Adult male Sprague Dawley (SD) rats (6 weeks old, 180–220 g) were purchased from Shanghai SIPPR-Bk Lab Animal Co., Ltd. and maintained in specific pathogen-free laboratory animal facilities of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. All rats were acclimated to the facility for seven days before in vivo experiments. All animal experiments complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were reviewed and approved by the Institution of Animal Care and Use Committee (IACUC) of the Ninth People's Hospital Affiliated to Shanghai Jiao Tong University School of Medicine. All animals were housed in individual cages under a 12-h light/dark cycle at room temperature (23 ± 1 °C) with free access to food and water.
Establishment of rat PTJC model
The number of surgically treated animals was calculated as 6 per experimental group. The PTJC model was established based on previous research . Briefly, rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg), placed in supine position, and prepared for surgery under aseptic conditions. The fur was shaved from the surgical site of the right knee joint, and the skin was disinfected with chlorhexidine. A 15-mm midline skin incision was made, and a lateral parapatellar arthrotomy was performed. The patella was reflected medially and the femoral condyles were exposed. Two cortical windows 1.5-mm in diameter were made from the non-articulating cartilaginous regions of the medial and lateral femoral condyles using a 1.5-mm drill bit. The anterior/posterior cruciate ligament was sequentially incised and the knee joint hyperextended to − 45° to disrupt the posterior capsule. The right knee joint was immobilized at approximately 135° of flexion with a 0.5-mm steel wire. The muscles and skin were sutured with silk threads after the patellofemoral joint reduction. For drug delivery, 10 µL of 100 mM 4-IPP (TOCRIS, Bristol, UK) or vehicle (normal saline) was injected into the joint cavity. After the surgery, the rats received sodium salicylate (150 mg/kg) for pain control, and unrestricted daily activity in cages was permitted. Rats were euthanized at 0, 1, 3, 7, and 14 days, the internal fixation was removed, and the range of motion (ROM) of the knee joint extension was measured using a mechanical goniometer (arthrometer) after myotomies of the trans-articular muscles, as has been described previously . Briefly, the lateral femoral condyle acted as the center of rotation and was pinned, while the proximal femur and distal tibia were attached to the two arms of the arthrometer. During motion, the two femoral points were fixed while the distal tibia could accommodate for translation at the knee joint. The reproducibility to repeated measures was within ± 4°. Conditions of temperature (21 °C) and angular velocity (3°/sec) were standard. ROM measurements in flexion and extension were performed at the torques of 667 g/cm. All measurements were made within 15 min after euthanasia. Posterior joint capsules were collected after ROM measurement for subsequent assays.
Primary joint capsule fibroblasts culture
To isolate primary joint capsule fibroblasts, SD rats (male, 4 weeks old) were sacrificed by an anesthesia overdose and disinfected in 75% alcohol for 10 min. Posterior joint capsule tissues were washed with minimum essential medium (MEM; Invitrogen, Carlsbad, CA, USA). Sub-sectioned tissues were placed in tissue culture plates containing Dulbecco's minimum essential medium (DMEM; HyClone, Logan, USA) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin (Gibco), and 100 µg/mL streptomycin (Gibco) in a 5% CO2 incubator at 37 °C to obtain joint capsule fibroblasts . After 3–5 days, fibroblasts started to migrate from the sub-sections. The tissues were removed when the culture reached approximately 90% confluence. The medium was changed every two days. Primary joint capsule fibroblasts were cultured to passages 2 to 4 and identified by immunofluorescence staining with vimentin (Abcam, Cambridge, UK, 1:1000) before used in subsequent experiments.
Samples of the posterior joint capsule collected at 0, 1, 3, 7, and 14 days following injury or primary joint capsule fibroblasts treated with various drugs were lysed in RIPA buffer (Beyotime, Shanghai, China) supplemented with 1 mM PMSF (Beyotime). Protein concentration was detected using the Bradford method to ensure equal loading. Western blots were performed as described previously . The relative intensities of protein bands were normalized to those of β-actin. The following antibodies were used: β-actin (ProteinTech, Wuhan, China, 1:5000), MIF (Abcam, 1:1000), TGF-β1 (Abcam, 1:1000), α-smooth muscle actin (α-SMA, Abcam, 1:1000), Collagen Ⅰ (Abcam, 1:1000), CD74 (Santa Cruz, CA, 1:100), p-ERK/ERK (Cell Signaling Technology, Danvers, MA, 1:1000), p-P38/P38 (Cell Signaling Technology, 1:1000), p-JNK/JNK (Cell Signaling Technology, 1:1000). Secondary antibodies included goat anti-rabbit IgG or goat anti-mouse IgG (Invitrogen, 1:30000).
Cell lysates were harvested and centrifuged at 14,000 rpm for 20 min to remove the debris after primary joint capsule fibroblast treatment with 2 µg/mL MIF (ProSpec, USA) for 24 h. Protein concentration in the supernatant was quantified using the Protein Assay Kit II (Bio-Rad, Inc., California, USA). For immunoprecipitation analysis, total cell lysates (500 µg) were precleared with protein A plus G-Sepharose (Beyotime) before incubation with specific antibodies at 4 °C, followed by addition of protein A plus G-Sepharose. After several washes, samples were boiled and analyzed by immunoblotting using anti-MIF or anti-CD74 antibody.
RNA from different groups of primary joint capsule fibroblasts were extracted using TRIzol (Invitrogen) according to the manufacturer's instructions. Next, 1 µg of total RNA per sample was used to synthesize cDNA using the Omniscript reverse transcription kit (QIAGEN, Dusseldorf, Germany), and qPCR was carried out using SYBR® Premix Ex Taq™ (Takara Bio, Dalian, China) on a real-time PCR system (Applied Biosystems). We normalized gene expression to Gapdh levels and calculated the relative levels using 2−ΔΔCt. All reactions were performed in triplicates. The primers were designed and synthesized by Sangon Biotech (Shanghai, China) as follows: Gapdh: forward primer 5ʹ-ACA GCA ACA GGG TGG TGG AC-3ʹ, reverse primer 5ʹ-TTT GAG GGT GCA GCG AAC TT-3ʹ; Mif: forward primer 5ʹ-CTT GGG TCA CAC CGC ACT TA-3ʹ, reverse primer 5ʹ-TCG CTC GTG CCA CTA AAA GT-3ʹ; Cd74: forward primer 5ʹ-CAT CGG GCT CAC AGG TTT GG-3ʹ, reverse primer 5ʹ-CTG GTG GCT CTG CTC TTG GC-3ʹ; Tgf-β1: forward primer 5ʹ-AGC AAC AAT TCC TGG CGT TAC-3ʹ, reverse primer 5ʹ-TGT ATT CCG TCT CCT TGG TTC A-3ʹ.
Following treatment with 2 µg/mL MIF in the presence or absence of 50 µM 4-IPP for 24 h, primary joint capsule fibroblasts were fixed in 4% paraformaldehyde with 0.1% Triton X-100 (Sigma, St Louis, MO, USA) and subsequently incubated with 4% goat serum to block nonspecific binding. Then, cells were incubated with anti-TGF-β1 (Servicebio, Wuhan, China, 1:100) primary antibodies overnight. Fluorescent secondary antibodies of FITC-labeled goat anti-rabbit IgG (Sigma, 1:400) were used to visualize the corresponding subsets. Cells were then stained with 4,6-diamidino-2-phenylindole (DAPI; Sigma, 1:4000) and phalloidin (Abcam, 1:1000), followed by observation under a confocal fluorescence microscope (Leica, Germany).
Knee joint samples were collected from experimental models at different time points, fixed in 4% paraformaldehyde, decalcified in 10% EDTA, and then embedded in paraffin. The knee joint specimens were sagittally sectioned (5 µm) and processed with hematoxylin-eosin (HE) and Masson trichrome staining. For immunohistochemical staining, the sections were co-incubated with antibodies against MIF (Abcam, 1:100), TGF-β1 (Servicebio, 1:100), and vimentin (Abcam, 1:1000). The sections were further incubated with FITC-labeled goat anti-mouse IgG (Gibco, 1:400), Cy3-labeled goat anti-rabbit IgG (Sigma, 1:400), and DAPI (Sigma, 1:4000). Zeiss LSM710 confocal microscope was used for confocal imaging of samples at an original magnification × 40. Confocal images were prepared with Zen software. All images were taken from similar areas of the posterior joint capsule.Three sequential specimens in each group were measured.
Cell viability assay
Primary joint capsule fibroblasts were seeded at a density of 2 × 104 cells/well. Cells were treated with different concentrations of MIF (0–2.5 µg/mL) or TGF-β1 (ImmunoClone, Houston, USA, 0–100 ng/mL) for 24 h. Then, 10 µL of Cell Counting Kit-8 (CCK-8; Dojindo, Kumamoto, Japan) was added to the cells and incubated for an additional 2 h at 37 °C. Cell viability was measured spectrophotometrically at 450 nm. Assays were performed three times using triplicate wells.
Sequencing of mRNA and bioinformatics analysis
Total RNA of primary joint capsule fibroblasts following treatment with 2 µg/mL MIF for 24 h or 48 h was extracted using the mirVana miRNA Isolation Kit (Ambion, Texas, USA) and then selected by RNA Purification Beads (Illumina, California, USA) for library construction and RNA-seq analysis. The library was constructed using the Illumina TruSeq RNA Sample Prep Kit v2 and sequenced by the Illumina HiSeq-2000 for 50 cycles. High-quality reads that passed the Illumina quality filters were used for sequence analysis. Sequencing outcomes were normalized with Reads Per Kilobase per Million mapped reads (RPKM). Differentially expressed genes (DEGs) were designated according to criteria of fold change > 2 and false discovery rate < 0.05 compared to the control. Gene functions were annotated by Blastx against the NCBI database or the AGRIS database with an E-value threshold of 10− 5. Gene ontology (GO) classification was obtained by WEGO via GO id annotated using the Perl and R programs. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways were assigned to the sequences using the KEGG Automatic Annotation Server (KAAS) online.
All results were expressed as the mean ± standard deviation after analysis by the SPSS 22.0 statistical software (SPSS Inc., Chicago, IL, USA). Parametric data were analyzed by Student's t-test or one-way analysis of variance (ANOVA) followed by post-hoc Tukey's test to compare two groups. A level of p < 0.05 was considered statistically significant.