A total of 50 Chinese patients with RA who fulfilled the 1987 revised criteria of the American College of Rheumatology (ACR) (28) or the 2010 ACR/European League against Rheumatism classification criteria for RA (29) were recruited from the Department of Rheumatology and Orthopedics of the Hunan Cancer Hospital (Changsha, China). All patients presented with active disease, described as the presence of a 28-Joint Disease Activity Score (DAS28) of ≥3.2. Age, gender or disease duration did not differ among the patients with RA and healthy controls. In total, 11 RA patients were male and 39 were female, with a mean age of 59 (49–64) years. In the healthy control group, 14 patients were male and 26 were female, with a mean age of 36 (29–48) years. The date range of assessment/sample collection for both groups was from June 2016 to October 2018. The present study was conducted in compliance with the Helsinki Declaration principles. All patients provided written informed consent

The clinical data of all patients with RA were collected at baseline, including the 28-joint tender and swollen joint counts (28TJC and 28SJC, respectively), patient and provider global assessment of disease activity (PtGA and PrGA, respectively) scores, visual analog scale score for pain, Chinese language version of the Stanford Health Assessment Questionnaire (HAQ) score (30), erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) level, rheumatoid factor (RF) level and anti-cyclic citrullinated peptide (anti-CCP) antibody level. Disease activity was assessed using the DAS28 with four variables, including CRP level [DAS28 (4)-CRP] (31).

Heparinized whole blood was collected from 50 patients with RA and 40 healthy controls (HCs). PBMCs were separated via Ficoll/Paque density gradient centrifugation at 400 × g for 30 min at 18–20°C. Cells were then re-suspended in α-Minimal Essential Medium with 10% fetal bovine serum (Gibco; Thermo Fisher Scientific, Inc.) and plated in a 37°C humidified incubator with 5% CO₂. On the following day, the suspended cells were removed and washed thoroughly, and the adherent cells were collected and plated. PBMCs were first treated with various concentrations (0, 50 and 100 µM) of LCA for 60 min, and then treated with LPS (100 ng/ml) for 12 h, mRNA was extracted, and the culture medium was collected for ELISA analysis.

The PBMCs (8×10⁵ cells/well) were plated on 24-well culture plates with coverslips, fixed in 4% paraformaldehyde for 20 min at room temperature, permeabilized with 0.2% Triton X-100 for 10 min at room temperature, and blocked in PBS containing 3% bovine serum albumin (Abcam) for 30 min at 37°C. The cells were then incubated in PBS containing rabbit anti-human polyclonal antibody against TGR5 (10 µg/ml; ab72608; Abcam) overnight at 4°C, and subsequently incubated with Alexa Fluor^® 633-conjugated goat anti-rabbit immunoglobulin G secondary antibodies (1:1,000; cat. no. A-21071; Invitrogen; Thermo Fisher Scientific, Inc.) for 1 h at 37°C. Subsequently, DAPI (Sigma-Aldrich; Merck KGaA) was used to stain the cell nuclei at a concentration of 1.43 µM for 3 min at room temperature, and cells were mounted using ProLong^® Gold Antifade Reagent (cat. no. P36934; Invitrogen; Thermo Fisher Scientific, Inc.). The images were acquired using a 160 Zeiss LSM 510 Confocal Imaging system (Zeiss GmbH).

Total RNA was extracted from PBMCs using the RNAiso Plus reagent (Takara Bio, Inc.) according to the manufacturer's protocols. First-strand complementary DNA (cDNA) was synthesized from each total RNA sample using a PrimeScript 1st Strand cDNA Synthesis kit (Takara Bio, Inc.) at 37°C for 15 min, 85°C for 5 sec. qPCR was performed using the QuantiTect™ SYBR Green PCR kit (Takara Bio, Inc.) on the Roche LightCycler480 sequence detector system (Roche Diagnostics) according to the manufacturer's protocols. The qPCR conditions were as follows: Denaturation at 95°C for 30 sec, then 95°C for 5 sec and 60°C for 30 sec for 40–50 cycles. The sequences of the primers used in the study are presented in Table I, and these were synthesized by Invitrogen (Thermo Fisher Scientific, Inc.). PCR amplification of the housekeeping gene β-actin was performed for each sample as a control for sample loading. Normalization and quantification of the PCR signals was performed by comparing the cycle threshold value of the gene of interest, in triplicate, with β-actin. The fold change in gene expression relative to the control was calculated using the 2^−ΔΔCq method (32). Data are presented as the mean ± standard deviation of three independent experiments and analyzed with the LightCycler480 software release 1.5.0 (Roche Diagnostics).

PBMCs were collected and lysed to obtain proteins using a RIPA buffer (Cell Signaling Technologies, Inc.), whereas nuclear protein was separated using a NE-PER™ Nuclear and Cytoplasmic Extraction kit (Pierce; Thermo Fisher Scientific, Inc.). Protein concentration was measured using bicinchoninic acid Protein Assay kit from Bio-Rad Laboratories, Inc. Proteins (50 µg/lane) were separated by 12% SDS-PAGE under denaturing conditions, and were electrotransferred to nitrocellulose membranes. The membranes were then blocked with 5% non-fat milk in 0.1% TBS-Tween 20 for 1 h at room temperature, and were probed with antibodies against TGR5 (cat. no. ab72608; Abcam), phosphorylated (p)-IκB kinase α (IκBα; cat. no. 2859; Cell Signaling Technologies, Inc.), total (T)-IκBα (cat. no. 9249; Cell Signaling Technologies, Inc.) and p-NF-κB (cat. no. 3033; Cell Signaling Technologies, Inc.; all 1:1,000); GAPDH (cat. no. 5174; Cell Signaling Technologies, Inc.; 1:1,000) and histone H3 (cat. no. 9275; Cell Signaling Technologies, Inc.; 1:2,000) overnight at 4°C. For the detection of proteins, membranes were then incubated for 1 h at room temperature with horseradish peroxidase-conjugated secondary antibody (cat. no. E030120-01; EarthOx Life Sciences; 1:5,000). Immunoreactive bands were visualized using an enhanced chemiluminescence reagent (EMD Millipore). Protein bands were semi-quantified via densitometric analyses using a G:BOX Gel & Blot Imaging Series (Syngene Europe) and ImageQuant LAS500 (GE Life Sciences). Phosphorylated protein expression was normalized to total protein expression, and expressed as a fold change relative to the untreated control. Data are presented as the means ± standard deviation of three independent experiments.

The quantities of TNF-α (cat. no. DTA00D, R&D Systems, Inc.), IL-1β (cat. no. DLB50, R&D Systems, Inc.), IL-6 (cat. no. D6050, R&D Systems, Inc.) and IL-8 (cat. no. D8000C, R&D Systems, Inc.) in culture supernatants were measured via ELISA according to the manufacturer's protocols. The quantities of TNF-α (cat. no. MTA00B, R&D Systems, Inc.), IL-1β (cat. no. MLB00C, R&D Systems, Inc.), IL-6 (cat. no. M6000B, R&D Systems, Inc.) and IL-8 (cat. no. LS-F55769, LifeSpan BioSciences, Inc.) in mouse blood samples were measured via ELISA according to the manufacturer's protocol. Briefly, PBMCs were subjected to different treatments. Then, mouse blood samples were centrifuged at 1,006.2 × g for 15 min to separate the blood plasma at room temperature. The culture supernatant or the blood plasma were mixed with the assay buffer and added to anti-TNF-α, IL-1β, IL-6 and IL-8 antibody-coated wells. Horseradish peroxidase-conjugated anti-human TNF-α, IL-1β, IL-6, and IL-8 monoclonal antibodies were added and incubated at 37°C for 2 h, followed by incubation with colorimetric (tetramethylbenzidine) solution for a further 10 min. The relative absorbance was measured at 450 nm, and three independent experiments were performed for each condition.

This study was conducted on male DAB/1J mice (age, 6–8 weeks; 18–22 g) obtained from Beijing HFK Bioscience Co., Ltd. A total of 30 mice were divided into three equal groups: Negative controls (NCs), experimental arthritis controls not treated with LCA and experimental arthritis group treated with LCA (10 mg/kg/day; Sigma-Aldrich; Merck KGaA). The NCs did not have CIA. The mice were housed in a room on a 12-h light/dark cycle under specific pathogen-free conditions, at a temperature of 22–26°C and relative humidity of 55–65%. All experiments were conducted in compliance with the National Institutes of Health guidelines for the care and use of laboratory animals (33). The study was approved by the Institutional Animal and Clinical Committees of Hunan Cancer Hospital.

CIA was induced according to the previously described method by Brand et al (34) in the experimental arthritis controls not treated with LCA and experimental arthritis group treated with LCA mouse groups. Briefly, native chick CII (Chondrex, Inc.) was dissolved in 10 mM acetic acid to a 1 mg/ml concentration, and was emulsified in an equal volume of Freund's complete adjuvant (Sigma-Aldrich; Merck KGaA). Each mouse received two intradermal injections of emulsion (0.1 ml) containing CII (1 mg/ml) and Freund's complete adjuvant at two separated sites tail on days 1 and 21.

For LCA treatment, LCA was dissolved and freshly diluted in PBS. PBS and LCA (10 mg/kg/day) were orally administered via a gastric tube once a day from day 21 to day 42 following the first immunization. Both NCs and experimental arthritis controls not treated with LCA were treated with PBS.

The arthritis score was assessed once every 3 days following the initiation of LCA treatment. The severity of arthritis was measured in a double manner using the semi-quantitative clinical scoring system described by Yoo et al (35) (0, no arthritis; 1, definite erythema and swelling of the ankle or one digit; 2, two or more joints involved or mild erythema and swelling of the entire paw; 3, erythema and swelling extending from the ankle to the metatarsal joints of the entire paw and all digits; and 4, ankylosing deformity with severe joint erythema and swelling). The arthritis score for each mouse was the sum of all paw scores (yielding a score between 0 and 16). On day 42, the mice were sacrificed and blood was collected for serum separation.

Statistical analyses were performed using SPSS 13.0 software (SPSS, Inc.). For categorical variables, data are presented as frequencies and percentages. For continuous variables, data are presented as the means ± standard deviation, or the median and interquartile range. Wilcoxon rank-sum test was used to compare the expression of TGR5 in the PBMCs of patients with RA and HCs, and the arthritis score in the experimental arthritis controls and experimental arthritis controls treated with LCA. One-way or Welch's ANOVA followed by least significant difference post-hoc tests were performed to compare the expression of TNF-α, IL-1β, IL-6 and IL-8 in the culture supernatant or the blood plasma, and the expression of p-NF-κB and p-IκBα in the PBMCs treated with various LCA or not treated with LCA. Spearman's rank order correlation test was used to assess the correlation between TGR5 expression in RA PBMCs and clinical parameters. P<0.05 was considered to indicate a statistically significant difference.

Table II presents the baseline demographic and clinical features of individuals in the study. The gender ratio did not differ between the patients in the RA and HC groups. Among the patients with RA, 48% (24/50) had not previously taken corticosteroids or disease-modifying anti-rheumatic drugs (DMARDs). The majority of patients had taken only Chinese herbal medicine and/or painkillers to relieve arthralgia. At recruitment, 18% (9/50) had taken corticosteroids alone. The remaining 34% (17/50) received treatment with one or more DMARD, including methotrexate, leflunomide, sulfasalazine, hydroxychloroquine and etanercept.

The mRNA expression levels of TGR5 in PBMCs were evaluated in 50 patients with RA and 40 HCs. The expression of TGR5 mRNA in the PBMCs of patients with RA was significantly decreased compared with in the HCs (0.53±0.58 vs. 1.49±0.83; P<0.001; Fig. 1A). Additionally, the expression of TGR5 mRNA in the PBMCs of patients with RA with a high DAS28 was significantly decreased compared with in patients with a low DAS28 (0.35±0.46 vs. 0.81±0.65, respectively; P=0.002; Fig. 1B). The expression of TGR5 mRNA in the PBMCs of the patients with RA with either a high or low DAS28 was also significantly lower than that of the HCs (0.81±0.65 vs. 1.49±0.83, P<0.001; 0.35±0.46 vs. 1.49±0.83, P=0.002; Fig. 1B). The number of patients with RA with a DAS28 score of ≥5.1 was 30. Of these patients, 50% (15/30) had never taken corticosteroids or DMARDs. At recruitment, 20% (6/30) had taken corticosteroids alone. The remaining 30% (9/30) received treatment with one or more DMARDs, including methotrexate, leflunomide, sulfasalazine, hydroxychloroquine or etanercept. Patients did not exhibit any obvious side effects after taking DMARDs. Immunofluorescence staining revealed markedly reduced expression of TGR5 in the PBMCs of patients with RA compared with in the PBMCs of HCs (Fig. 1C).

Spearman's rank order correlation test revealed significant correlations between the mRNA expression levels of TGR5 in PBMCs and the CRP level (r=−0.429, P=0.002), and the DAS28 (r=−0.383, P=0.006) of patients (Fig. 2). There was no significant correlation between TGR5 mRNA expression and 28TJC, 28SJC, PtGA score, PrGA score, HAQ score, RF level, anti-CCP level, ESR, the simplified disease activity index and the clinical disease activity index (respectively: r=0.043, P=0.763; r=−0.072 P=0.611; r=−0.011, P=0.939; r=0.185, P=0.188; r=0.172, P=0.223; r=0.049, P=0.732; r=0.188, P=0182; r=0.067, P=0.642; r=−0.099, P=0.544; and r=−0.106, P=0.455).

LPS is a potent endotoxin involved in the progression of RA diseases (36). LPS treatment (100 ng/ml) for 12 h markedly increased the mRNA and protein expression levels of TNF-α, IL-1β, IL-6 and IL-8 in the PBMCs of patients with RA by between 3- and 10-fold (Fig. 3). Pretreatment of PBMCs with LCA (50 and 100 µM) for 60 min significantly inhibited LPS-induced TNF-α, IL-1β, IL-6 and IL-8 mRNA expression and protein release into the supernatant in a concentration-dependent manner (Fig. 3).

To further investigate the mechanism underlying the effects of LCA on the inhibition of LPS-induced proinflammatory cytokine production, NF-κB and IκBα signaling was measured via western blot analysis. LCA treatment significantly decreased the phosphorylation of NF-κB and IκBα, but not the total protein levels of IκBα (Fig. 4).

Mice with CIA have been frequently utilized to investigate arthritic diseases, as they possess various pathological features similar to those of human RA (37). There was no macroscopic evidence of paw erythema or edema in the normal blank control mice. As presented in Fig. 5A, arthritis symptoms in the mice with CIA progressed and maintained a high intensity from day 21 to day 42 following immunization; this was notably relieved following treatment with LCA (10 mg/kg/day). From day 30, the arthritis score was significantly decreased in the LCA treatment group compared with in the CIA model group (P<0.05 or 0.01).

To demonstrate its anti-inflammatory effects in vivo, LCA was evaluated for its inhibition of the production of proinflammatory cytokines in mice with CIA. Cytokines, such as TNF-α, IL-1β, IL-6 and IL-8, are associated with the progression and severity of arthritis (16). On day 42, the mice were sacrificed, and blood was collected for serum separation and subsequent ELISA detection. As presented in Fig. 5B, the serum levels of the proinflammatory cytokines TNF-α, IL-1β, IL-6 and IL-8 were significantly increased in the non-treated CIA mice compared with in the non-arthritic group. The increased TNF-α, IL-1β, IL-6, and IL-8 production was significantly suppressed by LCA treatment compared with in the non-treated mice with CIA.