Synovial fluid collection
Synovial fluids (SF) were obtained from healthy people (undergone total knee replacement after knee trauma), OA patients and RA patients who had undergone therapeutic arthrocentesis at the First Affiliated Hospital of Anhui Medical University. All patients have signed informed consent. SF samples were transferred to heparin-treated tubes and centrifuged at 2000 rpm for 10 min at 4 ℃ to exclude cells and debris. Then the supernatants were collected and filtered via a 0.2 μm pore size membrane to remove remaining macromolecules and stored at -80 ℃ for future use.
UC-MSCs (from one independent donor) were provided by Nanjing Kangya Biological Technology Co., Ltd. Cells were thawed at 37 ℃ within 1 min, then seeded into 25-cm2 culture flasks and cultivated in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12, BI, Israel) containing 10% of fetal bovine serum (FBS, BI, Israel) and 1% of penicillin-streptomycin (Gibco). Cells were passaged at a split ratio of 1:3 when the confluency reached 80%. Fresh medium was replaced every 3 days, and cells between passages 3 and 6 were used in the subsequent experiments.
Phenotype of UC-MSCs
At the end of the third passage, cells were collected and washed with PBS containing 1% of bovine serum albumin (BSA; Sigma Aldrich, USA). Then, cells were resuspended with 200 μL PBS before incubating with FITC/PE/APC-conjugated mouse anti-human antibodies (CD11b, CD34, CD45, CD73, CD90 or CD105) (Biolegend, USA) for 30 mins in the dark at 4 ℃. Then, the cells were washed two times with PBS to remove unbound antibody and resuspended with 300 μL of PBS for flow cytometry analysis.
Trilineage differentiation potential of UC-MSCs
The multi-directional differentiation capabilities of UC-MSCs were detected using commercial MSCgo™ differentiation kits (BI, Israel). Briefly, UC-MSCs were seeded in a 48-well plates at the density of 1×104 per well and cultured with adipogenic, osteogenic, or chondrogenic differentiation induction medium. The induction medium was changed every 3 days. On day 21, cells were fixed and stained with Oil Red O for adipocytes, Alizarin Red S for osteocytes, and Alcian Blue for chondrocytes (Servicebio, China).
To determine the effect of SF on the chondrogenic differentiation of UC-MSCs, cells were seeded at a density of 3×104 cells/well in 24-well plates in the growth medium. At 80% confluence, cells were serum-starved overnight and the medium was replaced by the chondrogenic differentiation medium containing one of the following treatments: OA SF or RA SF was added to the media in a 20% or 40% ratio. The medium was replaced every 3 days, and Alcian blue staining was performed after 21 days of chondrogenic induction.
KYN treatment of UC-MSCs
To determine the effect of KYN on the chondrogenic differentiation of UC-MSCs, cells were seeded in 24-well plates with the growth medium. When the confluence is about 80%, cells were serum-starved overnight, using the chondrogenic differentiation medium containing 100 or 200 μM of KYN (Sigma, USA) to replace the medium. The medium was replaced every 3 days, and Alcian blue staining was performed after 21 days of chondrogenic induction.
The effect of SF or KYN on cell activity of UC-MSCs was measured by Cell Counting Kit 8 assay (CCK-8, Beyotime, China), cells were seeded in 96-well plates at an initial density of 1 ×103 cells/well and serum-starved overnight, then cells were cultured in DMEM/F12 containing 10% FBS and treated with SF or KYN for 7 days. At the indicated time-points (day 1, 3, 5 or 7), cells were incubated with CCK-8 solution (10 μl/well) at 37 °C for 1 h. Each time point included six replicate wells, followed by detecting the optical density at a wavelength of 450 nm by an enzyme-labeling instrument (BioTek Elx, Tecan, USA).
For AHR knock down, the short hairpin RNA targeting AHR (shAHR) was packaged into a lentiviral vector and constructed by Gene Chem Co. (Shanghai, China). The lentiviral vector containing a scramble sequence served as negative control (shNC). The sequences of shRNA were as below: shAHR: 5′-GCATAGAGACCGACTTAAT-3′; shNC: 5′-TTCTCCGAACGTGTCACGT-3′. Lentivirus transfection was performed following the manufacturer's instructions. The antibiotic selection which was conducted by adding puromycin (5 μg/mL, Sigma-Aldrich) and fluorescent cell sorting were employed to select stable knockdown cells.
RNA isolation and Real-time quantitative polymerase chain reaction (qRT-PCR)
According to the manufacturer's instructions to use Trizol Reagent (Thermo, USA) to extract the total RNA. The reverse transcription was performed using HiScript® II Q RT SuperMix for qRT-PCR (Vazyme Biotech, China) to synthesize cDNA. QRT-PCR was performed using AceQ® qRT-PCR SYBR Green Master Mix (Vazyme Biotech, China) on a 7500 Real-Time PCR Detection System (Applied Biosystems, USA) with gene-specific primers. Primers were synthesized by Sangon Biotech (China), the sequences of primers were as follows: AHR (forward 5'-CAGTGGTCCCAGCCTACAC-3' and reverse 5'-GACTGGCGTAGGTGATGTTG-3'), CYP1A1 (forward 5'-CTCAGTACCTCAGCAGCCAC-3' and reverse 5'-TTCTTCAGGCCTTTGGGGAC-3'), CYP1B1 (forward 5'-GACGCCTTTATCCTCTCTGCG-3' and reverse 5'-ACGACCTGATCCAATTCTGCC-3'), SOX-9 (forward 5'-GGACTTCTGAACGAGAGCGAGA-3' and reverse 5'-CGTTCTTCACCGACTTCCTCC-3'), COL2A1 (forward 5'-TGCATGAGGGCGCGGTA-3' and reverse 5'-GGTCCTGGTTGCCGGACAT-3'), Aggrecan (forward 5'-ACATTGTGGGGCTAGAACGA-3' and reverse 5'-CAGGAGGCTGCACAAGTTTT-3'), and GAPDH (forward 5'-ATGTTGCAACCGGGAAGGAA-3' and reverse 5'-AGGAAAAGCATCACCCGGAG-3'). GAPDH was regarded as a reference gene. The relative expression levels of the mRNAs in the groups were analyzed using the 2-ΔΔCT method.
Western blot analysis
The total cellular protein was obtained after lysis and the concentration of protein was measured by the BCA protein assay kit (BioChannel Biotechnology, China). The cell lysates were centrifuged and the supernatants were denatured by boiling for 10 min with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis buffer. For Western blot, protein samples were separated by 10% SDS-polyacrylamide gel electrophoresis and transferred on polyvinylidene fluoride membrane (Millipore, USA). They then incubated with the following primary antibodies against: AHR (1:800, Santa, USA), SOX-9 (1:800, Santa, USA), COL2A1 (1:600, Proteintech, USA) and β-actin (1:10000, Cell Signaling Technology, USA). Afterwards, membranes were washed and treated with appropriate HRP-conjugated secondary antibodies and developed by electrochemiluminescence (ELC, Thermo, USA). The relative band intensity was measured using ImageJ analysis software.
Cells grown on the glass coverslips were washed with ice-cold PBS, fixed with 4% formaldehyde, permeabilized with 0.2% Triton X-100, and then blocked with 5% bovine serum albumin. Then, the cells were incubated with appropriate primary antibodies at the following dilutions: anti-AHR (1:200, Santa, USA), anti-SOX-9 (1:200, Santa, USA), anti-COL2A1 (1:150, Proteintch, USA) overnight at 4 °C. The following day, cells were treated with fluorescence-labeled secondary antibodies as follows: anti-mouse IgG 488 (1:200, Biologend, USA), anti-goat IgG 594 (1:200, Biologend, USA) and anti-mouse IgG 647 (1:200, Biologend, USA) for 2 h at room temperature away from light. After PBS wash, nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI, Sigma, USA) for 10 min and anti-fluorescence quencher was added. Images were taken with a confocal microscope (Leica, TCS SP8, Germany).
Alcian blue staining
To induce chondrogenic differentiation, UC-MSCs were cultured in chondrogenic differentiation medium. At day 21 after chondrogenic induction, cells were washed twice with PBS, fixed with 4% formaldehyde solution for 30 min, and stained with Alcian Blue (1% in acetic acid, pH 2.5) overnight. Then, the cells were washed 3 times with 0.1N HCL and imaged using a microscope. In addition, the content of total sulfated GAG in chondrocyte spheroids was quantified. Briefly, chondrocyte spheroids were treated with 8 M guanidine HCL containing 0.05 M acetate, pH 5.8 and proteinase inhibitors overnight. Then, the absorbance of eluent was measured at 600 nm using a microplate reader as previously described.
All animal protocols were performed in accordance with the laboratory animal care and use guidelines and approved by the Animal Ethics Committee of institute of Clinical Pharmacology Laboratory in Anhui Medical University. 32 male Sprague-Dawley (SD) rats (approximately 12 weeks old) purchased from SPF Biotechnology Co. (Beijing, China) and housed in a specific pathogen-free (SPF) animal laboratory with 12:12 hours light/dark cycle and controlled temperature environment (23-25 °C). After 1 week of acclimatization, according to previous research, the rat OA model was established by completely transecting the medial collateral ligament and medial meniscus, removing the meniscus and cutting off the anterior cruciate ligament without damaging the tibial surface. After surgery, all rats received intramuscular injection of penicillin sodium (10 mg/kg) for 3 days after the operation to prevent infection. Then rats were randomly divided into four groups: (1) sham group (every rat received articular cavity injection of normal saline when the OA group was given an injection; 100 μl; n=8); (2) OA group (every rat received articular cavity injection of normal saline on the 8th week after surgery; 100 μl; n=8); (3) OA+shNC-UC-MSCs (every rat received articular cavity injection of shNC-UC-MSCs when the OA group was given an injection; 100 μl; 5×107 cells/mL; n=8); (4) OA+shAHR-UC-MSCs group (every rat received articular cavity injection of shAHR-UC-MSCs when the OA group was given an injection; 100 μl; 5×107 cells/mL; n=8). Four weeks after injection, rats were sacrificed by and the knee samples were harvested to evaluate disease progression.
To evaluate the severity of OA before the animals were euthanized, X-ray examination was performed on knee joint samples to observe the osteophyte formation, joint space and cartilage damage. Radiographic grading was based on Kellgren-Lawrence scoring system. Radiographic scoring was performed by a single surveyor after training was provided by an experienced surgeon.
Histology and immunohistochemical analysis
After the knee joints were opened and disarticulated, the gross morphological lesions on the rat tibial plateaus were visualized and cartilage lesions and fibrillation were quantified according to the Osteoarthritis Research Society International (OARSI) guidelines. Collecting knee joint samples and removing the tibiofemoral joints, the remaining femoral condyles were fixed in a neutral buffer of formalin (containing 4% formaldehyde) for 24 h. The fixed femoral condyles were decalcified in EDTA for 3 weeks and embedded in paraffin. Tissues were embedded in paraffin and sectioned into a 5-μm-thick section. The serial sections were obtained from the medial and lateral compartments at 200-μm intervals. The selected sections were deparaffinized in xylene, rehydrated through a graded series of ethanol washes, and followed by Safranin O/Fast Green staining (Servicebio, China). The cartilage degeneration was assessed by Mankin's score to assess the cartilage degeneration. OARSI scoring and Mankin's scoring were performed by three independent blinded observers.
For immunohistochemistry (IHC), the deparaffinized sections were soaked in EDTA (pH 9.0) for antigen retrieval by a microwave method. The sections were placed in a 3% hydrogen peroxide solution and incubated at room temperature for 25 min in the dark, followed by blocking with 5% BSA at room temperature for 30 min. Then, the sections were incubated with primary antibody Collagen II (Abcam, UK), and Aggrecan (Abcam, UK) at 4 °C overnight, followed by the secondary antibody (Abcam, UK) at room temperature for 60 min the next day. After extensive washing, 3,3′-diaminobenzidine (DAB)-peroxidase substrate and hematoxylin solution (Servicebio, China) was added.
KYN determination in synovial fluifd by high-performance liquid chromatography (HPLC)
KYN concentrations were measured by high-performance liquid chromatography (HPLC) as previously described. Briefly, frozen samples were thawed immediately prior to investigation. Protein was precipitated with trichloroacetic acid, mixed, and centrifuged at 12000 rpm and 4°C. For the measurement, 10 μl of clear supernatant was injected into the HPLC and using Agilent TC-C18 columns (250 mm length, 5 μm grain size) for separation. Kynurenine was detected by a fluorescence detector (Agilent, G1315D) at a wavelength of 360 nm. Data were recorded by the Agilent ChemStation software.
SPSS v. 25.0 software was used for the statistical analysis, and multiple–factorial analysis of variance (ANOVA) was applied for the comparison among multiple groups. All experiments were repeated through three independent batches and results were presented as mean ± SEM, A P value < 0.05 was considered statistically significant. Cell culture samples and animals were randomly assigned to each group with about equivalent numbers in each group, and all samples were analyzed in a blinded manner.