Bovine type II collagen (CII) was purchased from the Chondrex, Inc. (Redmond, WA, USA), while Complete Freund's Adjuvant (CFA) and Incomplete Freund's Adjuvant (IFA) were purchased from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). Interleukin (IL)-1β, interleukin-6 (IL-6), IL-10, IL-12 and IL-17A ELISA kits were purchased from Invitrogen; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). RPMI-1640 medium and fetal bovine serum (FBS) and trypsinase were obtained from Gibco (Thermo Fisher Scientific, Inc.). A Cell Counting kit-8 kit, BCA protein assay reagent and horseradish-peroxidase (HRP)-conjugated secondary antibodies (cat. no. A0208) were purchased from Beyotime Institute of Biotechnology (Haimen, China). Tumor necrosis factor (TNF)-α (cat. no. RAB0477), dimethyl sulfoxide (DMSO) and IκB (cat. no. SAB1305978) were purchased from Sigma-Aldrich; Merck KGaA. JAK1 (cat. no. ab133666), JAK3 (cat. no. ab203611), STAT3 (cat. no. ab119352), nuclear factor (NF)-κB p65 (Cytosolic; cat. no. ab19870), and β-actin (cat. no. ab8226) antibodies were purchased from Abcam (Cambridge, MA, USA). All other reagents used were of analytical grade.

Male Wistar rats (5–6 weeks old, 170–180 g; n=40) were purchased from the Experimental Animal Center of Kunming Medical University (Kunming, China). Animals were housed at 21±1°C and 30–70% humidity under a 12 h light/dark cycle with free access to standard pellet food and water. All animal experiments in the present study were performed in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals (12), and the experimental protocols were approved by the Animal Care and Use Committee of the First Affiliated Hospital of Kunming Medical University (approval no. KMUH-2016023).

The RA-derived fibroblast-like synoviocyte MH7A cell line was obtained from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). MH7A cells were cultured in RPMI-1640 medium with 10% FBS, 1% penicillin and 1% streptomycin in a 5% CO₂ humidified atmosphere at 37°C.

Dried rhizomes of C. orchioides were purchased from Beijing Tongrentang (Beijing, China) and identified by the department of Traditional Chinese Medicine, The First Affiliated Hospital of Kunming Medical University (Kunming, China). According to previously published protocols (13,14), the rhizome of C. orchioides was powdered and then extracted using 75% aqueous ethanol by reflux three times. Following this, the filtrates were concentrated under 50°C in vacuum, and were partitioned continuously with petroleum ether, ethyl acetate, and n-butanol. The ethyl acetate fraction mentioned above was eluted via silica-gel (100–200 mesh) with petroleum ether-acetone (15:1, 10:1, 5:1, 2:1, 1:1) to obtain five sub-fractions (F1-F5). By using a series of chromatographic techniques, including silica gel column chromatography and Sephadex LH-20 chromatography (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), curculigoside was extracted from the F3 fraction (13). In addition, curculigoside (Fig. 1) was identified by ¹H-nuclear magnetic resonance (NMR), ¹³C-NMR and previously reported NMR data according to methods described previously (13,14).

To investigate the potential anti-arthritic effects of curculigoside, a total of 40 rats were divided into the following four groups (n=10): Normal (not immunized and treated with 10 ml/kg/day saline), control (immunized and treated with 10 ml/kg/day saline), positive [immunized and treated with methotrexate (MTX); 1 mg/kg, three times a week] and curculigoside group (immunized and treated with 50 mg/kg curculigoside).

The CIA rat model was prepared according to previously reported methods, with minor modifications (15,16). Briefly, CII was dissolved in 0.1 mM acetic acid to achieve a final concentration of 4 mg/ml. Then, the CII solution was emulsified with an equal volume of CFA. Rats were initially immunized by subcutaneous injection of CII emulsion at the tail root (100 µl/rat). After 7 days, the rats were immunized by CII again, emulsified by an equal volume of IFA at the same location (100 µl/rat). After approximately 10 days from the initial immunization, rats evidently exhibited RA symptoms at the toe joint, including observable inflammatory reactions, erythema and swelling.

At 10 days after the initial immunization with CII, rats were orally treated with either saline, MTX or curculigoside (50 mg/kg/day). During the experiment, the rats body weight and paw volume were measured by using a PV-200 Plethysmometer (Paw Volume) Meter (Techman Soft, Chengdu, China) every 5 days. In addition, the arthritis indices of rats were measured every 3 days, using the following ordinal scale: 0, no obvious signs of arthritis; 1, one joint affected (swelling and erythema); 2, two joints affected; 3, three joints affected; 4, three joints affected and maximal erythema and swelling (17). After 30 days of drug treatment, rat weight was recorded (for the normal and curculigoside treatment groups, rats weighed 320–340 g; for the control and MTX groups, rats weighed 280–300 g), then rats were sacrificed by decapitation under aesthesia with sodium pentobarbital (35 mg/kg; intraperitoneal injection). Next, blood was collected from abdominal aorta. The spleen and thymus were dissected from each mouse to determine the ratio (mg/g) of thymus or spleen wet weight to body weight.

Serum samples were prepared and centrifugation 15 min (1,800 × g) at 4°C, and were stored at −80°C until analysis. Then, serum TNF-α (cat. no. RAB0477), IL-1β (cat. no. BMS6002), IL-6 (cat. no. BMS603-2), IL-10 (cat. no. 88-7105-88), IL-12 (cat. no. BMS616) and IL-17A (cat. no. BMS6001) were detected by using commercial ELISA kits according to the manufacturer's protocol and instruction (Invitrogen; Thermo Fisher Scientific, Inc.).

Effects of curculigoside on cell viability were determined by Cell Counting kit-8 according to the manufacturer's protocol. MH7A cells (5×10³ cells/well) were seeded in 96-well plates and incubated with various concentrations of curculigoside (1, 2, 4, 8, 16, 32 and 64 µg/ml) for 12, 24, 36, 48 or 72 h. CCK-8 solution was added to each well and incubated for another 1 h at 37°C. Optical density (OD) was measured at 450 nm using a 96-well plate reader (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Results were reported as a percentage of DMSO control cells.

JAK1, JAK3, STAT3, NF-κB p65 (C) and IκB expression was measured in MH7A cells by western blotting. Following treatment with various concentrations of curculigoside (4 and 16 µg/ml) or vehicle (DMSO) in the presence of 10 ng/ml TNF-α for 36 h, Cells (5×10⁶) were harvested and homogenized with lysis buffer for 10 min and centrifuged at 4°C for 5 min (10,000 × g). Total protein was extracted from cells using the cell lysis buffer for western blotting and IP (cat. no. P0013; Beyotime Institute of Biotechnology), in addition, the cytoplasmic protein was extracted by using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (cat. no. 78833; Thermo Fisher Scientific, Inc.). The protein concentration was determined with a bicinchoninic acid protein assay. Subsequently, 40 µg total proteins in each sample was separated by 12% SDS-PAGE and blotted onto polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% fat-free dry milk in 1X TBST (containing 0.1% Tween-20; Beyotime Institute of Biotechnology; cat. no. P0233) at room temperature for 2 h. Thereafter, proteins on the PVDF membranes were probed with JAK1 (dilution 1:1,000), JAK3 (dilution 1:1,000), STAT3 (dilution 1:1,000), NF-κB p65 (C) (dilution 1:1,000), IκB (dilution 1:1,000) and β-actin (dilution 1:2,000) antibodies at 4°C for 12 h, followed by incubation with corresponding horseradish peroxidase-conjugated secondary antibodies (1:1,000; cat. no. A0208) for 2 h at 37°C. Finally, immunoreactive bands were visualized with enhanced chemiluminescence detection reagents (Beyotime Institute of Biotechnology; cat. no. P0018A) and analyzed using the ImageQuant LAS 4000 Imaging system (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA). Protein expression was normalized to β-actin.

All data are presented as the mean ± standard deviation of three independent experiments, which were performed in triplicate. Statistical analyses were performed via one-way ANOVA followed by Dunnett's test using SPSS 19.0 software package (IBM Corp., Armonk, NY, USA). P<0.05 was considered to indicate a statistically significant difference.

The paw swelling and arthritis score of rats in each treatment group was determined to evaluate the therapeutic effects of curculigoside on RA. As shown in Fig. 2, significant RA symptoms were observed in the control CIA rats when compared to normal rats at 24 days, including paw swelling (P<0.01) and higher arthritis score (P<0.01). Following treatment with MTX, paw swelling and arthritis scores were reduced significantly, compared with the control group (P<0.01). In addition, curculigoside (50 mg/kg) also markedly decreased paw swelling and arthritis scores of CIA rats (P<0.01), compared with control rats.

The effects of curculigoside treatment on spleen and thymus indices in CIA rats were presented in Fig. 3. It was demonstrated that spleen and thymus indices in the control group were significantly higher than those of normal rats (P<0.01). Following treatment with curculigoside (50 mg/kg), spleen and thymus indices were decreased (P<0.01), compared with the control group.

The expression of TNF-α, IL-1β, IL-6, IL-10, IL-12 and IL-17A in serum following treatment were shown in Fig. 4. It was observed that TNF-α, IL-1β, IL-6, IL-10, IL-12 and IL-17A expression in control rats was significantly increased when compared to the normal group (P<0.01). The expression of these proteins in rat serum decreased significantly following treatment with 50 mg/kg curculigoside (P<0.01), compared with control CIA rats.

The effect of curculigoside on MH7A cell viability was detected with CCK-8 assays. As presented in Fig. 5, curculigoside exerted significant inhibitory effects on MH7A cell viability between 1 and 64 µg/ml. In addition, our results also showed that curculigoside at the concentrations of 4, 8 and 16 µg/ml possessed inhibitory effects on MH7A cell viability within 72 h.

Protein expression levels of JAK1, JAK3 and STAT3 in TNF-α stimulated MH7A cells were measured by western blotting. Compared to the TNF-α group, the protein expression of JAK1, JAK3 and STAT3 were significantly downregulated in the curculigoside-treated groups (4 and 6 µg/ml) (P<0.01; Fig. 6).

Furthermore, the effects of curculigoside on NF-κB p65 (cytosolic) and IκB expression was determined by western blotting in TNF-α stimulated MH7A cells. As presented in Fig. 7, the expression of NF-κB p65 (cytosolic) and IκB in TNF-α stimulated MH7A cells was downregulated, compared with normal MH7A cells. Following treatment with curculigoside (4 and 6 µg/ml) for 24 h, the expression of NF-κB p65 (Cytosolic) and IκB was significantly upregulated (P<0.01).