Interferon γ ifn γ

Manganese chloride tetrahydrate and ammonium ceric nitrate were obtained from Aladdin (Shanghai, China). Polyvinylpyrrolidone (PVP) and N, N-dimethylformamide (DMF, ≥ 99.5%) were obtained from Macklin (Shanghai, China). 2′,7′-Dichlorofluorescin diacetate (DCFH-DA) was provided by Beyotime (Shanghai, China). Dulbecco’s modified eagle medium (DMEM), penicillin, streptomycin, trypsin and cell counting kit-8 (CCK-8) were purchased from Meilunbio (Dalian, China). Foetal bovine serum (FBS) was obtained from Gibco (Guangzhou, China). Anti-CD86, anti-CD206, anti-CD3, anti-CD4, F4/80, and Foxp3 were obtained from BioLegend (San Diego, CA, USA). Lipopolysaccharide (LPS) and Interferon-γ (IFN-γ) were purchased from Sigma-Aldrich (Santa Barbara, CA, USA). The hematoxylin and eosin (HE) staining kit, catalase (CAT) activity assay kit, and superoxide dismutase (SOD) activity assay kit were purchased from Solarbio (Beijing, China). All chemicals and reagents were used as received without additional purification. All solutions were prepared with deionised (DI) water, which was purified through an 18-MΩ system (Millipore, USA).

To induce iNOS expression, subconfluent monolayers were cultured in serum-free medium for 24 h. Growth-arrested cultures were treated with pro-inflammatory cytokines, 100 U/ml interferon γ (IFN-γ) (Sigma-Aldrich, St. Louis, MO, USA), 10 ng/ml interleukin-1 α (IL-1α) (PeproTech, Inc., Rocky Hill, NJ, USA) and 25 ng/ml tumor necrosis factor-α (TNF-α) (R&D Systems, Minneapolis, MN, USA), pro-inflammatory cytokines and 0.1–5 mg/ml water extract of Cnidii Rhizoma or 0.5 mM 1400W (Sigma-Aldrich) in fresh medium without fetal bovine serum. After 48 h, the supernatants were collected and the cells were harvested and lysed as previously described (18 (link)).

To assess the effect of SVF or ADMSCs on macrophages polarization, 1 × 10⁶ ADMSCs or 3 × 10⁶ SVF were seeded on 11-mg TCP granules for 2 h as described above. 1 × 10⁶ THP-1 monocytes were seeded with 250 nM phorbol myristate acetate (PMA; monocytes were activated into macrophages) for 48 h in 24-well plates. These cell-loaded constructs and TCP granules were then transferred into 0.4-mm-pore size Corning transwell inserts (Sigma-Aldrich, Zwijndrecht, the Netherlands) and placed into the plates. Co-cultures were incubated for another 48 h. Wells supplemented with 100 ng/ml lipopolysachariden (LPS; Sigma-Aldrich, St Louis, MO, USA) and 20 ng/ml interferon-γ (IFN-γ; Sigma-Aldrich, St Louis, MO, USA) or 20 ng/ml interlukine-4 (IL-4; Sigma-Aldrich, St Louis, MO, USA) and 20 ng/ml interlukine-13 (IL-13; Sigma-Aldrich, St Louis, MO, USA) were set as control. The secreted tumor necrosis factor α (TNF-α) and transforming growth factor β (TGF-β) in the supernatants were evaluated using ELISA kits (e-Bioscience, San Diego, CA, USA) according to the manufacturer’s instructions.

PSB 777 ammonium salt, 2-Cl-IB-MECA, SCH 58261, and PSB 10 hydrochloride were purchased from Tocris Bioscience (Bristol, UK). Concentrated (10 mM) stock solutions prepared in DMSO were stored at −20 °C. In each experimental session, aliquots of concentrated solutions of compounds were thawed and conveniently diluted in the appropriate experimental solution. Lipopolysaccharides from Escherichia coli O111:B4 (LPS) and Interferon-γ (IFN-γ) were purchased from SigmaAldrich (St. Louis, MO, USA).

In the present study riparins was synthesized from substituted synthetic benzoic acids using methodology previously described by Barbosa-Filho and co-workers [19 ]. Riparin IV (chemical name N-[8′-(4′-methoxyphenylethyl)]-3,4,5-trimethoxybenzoylamide), is a synthetic amide modeled on the natural riparins isolated from Aniba riparina. Dexamethasone, indomethacin, complete Freund’s adjuvant (CFA), lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were obtained from Sigma Chemical Company (St. Louis, MO, USA). Diazepam and morphine were obtained from Cristália (Itapira, SP, Brazil). Riparins were dissolved in 5% Tween 80 in distilled water. indomethacin was dissolved in Tris HCl 0.1 M pH 8.0 plus distilled water. Dexamethasone was dissolved in ethanol 10% in distilled water, and remaining substances were dissolved directly in distilled water. Test compounds were administrated by intraperitoneal (ip) route 40 min before testing, and the control group only received vehicle.