Indole-3-propionic acid alleviates chondrocytes inflammation and osteoarthritis via the AhR/NF-κB axis

Animal experiments

A total of fifteen 8-week-old Wistar male rats were provided by the Hubei Provincial Centers for Disease Control and Prevention (Wuhan, Hubei, China). These rats were randomly divided into three groups, namely the sham group, ACLT group and IPA group, with 5 rats per group. The OA rat model in ACLT group and IPA group was established using anterior cruciate ligament transection (ACLT). The rats in the IPA group were then intraperitoneally injected with 20 mg/kg IPA (Aladdin, I103959) twice a week, and rats in the Sham and ACLT group were injected with 50 µL sterile saline as the control. The rats were sacrificed by an injecting of overdose pentobarbital sodium at the end of the 8th week. The animal experiments were performed according to the National Institutes of Health Guide for Care and Use of Laboratory Animals and approved by the Laboratory Animal Welfare & Ethics Committee of the Renmin Hospital of Wuhan University (Approval No: 20220103A).

Chondrocytes isolation and culture

We isolated cartilage from 8-week-old Wistar male rats to culture primary chondrocytes. The cartilage was cut into 3 mm pieces, then digested for 8 h in DMEM/F12 (Servicebio, G4610) medium containing 0.2% collagenase II (Servicebio, GC305014). Finally, chondrocytes were centrifuged at 1200 rpm for 8 min. The isolated chondrocytes were resuspended in DMEM/F12 medium supplemented with 10% fetal bovine serum (FBS, Gibco, 10270–106) and 1% penicillin-streptomycin (Servicebio, G4003) in a 5% CO2 and 37 °C incubator. Plasmids containing short hairpin RNA against AhR (shRNA-AhR) or a scrambled shRNA were obtained from Origene. Chondrocytes were treated with 10 ng/ml IL-1β alone, with IPA, or with shRNA. Transfection was performed by Lipofectamine 3000 (Invitrogen, L3000015) following the manufacturer’s protocol. Subsequent experiments were conducted 48 h after transfection.

Cell proliferation assay

The cytotoxicity of IPA on chondrocyte viability was quantitated by Cell Counting Kit-8 (CCK-8, Servicebio, G4103). Chondrocytes were seeded in 96-well plates at 5 × 103 cells per well. Then IPA was added at concentrations of 0, 10, 20, 40, 80, 160, 320 and 640 µM. 10 µL of CCK-8 solution was added to each well at 0, 24, 48, 72 and 96 h, and incubated at 37 °C for 2 h. The absorbance at a wavelength of 450 nm was determined using a microplate reader (EnVision, PerkinElmer, USA).

Real-time quantitative PCR (RT-qPCR)

The total cellular RNA was isolated by the RNA Isolation Kit (Beyotime, R0026) according to the manufacturer’s protocol. The RNA concentration was determined by Nanodrop (Thermo, USA). cDNA synthesis was performed from each 1 µg RNA using SweScript RT II Enzyme Mix (Servicebio, G3330) following the manufacturer’s protocol. RT-qPCR for mRNA was performed on LightCycler 480 (Roche Diagnostics, USA) with 2× Universal SYBR Green Fast qPCR Mix (Abclonal, RK21203). β-actin was used as an internal standard control and relative gene levels were detected by the 2−△△Ct method. The primer sequences are shown in Additional file 1: Table S1.

Western blot

The total proteins from chondrocytes were extracted using radio immunoprecipitation assay buffer, phenylmethanesulfonyl fluoride, phosphatase inhibitors and cotail. Chondrocyte cell lysis was performed for 30 min on ice, and ultrasonication and centrifugation were used to purify proteins. The concentration of proteins was measured by the BCA protein assessment kit. Then, the protein (30 µg) samples were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel. After transferring to PVDF membranes, the membranes were blocked using 5% nonfat milk and then incubated with primary antibodies against IL-6 (1:1000, Abmart, PY6087), iNOS (1:1000, Abclonal, A0312), COX-2 (1:1000, Abclonal, A3560), MMP-3 (1:1000, Abcam, ab52915), MMP-13 (1:1000, Proteintech, 18165-1), ADAMTS-5 (1:1000, Abcam, ab41037), AhR (1:1000, Proteintech, 67785-1), IKKβ (1:1000, Cell Signaling Technology, #2684), p-IKKβ (1:1000, Cell Signaling Technology, #2694), IκBα (1:1000, Abmart, T55026), p-IκBα (1:1000, Abmart, TP56280), p65 (Cell Signaling Technology, 4764), p-p65 (Cell Signaling Technology, 3031) or β-actin (1:3000, Servicebio, GB11001) overnight at 4 ℃. Next, after TBST washed three times, the membranes were incubated with HRP-conjugated secondary antibodies for 2 h at room temperature. After being washed using the TBST buffer, the membrane was incubated with ECL solution and exposed using a Bio-Rad scanner (California, America). Finally, the densitometric analysis was performed using Image-J software.

Immunofluorescence

Chondrocytes were fixed in 4% paraformaldehyde for 15 min, and washed with PBS three times. The normal goat serum (Servicebio, WGAR1009) was used to block chondrocytes for 30 min at room temperature. The chondrocytes were incubated with primary antibodies against aggrecan (1:500, Servicebio, GB11373) and collagen-II (1:200, Servicebio, GB11021) at 4 °C for 24 h. Then chondrocytes were incubated with fluorescein-conjugated goat anti-rabbit IgG (1:600, Servicebio, GB21301) at 37 °C for 1 h. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI). The images were acquired by an inverted fluorescence microscope. Cellular fluorescence intensity was quantified by evaluating positive cells using Image-J software.

ELISA

Chondrocytes were incubated with 10 ng/ml IL-1β supplemented alone or with IPA or with shRNA-AhR for 24 h. Nitric Oxide (NO), TNF-α, IL-6 and PGE2 expression levels in supernatants were evaluated. Besides, TNF-α, IL-6 and PGE2 expression levels in rat serum were measured. The concentration of NO was assessed using Griess reagent (Beyotime, S0024). The concentration of TNF-α (FineTest, ER1393), IL-6 (elabscience, R0015c) and PGE2 (FineTest, ER1800) levels were measured using ELISA Kits according to the manufacturer’s protocol.

Bioinformatics data mining

To analyze the binding affinities and modes of interaction between IPA and AhR, AutodockVina 1.2.2, a silico protein-ligand docking software, was employed (http://autodock.scripps.edu/) (Morris et al. 2008). The molecular structure of IPA (PubChem CID: 3744) was retrieved from PubChem Compound (https://pubchem.ncbi.nlm.nih.gov/) (Wang et al. 2017). The 3D structure of AhR (PDB ID: 5NJ8; resolution: 3.3 Å) was downloaded from the PDB (https://www.rcsb.org/). For docking analysis, both protein and molecular files were converted into PDBQT format with all water molecules excluded and polar hydrogen atoms added. The grid box was placed in the center to cover the protein’s domain and accommodate free molecular movement. Molecular docking and visualization were performed by Autodock Vina 1.2.2.

The molecular functional network map of canonical pathways, including physical interactions, co-expression, predicted networks, co-localization, genetic interactions, pathway and shared protein domains of AhR and NF-κB were analyzed using GeneMANIA (http://genemania.org/) (Warde-Farley et al. 2010).

Histological analysis

Knee joints were isolated from rat and fixed with 4% formaldehyde. Calcium in the knee joints was removed using a decalcifying solution for 28 days. At last, the knee joints were embedded in paraffin and coronally sectioned. The sections were stained with safranin O-fast green or hematoxylin & eosin (H&E) and scored according to the Osteoarthritis Research Society International (OARSI) scoring system to determine the extent of cartilage deterioration (McAlindon 2012). Grade 0 was for intact surface and cartilage; Grade 1 for intact surface only; Grade 2 for surface discontinuity, Grade 3 for vertical fissures; Grade 4 for erosion, Grade 5 for denudation and Grade 6 for deformation.

Histopathological assessment of synovitis was evaluated by enlargement of the synovial lining cell layer and density of the cells. The total synovitis scores were assessed as in the previous study (Lewis et al. 2011). Enlargement of the synovial lining cell layer: 0 Point: Thickness 1–2 cells; 1 Point: Thickness 2–4 cells; 2 Points: Thickness 4–9 cells; 3 points: Thickness ≥ 10 cells. Density of the cells: 0 Point: Synovial stroma shows normal cellularity; 1 Point: Cellularity is slightly increased; 2 Points: Cellularity is moderately increased; 3 Points: Cellularity is greatly increased, pannus formation and rheumatoid-like granulomas might occur.

Immunohistochemistry was conducted with anti-IL-6 (1:100, Abmart, PY6087), anti-MMP-3 (1:50, Abcam, ab52915), anti-MMP-13 (1:200, Proteintech, 18165-1), anti-ADAMTS-5 (1:1000, Abmart, TD13268), anti-aggrecan (1:500, Servicebio, GB11373) and anti-collagen-II (1:100, Servicebio, GB11021).

Statistics

All results are presented as means with SD. Shapiro-Wilk normality test was used to perform the normality test. For the data with non-normal distribution, log-transformation was performed to transform the data to normal distribution. For the data with normal distribution, Student’s t-test (paired, two groups, equal variances), Welch’s t-test (paired, two groups, unequal variances) and one-way analysis of variance (ANOVA) followed by Bonferroni’s test (multiple groups) were administrated. For rat total synovitis scores and ACLT OARSI scores, a non-parametric test was applied. Statistical analyses were conducted using SPSS version 25 and data was plotted by GraphPad Prism software version 8.0.2). Differences were considered significant when p < 0.05.

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