IPT (C16H14O4, Purity ≥ 98%) was obtained from Mansite Bio-Technology Co., Ltd. (Chengdu, Sichuan, China). Recombinant tumor necrosis factor alpha (TNFα) was purchased from Peprotech (PeproTech, Inc., Rocky Hill, NJ, USA). Fetal bovine serum (FBS) and Dulbecco’s modified Eagle’s medium (DMEM) were obtained from Gibco (Gibco BRL, Grand Island, NY, USA). MTS reagents, 5-Ethynyl-2ʹ-deoxyuridine (EdU), lyophilized native chicken type II collagen (CII), Freund’s complete adjuvant (CFA) and DAPI solution were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Annexin V-FITC apoptosis detection kit was obtained from KeyGen Biotech, Co., Ltd. (Nanjing, Jiangsu, China). TRIzol reagent was from Invitrogen (Carlsbad, CA, USA). Antibodies against extracellular signal-regulated kinase (ERK), phosphorylated ERK (p-ERK), p38, phosphorylated p38 (p-p38), c-Jun N-terminal kinase (JNK), phosphorylated JNK (p-JNK) and p65 were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibodies against IκBα and GAPDH were obtained from Santa Cruz Biotechnology (Dallas, CA, USA). Enhanced Chemiluminescence (ECL) solution was purchased from Amersham Pharmacia Biotechnology (Piscataway, NJ, USA)
2.2. Isolation, culture and identification of arthritic FLS cells
Four female Wistar rats (160–180 g) were purchased from Shanghai Slac Laboratory Animal Co. Ltd. (Shanghai, China). A collagen-induced arthritis (CIA) rat model was established as our previous descriptions [18–20]. In brief, the rats were intradermally immunized with 1.5 mg CⅡ emulsified with an equal volume of CFA. Seven days later, a subcutaneous booster was given with half the amount of CⅡ emulsified with Freund’s incomplete adjuvant (IFA). The animal experiment was approval by the Experimental Committee of Nanjing Normal University (SYXK 2020-0047). The knee synovium of rats with CIA that had significant signs of disease was dissected and digested by 0.4% type II collagenase and 0.25% trypsin. The primary synovial cells were cultured in H-DMEM supplemented with 10% FBS (v/v), 100 U∙mL− 1 penicillin and 100 µg∙mL− 1 streptomycin at 37℃ in an atmosphere of 5% CO2. The FLS cells were identified by staining for vascular cell adhesion molecule-1 (VCAM-1), as our previous reports [18, 21]. Cells obtained from the 3 ~ 10th passages were used in the subsequent experiments.
2.3. Cell viability assay
To determine the effect of IPT on the viability of arthritic FLSs, the cells were seeded into 96well plates and subsequently treated with different concentrations of IPT (0, 5, 10, 20, 40, 80 and 160 µM) for 48 h. MTS/PMS complex was then added to each well and incubated for another 4 hours. The absorbance of each well was measured at a wavelength of 490 nm using a microplate reader (Model 680, BioRAD, Hercules, CA, USA).
2.4. Wound healing assay
Arthritic FLSs were cultured into 12-well plates and serum starved overnight. A linear scratch on the cell monolayer was formed using a sterile 200 ul pipette tip. After washing the suspended cell debris with PBS, the cells were pretreated with different concentrations of IPT (0, 1, 2.5, 5 and 10 µM) for 1 h, followed by stimulation with TNFα (50 ng∙mL-1) for 24 h. The effect of IPT on cell migration ability was measured by comparing the remaining cell-free area in the identical fields using ImageJ software.
2.5. Cell proliferation assay
Arthritic FLSs were cultured into 24-well plates, pre-treated with 2.5 µM IPT for 6 h, and then stimulated with or without 50 ng∙mL-1 TNFα for another 24 h. According to the manufacturer’s instructions, the cells were incubated with 10 µM EdU for 6 h and then fixed with methanol. The cell nuclei were stained with Hoechst 33342. The numbers of the proliferating cells and total nucleated cells were counted by Image Plus Pro software. The proliferation rate was calculated according to the follow formula: the proliferation rate = (number of proliferating cells / number of total nucleated cells) × 100%.
2.6. Apoptosis detection by flow cytometry
Apoptosis assay was performed using an Annexin V-FITC apoptosis detection kit according to the manufacturer's instructions. Briefly, arthritic FLSs were treated with different concentrations of IPT (0, 2.5, 5 and 10 µM) for 24 h and then suspended in binding buffer. The cells were stained with Annexin V and propidium iodide (PI) solution. Flow cytometric analysis was performed with FACScan (Becton Dickinson) with the CellQuest program.
2.7. RNA extraction and quantitative real-time PCR
Arthritic FLSs were treated with different doses of IPT (0, 1, 2.5, 5 and 10 µM) for 1 h and then stimulated with TNFα (50 ng∙mL-1) for 24 h. Total RNA was extracted using TRIzol reagent and cDNA was synthesized using random primers and oligdT primers. Quantitative real-time PCR amplification was performed using the following primer sets: IL-1β, 5′-ATGATGGCTTATTACAGTGGCAA-3′ (forward), 5′-GTCGGAGATTCGTAGCTGGA-3′ (reverse); IL-6, 5'-AACCTGAACCTTCCAAAGATGG-3' (forward), 5'-TCTGGCTTGTTCCTCACTACT-3' (reverse); IL-8, 5'-CATACTCCAAACCTTTCCACCCC-3' (forward), 5'- TCAGCCCTCTTCAAAAACTTCTCCA-3' (reverse); TNFα, 5′-ATACACTGGCCCGAGGCAAC-3′ (forward), 5′-CCACATCTCGGATCATGCTTTC-3′ (reverse); β-actin, 5’-CCACACTGTGCCCATCTACG-3' (forward), 5'-AGGATCTTCATGAGGTAGTCAGTCAG-3' (reverse). The PCR reaction conditions were as follows: 95°C denature for 30 s, followed by 95°C 10 s, and 60°C 30 s for 40 cycles. PCR was performed on Mastercycler ep realplex 2 systems (Eppendorf, Hamburg, Germany). The relative expression of each target gene compared with β-actin was calculated using the 2−ΔΔCt method.
2.8. Western blot analysis
Arthritic FLSs were treated with different concentrations of IPT (0, 1, 2.5, 5 and 10 µM ) and then stimulated with or without TNFα (50 ng∙mL-1) for 15 minutes. The cells were lysed with radioimmunoprecipitation assay (RIPA) lysis buffer and the lysate was collected by centrifugation. Protein was separated by SDS-PAGE electrophoresis and transferred to polyvinylidene fluoride (PVDF) membranes. The membranes were incubated with primary antibodies against p-ERK, p-JNK, p-p38, ERK1/2, p38, JNK, IκBα and GAPDH. Immunological responses were detected by ECL solution. Each independent experiment was repeated three times and grayscale was analyzed by ImageJ software.
2.9 Immunofluorescent staining for p65 localization
Arthritic FLS cells were cultured into 24-well plates containing sterile cover slips and treated with IPT (5 µM) for 4 h. After stimulation with TNFa (50 ng·mL− 1) for 30 minutes, the cells on cover slips were washed, fixed and permeabilized with 0.5% Triton-X 100. After blocking with 10% goat serum, the cells were incubated with NF-κB p65 antibody overnight. DAPI solution was used to stain nuclei. The nuclear translocation of p65 was imaged using a Nikon A1R resonance scanning confocal microscope with spectral detector (Nikon, Tokyo, Japan).
2.10. Statistical analysis
All data were expressed as the mean ± SD of results obtained from three or more experiments. Statistical comparisons were performed using one-way ANOVA, followed by Tukey’s post hoc analysis. P < 0.05 was considered statistically significant.