A total of 30 8 week-old female Sprague Dawley rats (200–250 g body weight) were purchased from The Experimental Animal Center of Jinzhou Medical University (Jinzhou, China). All rats were housed at 23±1°C, 50±5% humidity with a 12 h light/dark cycle and free access to food and water. The induction of type II collagen-induced arthritis was achieved as previously described (31), by the subcutaneous injection of 2 mg collagen (ModiQuest Research) per rat (n=10 in each group). Rats were treated with IL-1β (10 mg/kg, Sigma-Aldrich; Merck KGaA), PBS (control; equal volume) or anti-IL-1β (10 mg/kg, ACZ885, Sigma-Aldrich; Merck KGaA) by subcutaneous injection every 4 days for a total of seven times.

Rats were examined 28 days after collagen injection, and an arthritis score was assigned to each rat. The arthritis scores of experimental rats were evaluated using a scale of 0–2 for each paw, with a maximum total score of 8, as previously described (32). A score for each paw was assigned as follows: 0, normal paw; 0.25, 1–2 swollen toes; 0.5, 3–4 swollen toes; 0.75, slightly swollen footpad or ankle; 1, swollen footpad or ankle; 1.25, 1–2 swollen toes and swollen footpad or ankle; and 2.0, swollen toes and swollen footpad and ankle.

The tibias in experimental rats (n=5 per group) were fixed in 4% paraformaldehyde for 24 h, decalcified in 10% EDTA (pH = 7.4) for 5 days and embedded in paraffin. The tibias were cut into 4 µm tissue sections and then stained with 1% haematoxylin and eosin (H&E) for 15 min at room temperature. The tissue sections were imaged using a light microscope (TE2000S; Nikon Corporation).

Blood samples were collected from all rats 28 days after collagen injection. Samples were centrifuged at 4,000 × g for 15 min at 4°C. The circulating levels of TNF-α (cat. no. RTA00, R&D Systems, Inc.) and IL-17 (cat. no. HS170, R&D Systems, Inc.) were analyzed using ELISA kits according to the manufacturer's protocol.

Synovial membranes were collected from rats 28 days after collagen injection. Tissues were fixed with 4% paraformaldehyde at room temperature for 12 h. Paraffin-embedded tissue samples of synovial membranes were obtained and cut into 4 µm sections, deparaffinized and rehydrated using a descending alcohol series. Sections were prepared and epitope retrieval was performed using Tris-HCl buffer (cat. no. AP-9005-050; Thermo Fisher Scientific, Inc.) for 30 min at 37°C. Tissue sections were stained H&E (Sigma-Aldrich) for 15 min at room temperature. Sections were treated with 3% hydrogen peroxide for 15 min at 37°C and subsequently blocked with 5% BSA (Sigma-Aldrich; Merck KGaA) for 2 h at 37°C. Sections were washed with PBS and incubated with rabbit anti-rat IL-17 (1:1,000; ab193955; Abcam), TNF-α (1:1,000; ab109332; Abcam), ERK (1:1,000; ab32537; Abcam), phosphorylated ERK (pERK; 1:1,000; ab201015; Abcam) and STAT1 (1:1,000; ab2071; Abcam) at 4°C overnight. Sections were washed three times and incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG; 1:2,000; cat. no. 1706515; Bio-Rad Laboratories, Inc.) for 1 h at 37°C. Diaminobenzidine was used as substrate for the immunohistochemical reaction. Tissue sections were visualized at ×200 magnification using a confocal microscope (LSM780; Carl Zeiss AG).

The sfd-FLS line HIG-82 (American Type Culture Collection cat. no. 1832) was purchased from BeNa Culture Collection. sfd-FLSs were grown in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% FBS (Gibco; Thermo Fisher Scientific, Inc.) at 37°C with 5% CO₂. Cells were treated with IL-1β (1 mg/ml; cat no. SRP6551; Sigma-Aldrich; Merck KGaA), anti-IL-1β (1 mg/ml; cat no. PRS4877; Sigma-Aldrich; Merck KGaA) and/or NF-κB inhibitor (1 mg/ml; cat no. 481412; Sigma-Aldrich; Merck KGaA) for 12 h at 37°C for further analysis.

sfd-FLSs were seeded in 6-well plates at a density of 1×10⁴ cells/well in 2 ml RPMI-1640 supplemented with 10% FBS. Cells were cultured for 12 h and washed with PBS three times. NF-κB cDNA was cloned into a pcDNA3.1 plasmid (pcDNA3.1-NF-κB; Thermo Fisher Scientific, Inc.), and the empty plasmid pcDNA3.1 (Thermo Fisher Scientific, Inc.) served as control. sfd-FLSs were transfected with pcDNA3.1-NF-κB (5 µg) or empty pcDNA3.1 (5 µg) using Lipofectamine 2000 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Cells were harvested after 72 h for further analysis.

Total RNA was extracted from sfd-FLSs using the RNAeasy mini kit (Qiagen GmbH) according to manufacturer's protocol. RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit (Qiagen GmbH) at 42°C for 2 h according to on the manufacturer's instrument. All forward and reverse primers were purchased from Invitrogen (Thermo Fisher Scientific, Inc.) and are listed in Table I. qPCR was performed as follows: Initial denaturation at 95°C for 2 min, followed by 45 cycles of 95°C for 30 sec, 59°C for 30 sec and 72°C for 30 sec. The total volume of each reaction was 25 µl and contained 50 ng of cDNA, 200 µM dNTP, 2.5 units of Taq DNA polymerase (Takara Biotechnology, Co., Ltd.) and 200 µM primers using the SYBR^® Premix Ex Taq™ kit (Takara Biotechnology, Co., Ltd.). Relative mRNA expression levels were calculated using the 2^−ΔΔCq method (33). The results are presented as fold-change relative to the expression level of β-actin, used as internal control.

sfd-FLSs were homogenized using RIPA lysis buffer (Thermo Fisher Scientific, Inc.). Protein concentration was measured using a bicinchoninic acid assay kit (Thermo Fisher Scientific, Inc.). Subsequently, protein samples (20 µg in each lane) were separated by 12.5% SDS-PAGE. Protein were blotted on a nitrocellulose membrane and the membranes were incubated with primary antibodies anti-IL-17 (1:1,000; ab193955; Abcam), TNF-α (1:1,000; ab109332; Abcam), ERK (1:1,000; ab32537; Abcam), pERK (1:1,000; ab201015; Abcam), STAT1 (1:1,000; ab2071; Abcam), pSTAT1 (1:1,000; ab30645; Abcam) and β-actin (1:1,000; ab8226; Abcam) for 12 h at 4°C, after blocking with 5% BSA (Sigma-Aldrich; Merck KGaA) for 1 h at 37°C. Subsequently, the membranes were incubated with HRP-conjugated goat anti-rabbit IgG (1:5,000; cat. no. PV-6001; OriGene Technologies, Inc.) for 24 h at 4°C. The blots were visualized using an enhanced chemiluminescence detection system (cat. no. 32209; Pierce; Thermo Fisher Scientific, Inc.). Densitometric quantification was performed using Quantity-One software (version 1.2; Bio-Rad Laboratories, Inc.).

Data are presented as the mean ± SD. Differences were evaluated for significance using one-way ANOVA followed by Tukey's post hoc test. Data were analyzed using GraphPad Prism (version 6.0; GraphPad Software, Inc.). P<0.05 was considered to indicate a statistically significant difference.

The effects of IL-1β and of an IL-1β inhibitory antibody (anti-IL-1β) on inflammation were investigated in a rat model of rheumatoid arthritis. The results suggested that treatment with anti-IL-1β decreased the rheumatoid arthritis score, whereas treatment with IL-1β exacerbated rheumatoid arthritis in vivo (Fig. 1A). Histopathological analysis demonstrated decreased synovial hyperplasia and bone erosion in the anti-IL-1β group compared with the control and IL-1β-treated groups. Treatment with anti-IL-1β decreased the bone injury score, whereas IL-1β increased the bone injury score compared with the control group (Fig. 1B). Treatment with anti-IL-1β increased the total body weight compared with the control group (Fig. 1C). Anti-IL-1β treatment decreased the serum levels of IL-17 and TNF-α in the rheumatoid arthritis rats, whereas IL-1β treatment increased the serum levels of IL-17 and TNF-α (Fig. 1D). Furthermore, the present results indicated that treatment with anti-IL-1β downregulated the gene and protein expression levels of NF-κB, ERK and STAT1, whereas treatment with IL-1β exhibited the opposite effects (Fig. 1E and F).

The effects of anti-IL-1β on the expression levels of various inflammatory factors were analyzed in sfd-FLSs in vitro. The results suggested that treatment with anti-IL-1β decreased the mRNA and protein expression levels of the pro-inflammatory factors IL-17 and TNF-α in sfd-FLSs (Fig. 2A and B). By contrast, treatment with anti-IL-1β increased the expression levels of the anti-inflammatory factors IL-6 and IL-10 in sfd-FLSs (Fig. 2C and D). Treatment with IL-1β exhibited the opposite effects (Fig. 2).

The effects of anti-IL-1β on the NF-κB-mediated ERK/STAT1 signaling pathway were analyzed in sfd-FLSs in vitro. The results indicated that treatment of sfd-FLSs with anti-IL-1β decreased the mRNA expression levels and the protein phosphorylation of NF-κB, ERK and STAT1 (Fig. 3A and B). Conversely, treatment with IL-1β exhibited the opposite effects (Fig. 3A and B). Treatment with an NF-κB inhibitor (NF-κBIR) suppressed the IL-1β-mediated increase in the mRNA expression levels of NF-κB, ERK and STAT1 in sfd-FLSs (Fig. 3). Additionally, NF-κBIR inhibited the IL-1β-mediated increase in pNF-κB/NF-κB, p-ERK/ERK and pSTAT1/STAT1 protein expression ratios in sfd-FLSs (Fig. 3D). Conversely, NF-κB overexpression (NF-κBOR) suppressed the anti-IL-1β-mediated decrease in the mRNA expression and protein phosphorylation levels of NF-κB, ERK and STAT1 (Fig. 3E and F).

The mechanism underlying IL-1β-mediated inflammation was further investigated in sfd-FLSs. The results suggested that NF-κB inhibition suppressed the IL-1β-mediated increase in the mRNA and protein expression levels of IL-17 and TNF-α in sfd-FLSs (Fig. 4A and B). Similarly, NF-κB overexpression inhibited the anti-IL-1β-mediated decrease in the mRNA and protein expression levels of NF-κB, IL-17 and TNF-α in sfd-FLSs (Fig. 4C and D).