Reagents and supplies
On this examine, GNS have been synthesized utilizing the citrate discount methodology and obtained from Wuhan MICE Biotechnology Co. Ltd. The synthesis of GNS was carried out in two important steps. Initially, trisodium citrate was employed as a lowering agent and a stabilizer, whereas chloroauric acid was the gold precursor. This concerned the speedy addition of trisodium citrate to a boiling chloroauric acid answer in ultrapure water, adopted by continued boiling for 20 min to supply gold seeds. Within the subsequent step, the gold seeds acted because the core, with trisodium citrate because the stabilizer and ascorbic acid because the lowering agent. By various the amount of chloroauric acid, which acts because the gold precursor, gold shells of various thicknesses have been shaped, leading to GNS with a spread of particle sizes.
The GNS samples underwent ultrasonic dispersion in water and have been positioned in a possible pool to measure zeta potential utilizing a Zetasizer Nano ZS90. The morphological properties of the GNS, reminiscent of measurement distribution and homogeneity, have been analyzed with a scanning electron microscope (Hitachi Regulus SU8100). The typical nanoparticle measurement was used because the consultant measurement for the samples on this examine.
CIA mice mannequin institution and related remedies
Male DBA/1 J mice have been procured from GemPharmatech (Nanjing, China). All experiments have been carried out with gender- and age-matched DBA/1 J mice housed in a selected pathogen-free (SPF) atmosphere with a 12-h gentle/darkish cycle and advert libitum entry to meals and water. Experimental procedures have been carried out by the moral rules for animal care and use in China and authorised by the Military Medical College (Approval No. AMUWEC20232385). The CIA mannequin was established in line with the protocol by Jiang et al. [12]. Briefly, 6-week-old male DBA/1 J mice acquired a subcutaneous injection on the tail base on day 0 with blended emulsion of bovine kind II collagen (Cat#: 20,022, Chondrex Inc.) and full Freund’s adjuvant (CFA, Cat#: 7001, Chondrex Inc.), reaching a remaining focus of 1 mg/mL collagen and 0.5 mg/mL M. tuberculosis. Every mouse acquired 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, sustaining the collagen focus at 1 mg/ml, with every mouse receiving 100 µL.
All mice have been randomly assigned to 5 experimental teams: CIA mice handled with regular saline (RA + NS, n = 5), antibiotics (RA + ABX, n = 5), GNS (RA + GNS, n = 5), GNS together 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 physique weight (bw) /day from day 21 to day 42 within the RA + GNS group. RA + NS mice acquired an equal quantity of regular saline. The RA + GNS + CH group moreover acquired CH-223191 (Cat#: C303374, Aladdin) at 10 mg/kg bw/day through gavage. Procedures for FMT and ABX are detailed within the subsequent part.
A tryptophan metabolite combination, 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) through oral gavage each day from booster immunization till the top of this examine (500 mg/kg). Management mice (CT group) acquired an equal quantity of 0.5% CMC-Na.
FMT and ABX therapy
For FMT therapy, contemporary feces from the RA + GNS donor mice have been collected in sterile tubes, diluted with regular saline, and filtered utilizing sterile gauze to organize a fecal suspension, which was then administered to the recipient mice, FMT (RA + GNS) group through oral gavage (200 µl/mouse) till the top of this experiment. Notably, submit booster immunization, recipient mice acquired a broad-spectrum ABX therapy for 5 days to deplete intestine micro organism. This therapy 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 therapy, a steady ABX cocktail routine described above was administered orally each different day from booster immunization to the top of this examine.
Evaluation of arthritis severity
Arthritis severity was evaluated as beforehand reported [28]. Scientific scores, primarily based on the severity of arthritic limbs, have been assigned as follows: 0 = no signs, 1 = delicate swelling and erythema restricted to tarsals or ankle joint, 2 = delicate swelling and erythema spreading from ankle to tarsals, 3 = average swelling and erythema spreading to metatarsal joints, and 4 = extreme swelling encompassing the ankle and foot or ankylosing deformity. The scientific rating for every mouse was the sum of the scores for every paw. Scoring was carried out each three days throughout the remark interval.
Histological analysis
The bilateral hind paws of mice have been mounted in 4% paraformaldehyde, decalcified with 10% ethylenediaminetetraacetic acid (EDTA) for one week, and embedded in paraffin. These samples have been then deparaffinized, rehydrated, and stained with hematoxylin–eosin (H&E) and tartrate-resistant acid phosphatase (TRAP) stains. To evaluate potential GNS unwanted side effects, H&E staining was additionally carried out on liver and kidney tissue samples from the mice. The paw-scoring standards have been carried out in line with the strategy described by Jiang et al. [12].
Immunohistochemical evaluation
Paw tissue sections have been blocked with 5% BSA and incubated in a single day at 4 °C with main antibodies: anti-PTEN (Cat#: 60,300–1, Proteintech, 1:200), anti-p-p65 (Cat#: bs-3543R, Bioss, 1:200). Sections have been subsequently incubated with secondary antibodies for one hour at room temperature. To guage intestinal permeability, colonic tissue sections have been equally blocked and incubated in a single day with main antibodies towards ZO-1 (Cat#: PB9234, Boster, 1:200) and Claudin-1 (Cat#: A21770, ABclonal, 1:200), adopted by secondary antibody incubation.
Bone microstructure indexes evaluation
Mouse paws have been harvested and glued in 4% paraformaldehyde. Subsequently, ex vivo micro-computed tomography (micro-CT) was carried out utilizing a Bruker Micro-CT Skyscan 1272 system (Kontich, Belgium) at a decision of seven μm. The acquired photographs have been reconstructed utilizing Nrecon software program (Ver. 1.6.10, Kontich, Belgium) and additional processed with CT analyzer software program (Kontich, Belgium). A spherical area of bone tissue, with a diameter of 1.2 mm centered on the metacarpophalangeal joint, was designated because the area of curiosity for quantitative micro-CT evaluation. Moreover, 3D morphological evaluation was carried out utilizing CTvox 3D-model visualization software program (Bruker Micro-CT, Ver. 3.3.1.0). Bone morphometric parameters, together with bone mineral density (BMD), bone quantity fraction (BV/TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), and trabecular quantity (Tb.N), have been quantitatively assessed.
Elisa assay
The quantification of serum lipopolysaccharide (LPS) was investigated utilizing Mouse LPS ELISA Kits (Cat#: rxj202425M, Quanzhou Ruixin Biotechnology, Co., Ltd.). All operations have been carried out in line with the producer’s directions.
Intestinal permeability assay
We utilized Fluorescein isothiocyanate-dextran 4 (FD4, Cat#: 60,842–46-8, Sigma-Aldrich) to carry out 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 have been collected from mice 4 h later, centrifuged, and the ensuing serum was transferred to a contemporary centrifuge tube. The focus of FD4 within the serum samples was quantified utilizing a microplate system. The measurement outcomes and the FD4 focus customary curve have been entered into information evaluation software program to calculate the FD4 content material within the samples.
Fecal metabolomics profiling
Fecal metabolomics concerned getting ready 50 mg of every pattern in Eppendorf tubes, handled with 500 μL of pre-cooled (−40 °C) extraction answer (methanol: acetonitrile: water, 2:2:1, together with 0.1% formic acid and isotopically labeled inside customary). After 30 s of vortexing and 4 min of homogenization at 35 Hz, the combination was sonicated in an ice-water tub for five min, twice. Samples have been 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 beneath nitrogen and reconstituted in 80 μL of 0.1% formic acid answer. A remaining centrifugation at 12,000 rpm for 15 min at 4 °C produced a transparent supernatant for UHPLC-MS/MS evaluation. 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 quantity. Solvents have been 0.1% formic acid in water and acetonitrile. Mass spectrometry was carried out on a SCIEX Triple Quad™ 6500 + spectrometer, outfitted with an IonDrive Turbo V ESI interface. Information acquisition and MRM evaluation have been dealt with by SCIEX Analyst Work Station Software program (v1.6.3) and Sciex MultiQuant Software program (v3.0.3).
Fecal 16S rDNA gene sequencing and evaluation
Fecal samples from experimental mice have been gathered in sterile 2 mL cryovials and saved at −80 °C. DNA extraction from the RA + NS, RA + GNS, and RA + GNS + CH teams was carried out utilizing the CTAB methodology, as per the producer’s tips, and resuspended in 50 μL of Elution Buffer. Prokaryotic 16S fragments have been amplified beneath PCR situations: preliminary 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 remaining extension at 72 °C for 10 min. PCR merchandise have been confirmed by 2% agarose gel electrophoresis, purified with AMPure XT beads, and quantified utilizing the Qubit system. Amplicon libraries have been ready and evaluated for measurement and amount with the Agilent 2100 Bioanalyzer. Sequencing was carried out on the NovaSeq PE250 platform after library quantification. Sequences have been assigned to samples through distinctive barcodes, with barcodes and primers eliminated. Excessive-quality clear tags have been generated utilizing FLASH and fqtrim, and chimeric sequences have been eradicated with Vsearch. DADA2 dereplication produced a function desk and sequence information. Alpha and beta diversities have been calculated post-normalization utilizing the SILVA classifier. Alpha range indices (Chao1, Noticed species, Items protection, Shannon, Simpson) have been derived utilizing QIIME2. Beta range was assessed and visually represented utilizing R packages. Sequence alignment and annotation have been carried out with Blast and the SILVA database, respectively, adopted by extra analyses utilizing the R bundle.
Cell tradition and cell viability assay
MH7A cells have been bought from BeNa Tradition Assortment (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 answer (Cat#: C0222, Beyotime). Cells have been maintained at 37 °C with 5% CO2.
To measure the proliferation of MH7A cells, a Cell Counting Package-8 (Cat#: C0038, Beyotime) was utilized in line with the producer’s directions. 5 × 103 MH7A cells have been seeded into 96-well plates and cultured in a single day. Then they have been uncovered to completely different concentrations of IPA/IAA combination (0, 1, 5, 10 μM) for 12, 24, 36, or 48 h. 10 μL CCK-8 buffer was added to every nicely and incubated for 0.5 h at 37 °C with 5% CO2 in a humidified incubator. Wells with out cells however containing the CCK-8 reagent have been designated because the clean. Finally, the OD at 450 nm was measured.
Circulate cytometric evaluation
To investigate the impact of IPA + IAA combination on cell apoptosis, MH7A cells have been handled with IPA + IAA combination (10 μM). Then the cell apoptosis was detected through the use of the Annexin V-FITC/PI Apoptosis Detection Package (Cat#: MA0220, Meilun Bio). Briefly, 1 × 106 cells have been stained with 5 μL of annexin V-FITC and 10 μL PI. Apoptosis was analyzed utilizing a FACS move cytometer (CytoFLEX, USA) and analyzed with Circulate Jo software program (Circulate Jo, USA).
EdU staining
To guage the proliferation of MH7A cells, the EdU answer (10 μM) was added to 10% FBS DMEM for 2 hours. Then, the cells have been mounted with 4% paraformaldehyde (PFA) and permeabilized with 0.3% Triton X-100 for 15 min. The Elabscience® E-Click on EdU Cell Proliferation Imaging Assay Package (Cat#: E-CK-A377, Elabscience) was used to detect EdU-positive cells in line with the producer’s directions. The IX81 inverted fluorescence microscope (Olympus, Japan) was used to visualise and analyze the EdU-positive cells.
Transwell assay
To evaluate cell migration, a Transwell insert with an 8 μm pore measurement polycarbonate filter membrane (Corning) was utilized. For invasion, extra Matrigel (Corning) was diluted (1:6) and added to the floor of the higher chambers adopted by resting at 37˚C for two h. 5 × 103 MH7A cells handled with IPA + IAA combination (10 μM) or not have been seeded into the higher chambers in 2% FBS DMEM, whereas the decrease chambers have been loaded with 10% FBS DMEM. The next day, cells on the underside floor of the filter membranes have been stained with crystal violet staining answer (Cat#: C0121, Beyotime) for 10 min, washed 3 instances, and imaged utilizing the ECLIPSE Ni microscope (Nikon). The pictures have been then analyzed utilizing NIS-Parts D software program (Nikon).
RNA-sequencing assay
RNA extraction was carried out utilizing Trizol reagent (ThermoFisher, Cat#: 15,596,018) per the producer’s directions. RNA focus and high quality have been assessed with the Bioanalyzer 2100 system and RNA 6000 Nano LabChip Package (Agilent, USA, 5067–1511). Samples with an RNA Integrity Quantity (RIN) above 7.0 have been chosen for library preparation. From 5 μg of whole RNA, mRNA was purified utilizing Dynabeads Oligo (dT) (ThermoFisher, USA). The purified mRNA was fragmented at 94 °C for five–7 min with the Magnesium RNA Fragmentation Module (NEB, USA). These fragments have been transformed into cDNA utilizing SuperScript™ II Reverse Transcriptase (Invitrogen, USA). E. coli DNA polymerase I, RNase H, and dUTP Answer (NEB, ThermoFisher, USA) have been used to synthesize U-labeled second-strand DNAs. The strands underwent end-repair, adenylation, and ligation to listed adapters with a T-base overhang. Measurement collection of the ligation merchandise was achieved utilizing AMPure XP beads. Warmth-labile Uracil-DNA Glycosylase (NEB, USA) was utilized to U-labeled second-strand DNAs, adopted by PCR amplification: preliminary 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 remaining extension at 72 °C for five min. The cDNA library’s imply insert measurement was 300 ± 50 bp. Paired-end sequencing (2 × 150 bp, PE150) was carried out on the Illumina NovaSeq™ 6000 system, adhering to the producer’s protocol.
Immunofluorescence staining
MH7A cells have been cultured with TNF-α (10 ng/mL) within the presence or absence of IPA + IAA combination (10 μM) for 1 h earlier than therapy of VO-Ohpic (5 μM; Cat#: 675,848–25-6, MedChemExpress). Then the cells have been mounted with 4% PFA for 15 min and blocked in QuickBlock™ Blocking Buffer (Cat#: P0231, Beyotime) for 30 min. Cells have been washed thrice with PBS after which incubated with anti-p65 antibody (Cat#: bsm-33117 M, Bioss, 1:200) in a single day at 4 °C. The next day, the cells have been washed thrice with PBS earlier than being incubated with a CoraLite® Plus 594-conjugated secondary antibody (Cat#: RGAR004, proteintech, 1:200) and DAPI for 1 h at room temperature at midnight. The depth worth was analyzed through the use of the Picture J.
Western blot
The entire mobile lysates have been 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 combination of protease (Cat#: BL630B, Biosharp) and phosphatase (Cat#: P1081, Beyotime) inhibitors. Then, the cell lysates have been sonicated (20% energy, 5 s every time, 15 repetitions) and centrifuged at 12 000 rpm for 15 min at 4 °C. Equal quantities of whole proteins (40 μg) have been separated by SDS-PAGE (ready with 5% acrylamide within the stacking gel and 10% acrylamide within the separating gel) and transferred onto PVDF membrane. The first 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 in a single day at 4 °C. Subsequent day, the PVDF membranes have been washed 3 instances with TBST and incubated with a secondary antibody for 1.5 h at room temperature. Chemiluminescence detection was carried out with a BeyoECL Plus ECL package (Cat#: P0018S, Beyotime) and imaged with a Bio-Rad ChemiDoc™ Contact Imaging System. The sign depth of every protein band was measured with ImageJ.
Immunoprecipitation and immunoblotting
The MH7A cells have been lysed utilizing 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 have been collected and incubated with an anti-PTEN antibody (Cat#: sc-7974, santa cruz; 1:50) for two h at 4 °C. Protein A/G beads (Cat#: KM0134, DIA-An Biotechnology) have been then added to the cell lysates and incubated in a single day at 4 °C after being washed 4 instances with RIP binding buffer (1 × PBS, 1% TritonX-100, 0.01% NP-40, 5% Glycerol). To gather immune complexes, beads have been centrifuged and heated at 95 °C for five min in 2 × SDS loading buffer. Then, the immunoprecipitated complexes have been analyzed by Western blot utilizing anti-Ubiquitin (P4D1) antibody (Cat#: sc-8017, santa cruz; 1:1000).
Statistical evaluation
All quantitative information have been analyzed utilizing a selected software program (GraphPad Prism 7.0), with outcomes introduced as imply with customary deviation. Unpaired Scholar’s t-tests have been employed to evaluate the importance of variations between the 2 teams. Welch’s correction was utilized when the F check was important. The Kruskal–Wallis check was employed for bacterial taxonomic evaluations. Associations have been decided by way of Spearman’s rank correlation evaluation. Vital variations have been indicated by *P < 0.05; **P < 0.01; ***P < 0.001; and ****P < 0.0001. No important distinction was indicated by NS.
