Reagents and antibodies
IMP (purity>99%) was purchased from Yuanye (Shanghai, China). MIA and dimethyl sulfoxide were obtained from Sigma (St Louis, USA). Antibodies against NLRP3, ASC, Caspase-1, VEGF, TIMP1, TGF-β1 and HIF-1α were purchased from Abcam (Cambridge, UK). HRP-conjugated affinipure goat anti-rabbit IgG(H+L) (Proteintech Group, Inc., SA00001-2, 1:20000), HRP-conjugated affinipure goat anti-mouse IgG(H+L) (Proteintech Group, Inc., SA00001-1, 1:20000). ECL luminescent liquid (Shanghai Tianneng, 180-5001), protein marker (Shanghai Tianneng, 180-6003), BCA protein assay kit (Thermo Fisher, 23227). bovine serum albumin, Fetal bovine serum (FBS), Dulbecco’s modified eagle’s medium (DMEM), TRIzol and 0.25% trypsin-EDTA were purchased from Gibco (Life Technologies Corp., California, USA). TransStart Green qPCR SuperMix was obtained from Takara (Dalian, China). The primers and rat GAPDH Endogenous Reference were supplied by Sangon Biotech (Shanghai, China). Enzyme linked immunosorbent assays (ELISA) kits for IL-1β and IL-18 were supplied by Invitrogen (Life Technologies Corp., California, USA). All other chemicals were of reagent grade.
Animals
Thirty 3-month-old SD male rats, weight ranging from 250-290 g, 10 for each group (provided by Beijing Vital River Laboratory Animal Technology Co., Ltd.), were used for experimental KOA studies. Animals were maintained in a specific pathogen-free laminar-flow housing apparatus under controlled temperature, humidity, and 12 h light/dark regimen. All animal protocols were approved by the Animal Care and Use Committee of the Nanjing University of Chinese Medicine. All experiments were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals.
Induction of KOA and Drug Administration
The rat osteoarthritis model was made according to a previous literature [6]. Thirty rats were randomly divided into three groups (Normal group, MIA group, MIA+IMP group). For Normal group, injection of 0.9% saline into articular joint was performed; for MIA group, 2 mg MIA dissolved in 50 µl 0.9% saline; for MIA +IMP group, we chose 5 mg/kg/day as oral administration concentration from 2 weeks after injection as previously described [19]. IMP was dissolved in 0.5% carboxymethylcellulose sodium (CMC-Na), and 0.5% CMC-Na was given by intragastric administration alone in sham group and MIA group every day for 6 weeks until the rats were sacrificed. All rats were sacrificed after eight weeks’ post-injection and the synovial tissues were processed for histological analysis and further experiments.
Histopathological analysis
For hematoxylin and eosin (H&E) staining, synovial tissues were frozen and fixed in 4% paraformaldehyde, soaked in 0.5 M EDTA, embedded in paraffin for routine H&E staining and the paraffin blocks were sectioned at a thickness of 5 μm.
Isolation and primary culture of synovial fibroblasts
Primary rat synovial fibroblasts were obtained from additional normal rats. In brief, synovial tissues were washed 2-3 times with phosphate-buffered saline and then minced into pieces of 2-3 mm2 and digested in 0.1% collagenase type II (Sigma Aldrich, St. Louis, MO, USA) for 30 min. Following cell dissociation, the samples were filtered through a cell strainer. After dissociation, fibroblasts were pelleted by centrifugation at 1500 rpm for 4 min and cultured in DMEM supplemented with 10% FBS (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) and antibiotics (100 U/ml penicillin, 100μg/ml streptomycin). Cells were cultured at 37°C in a humidified 95% air and 5% CO2 atmosphere. Passages 3-6 of the synovial fibroblasts were used for the experiments.
Fibroblasts were stimulated with LPS (5 μg/ml) in DMEM for 6 h to stimulate the inflammatory response and activate the NLRP3 inflammasome. The fibroblasts exposed to DMEM with same volume of saline served as control. Before administration of LPS, IMP were used for 24 h or 48 h for continued experiments.
Synovial extraction and preservation in rats
After 14 days of modeling or 14 days of treatment, the rats were sacrificed by CO2 asphyxia method, and the rat knee joint hair was removed. The ligament was incised on both sides of the patellofemoral ligament. The upper edge of the humerus was transversely cut to the distal end of the quadriceps muscle until the femur. The ophthalmologist picked up the free tibia and its surrounding tissue and opened it to the distal end. The pale yellow translucent synovial membrane was seen. The synovial tissue was carefully cut with a surgical blade. Paraformaldehyde was preserved for pathological sectioning, and the rest was placed in a cryotube at -70°C.
Real-time PCR
RNA was isolated from synovial tissues and fibroblasts with Trizol (Invitrogen, CA, USA), respectively. The reverse transcription was performed by using a first strand cDNA synthesis kit (Takara, Otsu, Japan) according to manufacturer’s instructions. qPCR was performed using Premix Ex Taq SYBR-Green PCR (Takara) according to manufacturer’s instructions on an ABI PRISM 7300 (Applied Biosystems, Foster City, CA, USA).
Primer was designed and synthesized by Shanghai Biotechnology Service Co. Ltd. in accordance with the gene sequence in GenBank gene sequence design, together with Oligo v6.6. Sequences for primers shown in Table 1. The mRNA level of individual genes was normalized to GAPDH and calculated by the 2−ΔΔCt method.
Western blotting
Dissect the synovial tissue, weigh and mix with RIPA lysate. The samples were centrifuged at 15,000 r/min for 15 minutes at 4°C. The cultured fibroblasts were washed and lysed. Protein levels were then quantified using the BCA protein assay kit. Protein samples were electrophoresed in SDS-PAGE to separate protein bands. The protein was transferred from the gel to a PVDF membrane and blocked with 5% skimmed milk for 2 hours. PVDF membranes were incubated with polyclonal rabbit antibodies specific for NLRP3, caspase-1, ASC, TGF-β1, VEGF and TIMP-1 overnight at 4°C. The next day, the membrane was incubated with the secondary antibody for 2 hours. The bands were visualized using the ECL method, and ImageJ software was used to quantify the total gray value (average gray value × gray value area) of protein band to calculate the relative value of target protein.
ELISA
Peripheral serum of rats and culture supernatant of cells were respectively collected and centrifuged at 10,000 rpm for 20 min at 4°C, after which the levels of IL-1β and IL-18 were measured by ELISA kits. All steps were performed according to the manufacturer's instructions.
Statistical analysis
Statistical analysis was performed using GraphPad Prism 8.0 Software (San Diego, CA, USA). Data were represented as mean ± standard deviation. Group comparisons were assessed with the one-way ANOVA or student’s t-test for comparison of multiple columns. P < 0.05 (two-tailed) was considered statistically significant.