Cell culture

RA-FLSs were extracted from synovial tissues of RA patients and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, USA) supplemented with 20% fetal bovine serum (FBS; Corning, USA) and 1% penicillin-streptomycin (Gibco, USA). For consistency in experimental results, only RA-FLSs at passages 4–8 were utilized. The human embryonic kidney cell line 293T were obtained from American Type Culture Collection (ATCC, USA) and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Corning, USA) supplemented with 10% FBS (Corning, USA) and 1% penicillin-streptomycin (Gibco, USA). Both cell cultures were maintained at a standard condition of 37 °C in a humidified environment containing 5% CO2.

Cell viability assay

RA-FLSs were plated at a density of 2 × 103 cells per well in a 96-well plate. Following an overnight incubation with 100 µL of medium to facilitate cell attachment, the cells were treated with varying concentrations of propionate (Sigma, USA) to assess proliferation over a period of 6 days. Cell viability was determined using the Cell Counting Kit-8 (CCK-8) assay. This involved washing the cells with PBS, adding 10 µL of CCK-8 reagent to each well along with 100 µL of medium, and then incubating the plates for 1 h. Absorbance was measured at 450 nm using a PerkinElmer EnSpire® spectrophotometer at two-day intervals to monitor cell viability.78

Colony formation assay

RA-FLSs were plated at a density of 1 × 103 cells per well into 6-well plates and allowed to adhere overnight. Subsequently, the cells were treated daily with various concentrations of propionate for a duration of 7 days. Post-treatment, the cells underwent fixation with 10% formalin for 30 min, followed by staining with crystal violet for 20 min at room temperature. High-resolution digital images of the resulting colonies were captured using a camera system. Quantitative analysis of the colony count was performed utilizing ImageJ software.

Transwell migration assay

RA-FLSs were seeded at a density of 8 × 103 cells in 100 µL of serum-free medium into the upper chamber of a Transwell polycarbonate culture insert, 6.5 mm in diameter with an 8 µm pore size (BIOFIL, USA). These inserts were placed into 24-well plates containing 600 µL of medium supplemented with 20% FBS in the lower chamber. The assay plates were incubated for 24 h with or without varying concentrations of propionate. Post-incubation, the Transwell inserts were carefully removed, and the upper chamber was cleared of non-migratory cells using a cotton swab. The cells that had migrated to the underside of the membrane were fixed with 10% formalin for 20 min, stained with 0.2% crystal violet for 30 min, and subsequently visualized under a Nikon Eclipse Ts2 microscope. To quantify the migratory cells, four distinct non-overlapping fields were counted using ImageJ software.

Invasion assay

Matrigel Basement membrane matrix (Corning, USA) was prepared at a concentration of 300 µg/mL by dilution with PBS. This solution was then applied to the 8-µm pore size Transwell inserts. Each insert was coated with 100 µL of the diluted Matrigel and allowed to solidify at 37 °C for 30 min to form a uniform layer. Following polymerization, 8 × 10³ cells were seeded into the upper chamber of the inserts, using the same procedure as outlined for the migration assay. The assay plates were incubated for 48 h with or without varying concentrations of propionate. After the incubation period, the staining and imaging of the cells were performed using the identical protocol established for the migration assay.

Wound healing assay

RA-FLSs were grown to confluence in six-well plates prior to performing a wound healing assay to assess cell migration. To create a wound, a sterile 200 µL pipette tip was used to scratch the cell monolayer. Following the scratch, the cells were incubated in medium with or without various concentrations of propionate. Wound images were captured at the initial time of wounding (0 h) and then again at the 24 h mark, utilizing a Nikon Eclipse Ts2 microscope equipped with CapStudio-SC200C software. The wound area was quantified using ImageJ software. The healing process was evaluated by normalizing the wound area at 24 h to that at the initial time point (0 h).

Apoptosis assay by flow cytometry

RA-FLSs were plated in 6-well plates and exposed to varying concentrations of propionate daily for a period of three days. Post-treatment, cells were enzymatically detached and washed twice with PBS to ensure purity. Subsequently, cells were stained utilizing an Annexin V-FITC Apoptosis Detection Kit (Beyotime, CHN) to label apoptotic cells. Flow cytometric analysis was carried out using a BD flow cytometer to quantify the proportion of apoptotic cells. Data acquired from the flow cytometer were analyzed with the FlowJo software (BiotreeDB, USA) to interpret the apoptotic profiles.79

Real-time PCR

RNA was extracted employing the RNeasy kit (TransGen Biotech, China) following the manufacturer’s protocol. cDNA was synthesized using the PrimeScriptTM RT reagent Kit with gDNA Eraser Kit (TaKaRa, JAN). The primers were synthesized by Sangon Biotech Co., Ltd and listed in Table S1. For real-time PCR, a 20 µL volume of the final PCR solution was prepared by adding 5 µL of diluted cDNA product, 10 µL of 2 × Power SYBR® Green PCR Master Mix (TransGen Biotech, CHN), and 5 µL of each forward and reverse diluted primer. The amplification and detection were conducted on the Bio-Rad CFX 96 Touch System, with all samples run in technical triplicates to ensure data reliability. Quantitative data were generated and analyzed using the Bio-Rad CFX Manager software.

RNA sequencing and data analysis

RA-FLSs were seeded in 6-well plates. Following overnight incubation, the cells were treated with 1 mmol/L propionate or vehicle for 48 h. Total RNA samples were obtained by adding 1 mL of Transzol (Transgen, USA) for subsequent transcriptome sequencing by BGI Genomics. Briefly, sequencing libraries were generated after depleting ribosomal RNA, synthesizing cDNA, and ligating adapters using DNBSEQ Eukaryotic mRNA library (BGI Genomics, CHN). After cluster generation, the libraries were sequenced using the DNBseq platform (BGI Genomics, CHN) and 150 bp paired-end reads were generated. Raw data with adapter sequences or low-quality sequences were filtered using the SOAPnuke software (BGI Genomics, CHN). The resulting data was then converted and stored in the fastq format for subsequent analysis. To assess the quality of the data, FastQC version 0.11.9 was utilized to generate a quality control report. The raw sequencing reads were then aligned to the reference genome GRCh38 using HISAT2 version 2.2.1 and the aligned reads were sorted by coordinate using Samtools version 1.10. The gene expression counts were quantified using FeatureCounts version 1.10, and gene expression counts were normalized for differential analysis using DESeq2 version 4.2.2, with an FDR threshold of 0.05.

Sample preparation for proteomics

Protein extracts from RA-FLSs were prepared using the EasyPep Mini MS Sample Prep Kit (Thermo Fisher Scientific, USA).80 Initially, cells were lysed with the provided lysis buffer to extract proteins. A quantity of 100 µg of the protein extract was then transferred to a fresh microcentrifuge tube, and the volume was brought up to 100 µL using the same lysis buffer. Subsequent steps involved the addition of reduction and alkylation solutions to the protein sample. The mixture was gently mixed and subjected to incubation at 95 °C for 10 min to facilitate the reduction and alkylation processes. Post incubation, the sample was allowed to cool to room temperature. The reconstituted Trypsin/Lys-C Protease Mix was then introduced to the prepared protein sample, followed by incubation with shaking at 37 °C for 2 h, to achieve protein digestion. Upon completion of the digestion, the digestion stop solution was added to terminate the enzymatic reaction. Peptides were then purified using a peptide clean-up column. The samples were dried via vacuum centrifugation and subsequently reconstituted in a 0.1% formic acid aqueous solution, preparing them for LC-MS/MS analysis.

Nanoflow LC-MS/MS analysis

The Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, USA) was used in combination with an Easy-nLC 1000 ultrahigh-pressure liquid chromatography pump (Thermo Fisher Scientific, USA) for the LC-MS/MS analysis. Separation was achieved using a trap column and an analytic column with a spray tip, both filled with 3 µm/120 Å ReproSil-Pur C18 resins (Dr. Maisch GmbH, DE). The separation buffers were comprised of 0.1% formic acid in both water and acetonitrile. A fraction of the collected samples was initially introduced into the trap column with a 2 µL/min flow rate, and then, it was separated via the analytical column at a flow rate of 300 nL/min. The separation gradient was established as starting with 3%–7% acetonitrile over 2 min, increasing to 22% acetonitrile over the next 50 min, then to 35% acetonitrile in 10 min, surging to 90% acetonitrile within 2 min, maintaining at 90% for 6 min, dropping back to 3% acetonitrile in 2 min, and finally stabilizing at 3% acetonitrile for a duration of 13 min. Full MS scans were performed in an Orbitrap mass analyzer over m/z range of 395–1 205 with a mass resolution of 60 000. Data was processed and analyzed for DIA-Based proteomics using Spectronaut version 14.9 (Biognosys, CH).81

Plasmid constructs, lentivirus packaging and infection

pLKO.1-U6-EF1a-copGFP-T2A-puro were purchased from IGE BIO (CHN). Plasmid constructs were created in accordance with the protocol available on the Addgene website. The FOXK1 shRNA sequence utilized was as follows: CCATCAAGATCCAGTTCACGT. The shRNA sequence was synthesized by Sangon Biotech (CHN), and subsequently, it was digested with a restriction enzyme and ligated into the vector. All plasmids were confirmed by DNA sequencing. For lentivirus packaging and infection, HEK293T cells were seeded onto a 6 cm dish in a quantity of 6 × 105. The co-transfection procedure was conducted after a duration of twenty-four hours, utilizing 1 mg of target plasmids, 0.75 mg of psPAX2, and 0.25 mg of pMD2.G. The media of the culture was replaced six h following the transfection process. The initial batch of material was collected 48 h after transfection, without any further changes to the medium. After a period of medium refreshment, a further collection of media took place 72 h after the transfection. The two medium batches were combined and subjected to centrifugation at 1 250 r/min for five min to remove any cell debris. The resulting supernatant was then kept in 1 mL portions at a temperature of −80 °C. In the same manner, 6 × 105 RA-FLSs were placed in a 6 cm plate and, after 24 h, were given 1 mL of the thawed lentivirus solution, which was gently stirred to ensure thorough mixing. The medium was substituted 24 h subsequent to the introduction of the virus. The process of antibiotic selection was began 48 h after transfection, using a concentration of 2 mg/mL puromycin in the media. This selection continued until the majority of cells died, leaving only the resistant cells, which finally attained confluency. Following that, the cells were prepped for extended preservation in liquid nitrogen.

Western blotting

Western blot analysis was conducted as previously described.79 In brief, total protein levels were quantified and loaded onto an SDS-PAGE gel. The separated proteins were then transferred onto a PVDF membrane (Millipore, MA, USA) via the Bio-Rad Trans-Blot Turbo™ transfer apparatus (USA). Following blocking with 5% non-fat dry milk in TBST, the membrane was incubated overnight at 4 °C with primary antibodies against FOXK1 (1:1 000, Abclonal, CHN), STAT1 (1:1 000, proteintech, CHN), STAT2 (1:1 000, proteintech, CHN), HDAC3 (1:1 000, proteintech, CHN) and β-actin (1:2 000, Abclonal, CHN). Following this, the membrane was rinsed thrice using TBS-T and then incubated with suitable HRP-linked secondary antibodies for one h at room temperature. Chemiluminescent detection was subsequently carried out using the enhanced chemiluminescence kit (ABclonal, CHN) and the blots were visualized using the Tanon Multi5200 chemiluminescence imaging system (Tanon, Multi5200, CHN).

Immunoprecipitation and ubiquitination assay

The immunoprecipitation procedure was performed as previously described.82 5 × 104 RA-FLSs were incubated with propionate for 24 h and were lysed in lysis buffer (Thermo Scientific, USA) with a proteinase inhibitor cocktail. The lysate was centrifuged at 13 000 × g at 4 °C, and the resulting supernatant was incubated overnight at 4 °C with anti-FOXK1 primary antibody (1:100, Abclonal, CHN) or anti-HDAC3 (1:100, Abclonal, CHN). Subsequently, the mixture was attached to Protein A/G Magnetic Beads (Thermo Scientific, USA) at room temperature for 1 h. The beads are then washed five times extensively to remove non-specifically bound proteins by using DynaMag™-2 Magnet (Invitrogen, USA). The immunoprecipitated proteins were then prepared in loading buffer, heated at 100 °C for 10 min, and subjected to SDS-PAGE and western blot analysis using primary antibodies against acetylated-Lysine (1:1 000, PTM BIO, CHN), FOXK1 (1:1 000, Abclonal, CHN), HDAC3 (1:1 000, proteintech, CHN) and β-actin (1:2 000, Abclonal, CHN).

Mice

Male C57BL/6J and DBA/1J mice aged 6-8 weeks were obtained from GemPharmatech (CHN). All animal studies were performed at the Experimental Animal Center of the Southern University of Science and Technology. This facility guarantees a controlled environment free from pathogens, offers unrestricted access to food and water, maintains a consistent temperature of 22 °C, and follows a 12-h light/dark cycle. The study’s methods were approved by the Institutional Animal Care and Use Committees (IACUC) at Southern University of Science and Technology, guaranteeing adherence to recognized animal care criteria.

CIA mouse model

The CIA model was performed as previously described.83 In brief, an emulsion was formed by combining bovine type II collagen (Chondrex, USA) with an equal amount of Complete Freund’s Adjuvant (Chondrex, USA). The mice were administered a single subcutaneous injection near the base of their tail. The injection consisted of 100 µL of an emulsion containing 100 µg of collagen and 2 mg/mL of Mycobacterium tuberculosis. Two independent observers, who were unaware of the therapy groups, did the assessment of arthritis severity using clinical arthritic scoring. The scoring system encompassed a range of values from 0 to 4, where 0 denoted the absence of symptoms, 1, indicated the presence of redness and/or swelling in one joint, 2, indicated the presence of redness and/or swelling in more than one joint, 3, revealed redness and/or swelling over the entire paw, and 4, indicated the presence of severe deformity and/or ankylosis. The scoring of each paw of the mouse resulted in a maximum achievable score of 16 per mouse. Mice with a score of one or above were categorized as exhibiting arthritis.

FMT experiment

Fecal samples were obtained from both C57BL/6J and DBA/1J mice. These samples, immediately after collection, were suspended in a saline solution at a concentration of 30 mg of feces/mL saline, then thoroughly homogenized using a TissueLyzer (Powteq, CHN), and subsequently passed through a stainless-steel mesh with 25 µm openings. Following this, the fecal solutions were combined with 10% sterile glycerol (Beyotime, CHN), portioned into aliquots, and preserved at −80 °C for future use. DBA/1J mice received an oral dose of 200 µL of this fecal concoction twice weekly until the study concluded.

Bacteria strain administration

B. fragilis (ATCC #23745, USA) were cultured as per the guidelines provided by the manufacturer. DBA/1J mice were subjected to an antibiotic regimen via oral gavage, receiving 1 g/L of a combination of ampicillin, neomycin, and metronidazole. A week following this, the mice were administered the specified bacterial strain through gavage twice weekly for the duration of the study, with anaerobe basal broth (Solarbio, CHN) serving as the vehicle control. Furthermore, the functional profiles of the microbiota in C57BL/6J and DBA1/J mice were inferred from 16S rRNA gene sequencing data, utilizing the Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt2) software in conjunction with the MetaCyc database for analysis.84

Fecal DNA extraction, 16S rRNA gene sequencing, and data analysis

Microbial DNA extraction and 16S rRNA amplicon sequencing were performed by MAGIGENE. Following their preparation, the amplicon libraries were sequenced on the MiSeq system (Illumina, USA). The sequences obtained from the bacteria were then organized into Operational Taxonomic Units (OTUs) and matched to the Greengenes microbial gene database, ensuring a 97% sequence similarity threshold, employing the QIIME2 software (version 2022.11).85 OTUs present in fewer than 10 samples were removed. The final Biological Observation Matrix files were derived from mouse samples, with an average count of 81 255 per sample, and were used for further analyses. The summarization of bacterial taxonomy, assessment of microbial diversity through rarefaction analysis, and evaluation of compositional variations were performed using the designated script provided by QIIME2.85 PCA plots and a heatmap were generated based on the normalized bacterial abundance in R.

Collection of human samples

Plasma samples from RA patients and healthy control (HC) subjects were obtained from the Department of Laboratory Medicine at Peking University Shenzhen Hospital and the Department of Rheumatology at Guanghua Hospital, affiliated with Shanghai University of Traditional Chinese Medicine. The procedures for collecting and processing these samples were consistent across all participants. The demographic and clinical characteristics of the RA patients and HC subjects are detailed in Table S2. Prior to participation, informed consent was secured from all individuals involved in the study, which received ethical approval from the Clinical Ethics Committees at both Peking University Shenzhen Hospital and Guanghua Hospital of Shanghai University of Traditional Chinese Medicine.

Targeted metabolomics data acquisition and processing

Fecal and plasma SCFA contents were quantified following the established protocol with some modifications.86 Fecal samples were processed by extracting about 150 mg portions using a TissueLyzer with a 0.005 mol/L NaOH solution, which included 1 µg/mL of 2-methylbutyric acid as an internal standard (IS). After centrifuging these extracts at 13 200 r/min for 15 min, the supernatants were moved to 10-mL glass tubes. Plasma samples were prepared by combining 300 µL of the sample with 500 µL of a 0.005 mol/L NaOH solution containing the IS, mixed in a glass centrifuge tube. This was followed by the addition of 500 µL of a propanol/pyridine (PrOH/Py) solvent mix and 100 µL of perchloric acid (PCF), vortexed and sonicated for 1 min. The propyl derivatives extracted with hexane were then ready for GC-MS analysis. The analysis was performed on an Agilent Technologies GC/MS system, injecting 1 microliter of extract in a split mode (1:6), with separation on an Agilent HP-5ms column and helium as the carrier gas. The oven temperature began at 50 °C, increasing through a series of ramps to a final 290 °C, which was maintained for 8 min. The method’s precision, both intra- and inter-day, was maintained below 15%, and compound identification utilized the NIST 14.0 database.

Untargeted metabolomics data acquisition and processing

To prepare plasma samples for analysis, 100 µL of plasma was combined with 400 µL of a 1:1 acetonitrile:methanol extraction solution containing a mix of isotopically-labeled internal standards. The mixture was vortexed for 30 s, sonicated for 10 min in ice-water, and then incubated at −40 °C for 1 h. Post-centrifugation at 12 000 r/min for 15 min at 4 °C, the supernatant was transferred to a fresh glass vial for further analysis. A quality control (QC) sample was created by pooling equal volumes of supernatant from all samples. LC-MS/MS analysis utilized a Vanquish UHPLC system connected to a Q Exactive HFX Orbitrap mass spectrometer, with separations on a UPLC BEH Amide column. The mobile phase comprised 25 mmol/L ammonium acetate and ammonia hydroxide in water (pH 9.75) and acetonitrile. With a 2 µL injection volume and a 4 °C auto-sampler temperature, the system acquired data in information-dependent acquisition (IDA) mode via Xcalibur software. ESI source settings included sheath and auxiliary gas flows, capillary temperature, full MS and MS/MS resolutions, collision energy levels, and spray voltage for both positive and negative modes. Data conversion to mzXML was done using ProteoWizard, followed by peak processing with XCMS. Metabolite identification employed an in-house database with a 0.3 cutoff. MetaboAnalyst 5.0 facilitated metabolite enrichment and visualization through PCA, Orthogonal PLS-DA, and heatmaps. MetaMapp was used for metabolomic network analysis, visualized in Cytoscape 3.9.1.87

In vivo administration of SCFAs and etanercept

28 days following the initiation of arthritis, DBA/1J mice received treatments that included either 200 mmol/L sodium butyrate or 200 mmol/L sodium propionate (both from Sigma, USA) added to their drinking water, and/or were given intraperitoneal injections of 5 mg/kg etanercept (sourced from MCE, USA) twice every week until the conclusion of the study.

Histological analysis

Mouse hind paws were dissected, fixed overnight in 10% formalin at 4 °C, and subsequently decalcified in 10% EDTA solution at 4 °C over a period of three weeks. The tissues were then processed, embedded, and sectioned at a 5 µm thickness for H&E and Safranin O-Fast Green staining. Histopathological evaluation was conducted blindly, following previously established protocols.88 Images of the entire ankles were acquired using a NanoZoomer S60 Digital slide scanner C13210-01 (HAMAMATSU, JAN) and analyzed using NDP.view2 software (HAMAMATSU, JAN).88

Micro-CT analysis

Mouse ankle specimens were immersed in 10% formalin for fixation and subsequently placed in 70% ethanol in preparation for imaging. High-resolution micro-computed tomography (µCT) scanning was conducted using a Bruker Skyscan 1276 scanner in the USA, capturing images at a 10 µm resolution under settings of 60 kV/100 mA through a 0.5 mm aluminum filter. The scanned data were then reconstructed into three-dimensional representations with the aid of NRecon and DataViewer software (Bruker, USA). Further data analysis was executed using CTAn software, while CTvox software was employed to produce the 3D visualizations, all of which are products of Bruker, USA.

Immunofluorescence staining

To perform immunofluorescence staining on tissue sections, 5 µm thick slices were subjected to deparaffinization using xylene and progressively hydrated with gradient of ethanol to water. Then, the sections were subjected to antigen retrieval, permeabilized with 0.2% Triton X-100. Blocking was carried out with 5% BSA for 1 h before incubating overnight at 4°C with primary antibodies against FOXK1 (1:100), STAT1 (1:100), STAT2 (1:100), IL-6 (1:100, Abcam, UK), IL-1β (1:100, Abcam, UK) and MMP3 (1:100, Abcam, UK). The sections were then rinsed and incubated with either anti-mouse or anti-rabbit Alexa Fluor 488 secondary antibodies (1:200, Abcam, UK) for 1 h at room temperature. Finally, the sections were counterstained with DAPI (Beyotime, CHN) and images were acquired using a confocal fluorescence microscope (Leica, SP8, DE).

Serum biochemical assays

Blood samples were collected from mice after treatment via hepatic portal vein puncture. Serum was collected via centrifugation of blood samples in 12 000 × g at 4 °C for 30 min. Serum biochemical parameters including alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin (ALB), total protein (TP), and lgG were analyzed by a MS-480 Automatic Biochemistry Analyzer (Medicalsystem Biotechnology, CHN).

Statistical analysis

Data analysis was conducted using GraphPad Prism software. For assessing differences among multiple independent groups, one-way ANOVA followed by a post-hoc test was employed. Comparisons between two independent groups were made using the Student’s t test. For examining differences among groups categorized by two factors, two-way ANOVA with a subsequent post-hoc test was utilized. Data in the figures are represented as mean ± SEM, with levels of statistical significance denoted by *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.000 1. Sample sizes for in vivo studies were established based on power analysis beforehand. Mice were assigned to groups in a random and blinded manner, and any mice in poor health prior to the start of the studies were not included.