Reagents and materials
In this study, GNS were synthesized using the citrate reduction method and obtained from Wuhan MICE Biotechnology Co. Ltd. The synthesis of GNS was conducted in two main steps. Initially, trisodium citrate was employed as a reducing agent and a stabilizer, while chloroauric acid was the gold precursor. This involved the rapid addition of trisodium citrate to a boiling chloroauric acid solution in ultrapure water, followed by continued boiling for 20 min to produce gold seeds. In the subsequent step, the gold seeds acted as the core, with trisodium citrate as the stabilizer and ascorbic acid as the reducing agent. By varying the quantity of chloroauric acid, which acts as the gold precursor, gold shells of different thicknesses were formed, resulting in GNS with a range of particle sizes.
The GNS samples underwent ultrasonic dispersion in water and were placed in a potential pool to measure zeta potential using a Zetasizer Nano ZS90. The morphological properties of the GNS, such as size distribution and homogeneity, were analyzed with a scanning electron microscope (Hitachi Regulus SU8100). The average nanoparticle size was used as the representative size for the samples in this study.
CIA mice model establishment and relevant treatments
Male DBA/1 J mice were procured from GemPharmatech (Nanjing, China). All experiments were conducted with gender- and age-matched DBA/1 J mice housed in a specific pathogen-free (SPF) environment with a 12-h light/dark cycle and ad libitum access to food and water. Experimental procedures were performed by the ethical regulations for animal care and use in China and approved by the Army Medical University (Approval No. AMUWEC20232385). The CIA model was established according to the protocol by Jiang et al. [12]. Briefly, 6-week-old male DBA/1 J mice received a subcutaneous injection at the tail base on day 0 with mixed emulsion of bovine type II collagen (Cat#: 20,022, Chondrex Inc.) and complete Freund’s adjuvant (CFA, Cat#: 7001, Chondrex Inc.), reaching a final concentration of 1 mg/mL collagen and 0.5 mg/mL M. tuberculosis. Each mouse received 100 µL of this emulsion. A booster injection with an emulsion of collagen and incomplete Freund’s adjuvant (IFA, Cat#: 7002, Chondrex Inc.) was administered on day 21, maintaining the collagen concentration at 1 mg/ml, with each mouse receiving 100 µL.
All mice were randomly assigned to five experimental groups: CIA mice treated with normal saline (RA + NS, n = 5), antibiotics (RA + ABX, n = 5), GNS (RA + GNS, n = 5), GNS along with AhR antagonist CH-223191 (RA + GNS + CH, n = 5), and CIA mice underwent fecal microbiota transplantation (FMT) from the RA + GNS group (FMT (RA + GNS), n = 5). GNS was administered orally at 0.01 mg/g body weight (bw) /day from day 21 to day 42 in the RA + GNS group. RA + NS mice received an equal volume of normal saline. The RA + GNS + CH group additionally received CH-223191 (Cat#: C303374, Aladdin) at 10 mg/kg bw/day via gavage. Procedures for FMT and ABX are detailed in the next section.
A tryptophan metabolite mixture, comprising equal proportions of IAA (Cat#: I101072, Aladdin) and IPA (Cat#: I103959, Aladdin), dissolved in 0.5% CMC-Na, was administered to CIA mice (IPA + IAA group) via oral gavage daily from booster immunization until the end of this study (500 mg/kg). Control mice (CT group) received an equivalent volume of 0.5% CMC-Na.
FMT and ABX treatment
For FMT treatment, fresh feces from the RA + GNS donor mice were collected in sterile tubes, diluted with normal saline, and filtered using sterile gauze to prepare a fecal suspension, which was then administered to the recipient mice, FMT (RA + GNS) group via oral gavage (200 µl/mouse) until the end of this experiment. Notably, post booster immunization, recipient mice received a broad-spectrum ABX treatment for five days to deplete gut bacteria. This treatment included Vancomycin, 100 mg/kg bw/day (Cat#: MB1260, MeilunBio); Neomycin sulfate, 200 mg/kg bw/day (Cat#: N412785, Aladdin); Metronidazole, 200 mg/kg bw/day (Cat#: M109874, Aladdin); and Ampicillin Na, 200 mg/kg bw/day (Cat#: A105483, Aladdin). For the ABX treatment, a continuous ABX cocktail regimen described above was administered orally every other day from booster immunization to the end of this study.
Assessment of arthritis severity
Arthritis severity was evaluated as previously reported [28]. Clinical scores, based on the severity of arthritic limbs, were assigned as follows: 0 = no symptoms, 1 = mild swelling and erythema limited to tarsals or ankle joint, 2 = mild swelling and erythema spreading from ankle to tarsals, 3 = moderate swelling and erythema spreading to metatarsal joints, and 4 = severe swelling encompassing the ankle and foot or ankylosing deformity. The clinical score for each mouse was the sum of the scores for each paw. Scoring was conducted every three days during the observation period.
Histological evaluation
The bilateral hind paws of mice were fixed in 4% paraformaldehyde, decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for one week, and embedded in paraffin. These samples were then deparaffinized, rehydrated, and stained with hematoxylin–eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) stains. To assess potential GNS side effects, H&E staining was also conducted on liver and kidney tissue samples from the mice. The paw-scoring criteria were conducted according to the method described by Jiang et al. [12].
Immunohistochemical analysis
Paw tissue sections were blocked with 5% BSA and incubated overnight at 4 °C with primary antibodies: anti-PTEN (Cat#: 60,300–1, Proteintech, 1:200), anti-p-p65 (Cat#: bs-3543R, Bioss, 1:200). Sections were subsequently incubated with secondary antibodies for one hour at room temperature. To evaluate intestinal permeability, colonic tissue sections were similarly blocked and incubated overnight with primary antibodies against ZO-1 (Cat#: PB9234, Boster, 1:200) and Claudin-1 (Cat#: A21770, ABclonal, 1:200), followed by secondary antibody incubation.
Bone microstructure indexes assessment
Mouse paws were harvested and fixed in 4% paraformaldehyde. Subsequently, ex vivo micro-computed tomography (micro-CT) was performed using a Bruker Micro-CT Skyscan 1272 system (Kontich, Belgium) at a resolution of 7 μm. The acquired images were reconstructed using Nrecon software (Ver. 1.6.10, Kontich, Belgium) and further processed with CT analyzer software (Kontich, Belgium). A spherical region of bone tissue, with a diameter of 1.2 mm centered on the metacarpophalangeal joint, was designated as the region of interest for quantitative micro-CT analysis. Additionally, 3D morphological analysis was conducted using CTvox 3D-model visualization software (Bruker Micro-CT, Ver. 3.3.1.0). Bone morphometric parameters, including bone mineral density (BMD), bone volume fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular number (Tb.N), were quantitatively assessed.
Elisa assay
The quantification of serum lipopolysaccharide (LPS) was investigated using Mouse LPS ELISA Kits (Cat#: rxj202425M, Quanzhou Ruixin Biotechnology, Co., Ltd.). All operations were performed according to the manufacturer’s instructions.
Intestinal permeability assay
We applied Fluorescein isothiocyanate-dextran 4 (FD4, Cat#: 60,842–46-8, Sigma-Aldrich) to perform an in vivo permeability assay, thereby evaluating the integrity of the intestinal barrier. After oral administration of FD4 (40 mg/100 g BW), blood samples were collected from mice 4 h later, centrifuged, and the resulting serum was transferred to a fresh centrifuge tube. The concentration of FD4 in the serum samples was quantified using a microplate system. The measurement results and the FD4 concentration standard curve were entered into data analysis software to calculate the FD4 content in the samples.
Fecal metabolomics profiling
Fecal metabolomics involved preparing 50 mg of each sample in Eppendorf tubes, treated with 500 μL of pre-cooled (−40 °C) extraction solution (methanol: acetonitrile: water, 2:2:1, including 0.1% formic acid and isotopically labeled internal standard). After 30 s of vortexing and 4 min of homogenization at 35 Hz, the mixture was sonicated in an ice-water bath for 5 min, twice. Samples were precipitated at −40 °C for an hour, centrifuged at 12,000 rpm for 15 min at 4 °C, and a 320 μL aliquot of the supernatant was dried under nitrogen and reconstituted in 80 μL of 0.1% formic acid solution. A final centrifugation at 12,000 rpm for 15 min at 4 °C produced a clear supernatant for UHPLC-MS/MS analysis. The ACQUITY Premier system (Waters) with a Waters ACQUITY UPLC HSS T3 column (100 × 2.1 mm, 1.8 μm) was used at 40 °C, with the auto-sampler at 10 °C and a 5 μL injection volume. Solvents were 0.1% formic acid in water and acetonitrile. Mass spectrometry was conducted on a SCIEX Triple Quad™ 6500 + spectrometer, equipped with an IonDrive Turbo V ESI interface. Data acquisition and MRM analysis were handled by SCIEX Analyst Work Station Software (v1.6.3) and Sciex MultiQuant Software (v3.0.3).
Fecal 16S rDNA gene sequencing and analysis
Fecal samples from experimental mice were gathered in sterile 2 mL cryovials and stored at −80 °C. DNA extraction from the RA + NS, RA + GNS, and RA + GNS + CH groups was performed using the CTAB method, as per the manufacturer’s guidelines, and resuspended in 50 μL of Elution Buffer. Prokaryotic 16S fragments were amplified under PCR conditions: initial denaturation at 98 °C for 30 s, 32 cycles of 98 °C for 10 s, 54 °C for 30 s, 72 °C for 45 s, and a final extension at 72 °C for 10 min. PCR products were confirmed by 2% agarose gel electrophoresis, purified with AMPure XT beads, and quantified using the Qubit system. Amplicon libraries were prepared and evaluated for size and quantity with the Agilent 2100 Bioanalyzer. Sequencing was conducted on the NovaSeq PE250 platform after library quantification. Sequences were assigned to samples via unique barcodes, with barcodes and primers removed. High-quality clean tags were generated using FLASH and fqtrim, and chimeric sequences were eliminated with Vsearch. DADA2 dereplication produced a feature table and sequence data. Alpha and beta diversities were calculated post-normalization using the SILVA classifier. Alpha diversity indices (Chao1, Observed species, Goods coverage, Shannon, Simpson) were derived using QIIME2. Beta diversity was assessed and visually represented using R packages. Sequence alignment and annotation were performed with Blast and the SILVA database, respectively, followed by additional analyses using the R package.
Cell culture and cell viability assay
MH7A cells were purchased from BeNa Culture Collection (BNCC, BNCC371792) and cultured in Dulbecco’s Modified Eagle Medium (DMEM; Cat#: G4524, Servicebio) supplemented with 10% fetal bovine serum (FBS; Cat#: C04001-050X10, VivaCell), and 100 U/mL penicillin–streptomycin solution (Cat#: C0222, Beyotime). Cells were maintained at 37 °C with 5% CO2.
To measure the proliferation of MH7A cells, a Cell Counting Kit-8 (Cat#: C0038, Beyotime) was utilized according to the manufacturer’s instructions. 5 × 103 MH7A cells were seeded into 96-well plates and cultured overnight. Then they were exposed to different concentrations of IPA/IAA mixture (0, 1, 5, 10 μM) for 12, 24, 36, or 48 h. 10 μL CCK-8 buffer was added to each well and incubated for 0.5 h at 37 °C with 5% CO2 in a humidified incubator. Wells without cells but containing the CCK-8 reagent were designated as the blank. Ultimately, the OD at 450 nm was measured.
Flow cytometric analysis
To analyze the effect of IPA + IAA mixture on cell apoptosis, MH7A cells were treated with IPA + IAA mixture (10 μM). Then the cell apoptosis was detected by using the Annexin V-FITC/PI Apoptosis Detection Kit (Cat#: MA0220, Meilun Bio). Briefly, 1 × 106 cells were stained with 5 μL of annexin V-FITC and 10 μL PI. Apoptosis was analyzed using a FACS flow cytometer (CytoFLEX, USA) and analyzed with Flow Jo software (Flow Jo, USA).
EdU staining
To evaluate the proliferation of MH7A cells, the EdU solution (10 μM) was added to 10% FBS DMEM for two hours. Then, the cells were fixed with 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 15 min. The Elabscience® E-Click EdU Cell Proliferation Imaging Assay Kit (Cat#: E-CK-A377, Elabscience) was used to detect EdU-positive cells according to the manufacturer’s instructions. The IX81 inverted fluorescence microscope (Olympus, Japan) was used to visualize and analyze the EdU-positive cells.
Transwell assay
To assess cell migration, a Transwell insert with an 8 μm pore size polycarbonate filter membrane (Corning) was utilized. For invasion, additional Matrigel (Corning) was diluted (1:6) and added to the surface of the upper chambers followed by resting at 37˚C for 2 h. 5 × 103 MH7A cells treated with IPA + IAA mixture (10 μM) or not were seeded into the upper chambers in 2% FBS DMEM, while the lower chambers were loaded with 10% FBS DMEM. The following day, cells on the bottom surface of the filter membranes were stained with crystal violet staining solution (Cat#: C0121, Beyotime) for 10 min, washed 3 times, and imaged using the ECLIPSE Ni microscope (Nikon). The images were then analyzed using NIS-Elements D software (Nikon).
RNA-sequencing assay
RNA extraction was conducted using Trizol reagent (ThermoFisher, Cat#: 15,596,018) per the manufacturer’s instructions. RNA concentration and quality were assessed with the Bioanalyzer 2100 system and RNA 6000 Nano LabChip Kit (Agilent, USA, 5067–1511). Samples with an RNA Integrity Number (RIN) above 7.0 were selected for library preparation. From 5 μg of total RNA, mRNA was purified using Dynabeads Oligo (dT) (ThermoFisher, USA). The purified mRNA was fragmented at 94 °C for 5–7 min with the Magnesium RNA Fragmentation Module (NEB, USA). These fragments were converted into cDNA using SuperScript™ II Reverse Transcriptase (Invitrogen, USA). E. coli DNA polymerase I, RNase H, and dUTP Solution (NEB, ThermoFisher, USA) were used to synthesize U-labeled second-strand DNAs. The strands underwent end-repair, adenylation, and ligation to indexed adapters with a T-base overhang. Size selection of the ligation products was achieved using AMPure XP beads. Heat-labile Uracil-DNA Glycosylase (NEB, USA) was applied to U-labeled second-strand DNAs, followed by PCR amplification: initial denaturation at 95 °C for 3 min, 8 cycles of denaturation at 98 °C for 15 s, annealing at 60 °C for 15 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min. The cDNA library’s mean insert size was 300 ± 50 bp. Paired-end sequencing (2 × 150 bp, PE150) was performed on the Illumina NovaSeq™ 6000 system, adhering to the manufacturer’s protocol.
Immunofluorescence staining
MH7A cells were cultured with TNF-α (10 ng/mL) in the presence or absence of IPA + IAA mixture (10 μM) for 1 h before treatment of VO-Ohpic (5 μM; Cat#: 675,848–25-6, MedChemExpress). Then the cells were fixed with 4% PFA for 15 min and blocked in QuickBlock™ Blocking Buffer (Cat#: P0231, Beyotime) for 30 min. Cells were washed three times with PBS and then incubated with anti-p65 antibody (Cat#: bsm-33117 M, Bioss, 1:200) overnight at 4 °C. The following day, the cells were washed three times with PBS before being incubated with a CoraLite® Plus 594-conjugated secondary antibody (Cat#: RGAR004, proteintech, 1:200) and DAPI for 1 h at room temperature in the dark. The intensity value was analyzed by using the Image J.
Western blot
The total cellular lysates were obtained by lysing MH7A cells with RIPA buffer (25 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% SDS) and a mixture of protease (Cat#: BL630B, Biosharp) and phosphatase (Cat#: P1081, Beyotime) inhibitors. Then, the cell lysates were sonicated (20% power, 5 s each time, 15 repetitions) and centrifuged at 12 000 rpm for 15 min at 4 °C. Equal amounts of total proteins (40 μg) were separated by SDS-PAGE (prepared with 5% acrylamide in the stacking gel and 10% acrylamide in the separating gel) and transferred onto PVDF membrane. The primary antibody anti- NF-κB p65 (Cat#: bsm-33117 M-1, bioss, 1:1000), anti-Phospho-NFKB p65 (Ser276; Cat#: bs-3543R, bioss; 1:1000), anti-IkB-α (Cat#: BS3601, bioworld; 1:1000), anti-p-IkB-α (phospho-S32/S36; Cat#: BS4105, bioworld; 1:1000), anti-PTEN (Cat#: sc-7974, santa cruz; 1:1000), anti-p-PTEN (Cat#: sc-377573, santa cruz; 1:500) was added to the PVDF membranes and incubated overnight at 4 °C. Next day, the PVDF membranes were washed 3 times with TBST and incubated with a secondary antibody for 1.5 h at room temperature. Chemiluminescence detection was performed with a BeyoECL Plus ECL kit (Cat#: P0018S, Beyotime) and imaged with a Bio-Rad ChemiDoc™ Touch Imaging System. The signal intensity of each protein band was measured with ImageJ.
Immunoprecipitation and immunoblotting
The MH7A cells were lysed using IP lysis buffer (50 mM Tris–HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Triton X-100) and sonicated as described above. The lysates were collected and incubated with an anti-PTEN antibody (Cat#: sc-7974, santa cruz; 1:50) for 2 h at 4 °C. Protein A/G beads (Cat#: KM0134, DIA-An Biotechnology) were then added to the cell lysates and incubated overnight at 4 °C after being washed 4 times with RIP binding buffer (1 × PBS, 1% TritonX-100, 0.01% NP-40, 5% Glycerol). To collect immune complexes, beads were centrifuged and heated at 95 °C for 5 min in 2 × SDS loading buffer. Then, the immunoprecipitated complexes were analyzed by Western blot using anti-Ubiquitin (P4D1) antibody (Cat#: sc-8017, santa cruz; 1:1000).
Statistical analysis
All quantitative data were analyzed using a specific software (GraphPad Prism 7.0), with results presented as mean with standard deviation. Unpaired Student’s t-tests were employed to assess the significance of differences between the two groups. Welch’s correction was applied when the F test was significant. The Kruskal–Wallis test was employed for bacterial taxonomic evaluations. Associations were determined through Spearman’s rank correlation analysis. Significant differences were indicated by *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. No significant difference was indicated by NS.
