritis index through a stratified random sampling method and then subjected to vehicle (Veh; 0.25% sodium carboxymethyl cellulose, CMC-Na; CIA-Veh), CP-25 (C29H32O13S, MW: 620, 50 mg/kg/d, synthesized by Chemistry Lab of Institute of Clinical Pharmacology, Anhui Medical University, Anhui, China; CIA-CP-25), or methotrexate (MTX; 2 mg/kg/3d, MAOXIANG Pharm, Co., Ltd. Changchun, China; CIA-MTX) treatment for 21 days. Five noninjected rats served as normal controls. The body weight and clinical parameters, including the swollen joint count, arthritis index, volume of paw swelling, and global assessment, were evaluated and recorded every 3 days.

Histopathological examination of joints

After treatment, all rats were sacrificed, and the ankle joints were collected and fixed with formalin for 24 h prior to decalcification in 10% ethylenediaminetetraacetic acid. Four-micron slices of paraffin-embedded joints were stained with H&E, imaged with a 3D HISTECH panoramic scanner and analysed with CaseViewer software 2.4.0.119028 (3DHISTECH Ltd, Budapest, Hungary). Two independent observers evaluated the histological changes in joints, namely, synovial hyperplasia, bone erosion, pannus formation, cell infiltration and cartilage destruction. The pathological score ranged from 0 (no change) to 4 (severe change) based on the scoring standards described previously [14].

Epi measurement in joints

The Epi concentration in joint homogenates of CIA rats was measured using an enzyme-linked immunosorbent assay (ELISA) kit (Catalogue # CSB-E08678r, CUSABIO, Wuhan, China) according to the operation manual. The absorbance was measured at 450 nm using a Bio Tek ELx808 microplate reader (Lonza Group, Ltd, Basel, Switzerland).

Primary FLS culture and transfection

Rats were sacrificed and sterilized in 75% alcohol, and synovial tissues from the bilateral knees were collected under sterile conditions. After rinsing in 75% alcohol for 5 min and in PBS three times, the synovial tissues were cut into approximately 1 mm3 blocks and attached to the bottom of a culture flask in a cell culture hood. The flask was inverted for 4 h and was then turned upright for continuous culture. After FLSs were spread around the tissue blocks, the tissue blocks were removed, and the FLSs were detached by trypsin. Three to five generations of FLSs were used for the following experiments. For the indicated study, 0.1 μg of β2AR short hairpin RNA (shRNA) was added to 5 μl of Opti-MEM (Catalogue # 31985062, Thermo Fisher Scientific, Inc., Waltham, MA, USA), and 0.3 μl of Lipofectamine 2000 transfection reagent (Catalogue # 11668027, Thermo Fisher Scientific, Inc., Waltham, MA, USA) was added to 5 μl of Opti-MEM. The 2 solutions were mixed gently and incubated at room temperature for 2 min before being added to the cells. The small interfering RNA (siRNA) (50 nM) against βarr2, Gαs, and Gαs and the control siRNA (Sangon Co., Ltd, Shanghai, China) were mixed separately with PEI (Mirus, Madison, WI, USA) according to the manufacturer’s instructions and transfected into the cells for 24 h at 37 °C. Green fluorescence could be observed after 48 h of incubation, indicating the successful transfection of β2AR shRNA, βarr2 siRNA, Gαs siRNA and Gαi siRNA. The sequences of specific siRNA were listed in Table 1.

Table 1 The sequences of siRNA for βarr2, Gαs and GαiFLS viability assay

A cell counting kit-8 (CCK-8) assay was used to evaluate the viability of FLSs. Briefly, FLSs were seeded in a 96-well plate at 5 × 104 cells/well and cultured for 48 h under the indicated treatment conditions. Ten microlitres of CCK8 reagent (Catalogue # BS350A, Biosharp, Guangzhou, China) was added to each well 4 h before the end of the culture period, and the absorbance was measured at 450 nm on a Bio Tek ELX808 microplate reader (Lonza Group, Ltd, Basel, Switzerland).

Cell migration and invasion assays

Transwell plates were used to evaluate the migration and invasion of FLSs. A total of 5 × 104 FLSs in serum-free Dulbecco’s modified Eagle’s medium (DMEM) were seeded in the upper chamber of a transwell plate, and 500 μl of 10% serum DMEM was added to the bottom chamber. The cells were treated and cultured for 48 h, and the membrane in the upper chamber was washed with phosphate-buffered saline. The cells remaining in the upper chamber were removed by wiping, while the migrated FLSs were fixed with crystal violet solution and counted after photographing. The FLS invasion ability was measured using the same method but with a Matrigel coating on the membrane (Catalogue # 354234, Corning, NY, USA) in the upper chamber of the transwell plate.

Coimmunoprecipitation (Co-IP)

The interaction of β2AR with Gαs or Gαi was confirmed by co-IP as previously reported [15]. Normal or CIA FLSs or normal FLSs treated with ISO in the presence or absence of the GRK2 inhibitor CP-25 were lysed in NP40 immunoprecipitation buffer supplemented with protease inhibitor cocktails. The cell lysate supernatant was collected after centrifugation at 15,000 × g for 15 min at 4 °C, and the protein concentration was determined by a BCA protein assay kit (Catalogue #23225, Thermo Fisher Scientific Inc., Waltham, MA, USA). One milligram of protein was preincubated with 10 μl of Protein A/G PLUS-Agarose beads (Catalogue # sc-2003, Santa Cruz, CA, USA) and with 2 μg of mouse IgG as the control antibody for 1 h at 4 °C, and the precipitates were then collected by centrifugation at 1000 × g for 1 min at 4 °C. A portion of the supernatant was retained for input analysis. The precleared protein was then incubated with 10 μl of Protein A/G PLUS-Agarose beads preincubated with the anti-β2AR antibody (Catalogue # sc-570, Santa Cruz, CA, USA) overnight at 4 °C with rotation. The beads were then precipitated by centrifugation and boiled with 2 × sodium dodecyl sulfate (SDS) loading buffer, and Gαs, Gαi and β2AR were detected using Western blotting.

Western blotting

Proteins from lysed FLSs were collected as mentioned before, separated on a 10% SDS polyacrylamide gel and then transferred to a polyvinylidene fluoride membrane (Millipore Corporation, Billerica, MA). The membrane was blocked in Tris-buffered saline containing 0.05% Tween 20 (TBST) and 5% nonfat milk at 37 °C for 2 h, followed by incubation with a primary antibody against β1AR (1:1000, Catalogue # PA1-049, Thermo Fisher Scientific, Waltham, MA, USA), β2AR (1:600, Catalogue # sc-570, Santa Cruz Biotechnology, CA, USA), β3AR (1:500, Catalogue # YT0363, Immunoway, TX, USA), Gαs (1:500, Catalogue # sc-823, Santa Cruz Biotechnology, CA, USA), Gαi (1:500, Catalogue # sc-391, Santa Cruz Biotechnology, CA, USA), or GAPDH (1:5000, Catalogue # AF0911, Affinity Biosciences, Changzhou, China) overnight at 4 °C. After washing with TBST. The membrane was incubated with goat anti-rabbit IgG (H + L) horseradish peroxidase (HRP)-linked (1:10,000, Catalogue # S0001, Affinity Biosciences, Changzhou, China) or goat anti-mouse IgG (H + L) HRP-linked (1:10,000, Catalogue # S0002, Affinity Biosciences, Changzhou, China) at 37 °C for 2 h. To evaluate the cellular distribution of β2AR, membrane and cytosolic proteins were extracted with a membrane and cytosolic protein extraction kit (Beyotime Biotechnology, Shanghai, China). Enhanced Chemiluminescence Western Blotting Substrate (Catalogue # 32106, Thermo Fisher Scientific, Waltham, MA, USA) was applied for band detection on an ImageQuant LAS 500 Imager (GE Healthcare Systems, Chicago, IL, USA). Protein expression was semiquantified with ImageJ (version 1.42q, NIH) and was normalized to GAPDH expression.

Quantitative real-time PCR (qRT‒PCR)

Total RNA was extracted from FLSs using TRIzol reagent following the manufacturer’s protocol. Complementary DNA (cDNA) was then synthesized with a cDNA synthesis kit (Catalogue #: 634926, Takara Bio Inc., Otsu, Shiga, Japan), and the specific genes were then amplified from the cDNA templates in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with Fast SYBR Green Master Mix (Catalogue #: 4385612, Thermo Fisher Scientific, Waltham, MA, USA). The specific primers used for amplification of β1AR, β2AR, and β3AR were listed in Table 2. Changes in expression were calculated by normalization to the corresponding ACTIN levels with the 2−ΔΔCt method.

Table 2 Primers for β1AR, β2AR, and β3AR mRNA amplificationImmunofluorescence staining

Immunofluorescence staining was performed to detect the in situ expression and distribution of the indicated proteins as previously described [16]. FLSs were plated on coverslips and treated for 48 h before fixation with 4% paraformaldehyde for 30 min and permeabilization with 0.5% Triton for 15 min. Subsequently, the cells were blocked with 1% BSA for 30 min and incubated with primary antibodies, including anti-β1AR (1:600, Catalogue # sc-568, Santa Cruz Biotechnology, CA, USA), anti-β2AR (1:600, Catalogue # sc-570, Santa Cruz Biotechnology, CA, USA), anti-Gαs (1:500, Catalogue # sc-823, Santa Cruz Biotechnology, CA, USA), and anti-Gαi (1:500, Catalogue # sc-391, Santa Cruz Biotechnology, CA, USA) antibodies, overnight at 4 °C prior to 3 rinses and incubation with goat-anti-mouse Alexa Fluor 488 (1:200, Catalogue # 615–545-214, Jackson ImmunoResearch Inc., West Grove, PA, USA) or goat-anti-rabbit Alexa Fluor 555 (1:200, Catalogue # A-21428, Thermo Fisher Scientific Inc., Waltham, MA, USA) secondary antibodies for 1 h. Finally, coverslips were mounted with a mounting solution containing 4′,6-diamidino-2-phenylindole and then observed on a Leica TCS SP80 confocal microscope (Leica Microsystems, Wetzlar, Germany). β2AR coupling with Gαs and Gαi was semiquantified using the built-in colocalization analysis software module.

Fluorescence Resonance Energy Transfer (FRET)

A FRET assay was performed to detect intracellular cAMP production upon β2AR activation [17]. FLSs were seeded on coverslips in 24-well plates and transfected with 0.5 μg of regular pcDNA-Epac 3 (Reg-ICUE3) plasmid for 36 h. The cells were treated with ISO (final concentration of 1 μM), with or without CGP20712A (CGP, β1AR antagonist, 1 μM), ICI (β2AR antagonist, 1 μM), SR59230A (SR, β3AR antagonist, 100 nM), Bar (10 μM), a PKA inhibitor (PKI, 1 μM), CP-25 (1 μM), or PAR (1 μM) overnight before the end of the transfection period and stimulated acutely with ISO (100 nM), dobutamine (Dob;, β1AR agonist, 10 μM) or Ter (β2AR agonist, 10 μM). The fluorescence signals in both the 480 nm and 535 nm channels were recorded on a Leica TCS SP80 confocal microscope, and the intensity ratio of cyan fluorescent protein (CFP) to yellow fluorescent protein (YFP) was calculated at different time points. When the level of intracellular cAMP is increased, the CFP/YFP ratio is decreased.

Statistical analysis

Data were collected from three to five animals per group and were analysed with GraphPad Prism software (version 9, GraphPad Software, Inc., San Diego, CA, USA) and expressed as the means ± standard deviations (SDs). One-way analysis of variance (ANOVA) was used to determine the significance of differences among three or more groups. Two-way ANOVA was used to determine the significance of differences among three or more groups when time was also considered as a variable. Independent t tests were used for comparisons between 2 groups. p < 0.05 was considered to indicate a significant difference.