Aprotinin a

For the measurement of cytokine levels, the spinal cord, and sciatic nerve were collected at day 21 after PSL, in mice terminally anesthetized with halothane from each experimental group. The L4-L5 spinal segments and 1 cm sciatic nerve sample containing the lesion site (or comparable region of sham-operated mouse) were removed and rapidly frozen and stored at −80°C. Frozen tissues were later homogenized in ice cold phosphate-buffered saline (PBS; 100 mg tissue/ml) to which 0.4 M NaCl, 0.05% Tween 20, and protease inhibitors (0.1 mM PMSF, 0.1 mM benzethonium chloride, 10 mM EDTA, and 20 KI aprotinin A/100 ml) were added (Sigma). The samples were centrifuged for 10 minutes at 3000g, and a supernatant aliquot was frozen at −80°C for later quantification. Tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-10 (IL-10) levels were estimated using commercially available immunoassay ELISA kits for mice (R&D System, Minneapolis, MN, USA), according to the manufacturer's instructions. The results are expressed as picograms of cytokine per milligram of protein [26 (link)].

Colon fragments (100 mg for each mouse) were homogenized in 1 mL of PBS buffer containing 0.05% tween-20 (Vetec, Rio de Janeiro, Brazil), 0.1 mM phenylmethylsulfonyl fluorid (MP Biomedicals, Solon, Ohio, USA), 0.1 mM benzethonium chloride (Sigma-Aldrich), 10 mM EDTA (ethylenediaminetetraacetic acid) (Synth, Brazil) and 20 KIU aprotinin A (Sigma-Aldrich). Tissues mixtures were centrifuged (3.000× g, 10 min) and supernatants were collected for ELISA immunoassays using DuoSet ELISA kit (R&D Systems, Minneapolis, MN, USA). Plates (Nunc^®, Sigma-Aldrich) were coated with purified monoclonal antibodies anti IL-10, IL-1β, IL-12p70, IL-17, IFN-γ, TGF-β, TNF-α and IL-6, overnight at 4 °C. Plates (Nunc-Immuno Plates, MaxiSorp) were washed by TBS (Tris-buffered saline) and supernatant from homogenized colon tissues were added. Plates then were incubated overnight at 4 °C. After plates washing, biotinylated monoclonal antibodies against different cytokines were added to coated plates and incubated for 2 h at room temperature. The revelation was performed by adding 100 µl/well of a citrate buffer containing Orthophenyldiamine (Sigma-Aldrich) (1 mg/mL) and 0.04% (v/v) H₂O₂. Then, 2N H₂SO₄ solution was added to stop the reaction. The absorbance was measured at 492 nm using an ELISA reader (Bio-Rad, Philadelphia, PA, USA).

Dexamethasone, antagonist of glucocorticoid receptor R486, complete Freund’s adjuvant (CFA), phosphate buffered saline (PBS), Tween 20, phenylmethylsulphonyl fluoride (PMSF), benzamethonium chloride, EDTA, aprotinin A, Dulbecco's Modified Eagle's Medium (DMEM), and 3,3´,5,5´- tetramethylbenzidine (TMB) were obtained from Sigma Chemical Company (St. Louis, MO, USA). Diazepam and morphine were obtained from Cristália (Itapira, SP, Brazil). Dexamethasone was dissolved in ethanol (10% in normal saline). Braylin was dissolved in 50% propylene glycol plus saline, and remaining substances were dissolved directly in saline. Drugs were administrated by intraperitoneal (ip) route 40 minutes before testing, and the control group only received vehicle.

Western blot studies were performed as previously described (12 (link)). Briefly, total extract of L. infantum was obtained after growth of 1 × 10⁸ cells/ml until stationary phase. Cells were lysed using NP-40 lysis buffer supplemented with a protease inhibitor cocktail (2 mM AEBSF, 0.3 µM Aprotinina, 116 µM Bestatina, 14 µM E-64, 1 µM Leupeptina, and 1 mM EDTA; Sigma Adrich). After electrophoresis in a SDS-PAGE gel, samples were transferred to nitrocellulose membrane using the mini transblot electrophoretic transfer cell (Bio-Rad). Next, the membrane was blocked with 1% BSA prior to extensive washing in PBS Tween 0.1% (PBS-T). The blot was then incubated overnight with each respective primary antibody at a dilution of 1:250. Membranes were washed three times with PBS-T for 5 min and incubated with respective secondary antibodies conjugated to Alexa 488 (Santa Cruz Biotechnology). After washing three times with PBS-T for 5 min, the membranes were analyzed in the ChemiDoc MP (Bio-Rad).