Chemicals
Uric acid, sesame oil, and mast cell stabilizer ketotifen were purchased from Sigma-Aldrich (St. Louis, MO).
Animals
Male SPF Sprague–Dawley rats, weighing 200 to 250 g, were obtained from and housed in the Laboratory Animal Center of National Cheng Kung University. Rats were housed individually in a room with a 12-h light/dark cycle and central air conditioning (25°C, 70% humidity). Rats were allowed free access to tap water and were fed a rodent diet from Richmond Standard, PMI Feeds, Inc. (St. Louis, MO). The animal care and experimental protocols were in accordance with nationally approved guidelines. This study was reviewed and approved by the Laboratory Animal Center of National Cheng Kung University in Taiwan (NO. 102122).
Rat air-pouch model
Subcutaneous air pouches were produced under ketamine anesthesia. Twenty-four milliliters sterile air was injected subcutaneously through a 0.25 μm microfilter into the backs of the animals to create a pseudosynovial cavity (Ferrari et al. 1996; Nagase et al. 1998). A second air injection was given 3 days after, if needed, to keep the air pouch inflated.
MSU crystal preparation
Briefly, 8 g of uric acid was dissolved in 1.6 liter of boiling water containing 49 mL of 1 N NaOH. After adjusting the pH value to 7.4, the solution was cooled gradually at room temperature, and then stand overnight at 4°C. MSU crystals were recovered by centrifugation and dried by evaporative drying. Then, MSU crystals were sterilized by heating at 180°C for 2 h before experiments. After sonication, needle-shaped crystals (5–25 μm in length) were checked by using microscopy and then the MSU crystals were ready to used (Seegmiller et al. 1962). A Limulus amebocyte cell lysate assay was used to confirm the absence of endotoxin (less than 0.015 EU/ml) in the crystal preparation (Murakami et al. 2002).
Collecting pouch lavage and counting leukocyte number in lavage
The air pouch lavage was collected by injecting 5 mL of PBS into the pouch then the lavage fluids were slowly instilled and withdrawn three times. Lavage was centrifuged (350 × g for 10 min, 4°C) and the cell pellet was resuspended with red blood cell lysing buffer (Sigma). After washing with PBS, viable cells were counted using trypan blue dye exclusion.
Experimental design
Experiment I
Time course study of MSU crystal-induced acute inflammation in rat air pouches. Six rats were injected with MSU crystal (5 mg/rat, suspended in sterilized phosphate buffered saline, pH 7.4). The leukocyte infiltration was assessed 6 and 12 h after MSU injection.
Experiment II
The therapeutic effect of sesame oil on MSU crystal-induced acute inflammation. Rats were divided into five groups of six: Group I, negative control group; Group II, rats were injected MSU crystal (5 mg/rat) into air pouch; Group III-V, rats were given sesame oil 1, 2, and 4 mL/kg orally 6 h after MSU crystal, respectively. Pro-inflammatory mediators (TNF-α, IL-1β, and IL-6), leukocyte counts, neutrophil counts, and complement protein (C3a and C5a) levels were assessed in air pouch lavage 12 h after MSU crystal injection. In addition, mast cell counts in surrounding skin tissue were also assessed 12 h after MSU crystal injection.
Experiment III
Effects of sesame oil on nuclear factor (NF)-κB activation and IL-4 level in mast cells. Rats were divided into four groups of six: Group I, negative control group; Group II, rats were received sesame oil (4 mL/kg, orally) alone; Group III, rats were injected MSU crystal (5 mg/rat) into air pouch; and Group IV, rats were given sesame oil 6 h after MSU crystal injection. NF-κB activity and IL-4 levels in mast cells isolated from surrounding tissue were assessed 12 h after MSU crystal injection.
Experiment IV
Effect of mast cell stabilizer ketotifen on MSU crystal-induced acute inflammation. Rats were divided into five groups of six: Group I, negative control group; Group II, rats were injected MSU crystal (5 mg/rat) into air pouch; Group III-V, rats were given ketotifen orally (0.1, 1, and 10 mg/kg, respectively) 30 min before MSU crystal was given. A same dose of ketotifen was then given intravenously 15 min before MSU crystal. This dose regimen has been reported to avoid connective tissue mast cell degranulation (Kubes et al. 1993). Leukocyte numbers in pouch lavage were determined 12 h after MSU.
Measuring TNF-α, IL-1β, IL-4, and IL-6 levels in lavage and surrounding skin tissue
TNF-α, IL-1β, IL-4, and IL-6 were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits (DuoSet; R&D System, Minneapolis, MN). Briefly, a 96-well immunoassay plate was coated with 100 μL/well capture-antibody overnight at room temperature. After blocking step, lavage or skin tissue homogenate (tissue:water 1:10 w/v) was loaded into each well (100 μL/well) and were incubated at room temperature for 2 h. One hundred microliter of biotinylated rabbit anti-rat TNF-α, IL-1β, IL-4, and IL-6 antibodies were then added and incubated at room temperature for 2 h. After antibody capturing, streptavidin-conjugated horseradish peroxidase was added and was incubated at room temperature for 20 min. The peroxidase reaction was initiated by adding 100 μL of 3’,3’,5’,5’-tetramethylbenzidine/H2O2 (R&D systems) for 30 min, and then was stopped by adding 50 μL of 0.5 M H2SO4. The absorbance was measured at 450 nm using an enzyme-linked immunosorbent reader.
Leukocyte counts in pouch lavage
The leukocyte count in the lavage fluid was determined manually using a hemocytometer. Erythrocytes are lysed in hypotonic buffer to avoid interfering determination of leukocyte count.
Mast cell counts in inflammatory tissue
Briefly, skin tissue was fixed in 4% formaldehyde buffered with a phosphate solution (0.1 M [pH 7.4]) at room temperature. Tissue fragments were washed in phosphate buffer, dehydrated in graded concentrations of ethanol, and then embedded in paraffin. From each tissue, 4-μm-thin sections were cut, stained with toluidine blue, and examined under a light microscope.
Mast cell isolation
The skin tissue was cut into 5-cm pieces and washed in Hanks' balanced salt solution (HBSS) containing 5% fetal bovine serum (FBS). To remove epithelial cells, the tissue was washed and stirred in HBSS containing ethylenediaminetetraacetic acid (EDTA). Then, skin tissue was cut into 2- to 5-mm pieces and enzymatically digested in HBSS (containing 20% FBS and 1 mg/mL of collagenase) for 90 min at 37°C. After digestion, the cells in supernatant were collected. Mast cells were purified by using Percoll (Pharmacia Fine Chemicals, Uppsala, Sweden). Isotonic Percoll solutions were prepared by dissolving nine parts of Percoll with one part of a ten-fold concentrated Hanks solution. An aliquot of 0.75 mL of the cell suspension in Hanks solution was added to 3.5 mL Percoll isotonic solution, resulting in a final density of 1.110 (Hachisuka et al. 1988). Then, 0.5 mL Hanks buffer was layered on top of the solution and centrifuged at 125 × g for 15 min. The uppermost 2.5 mL were removed. The remaining volume containing the mast cells was washed twice in Hanks solution. More than 90% of the isolated cells were mast cells (Enerbfick & Sevensson 1980).
Assessing NF-κB activity in isolated mast cells
Nuclear protein extraction kit (Sigma) was used to isolate nuclear protein in skin tissue. NF-κB p65 was detected by the chemiluminescent NF-κB assay kits (Thermo Scientific, Inc, Rockford, USA). In brief, nuclear protein was loaded to the 96-well plate and bound to the biotin-Duplex. After incubation, the primary antibody and secondary antibody conjugated with HRP were added. Then chemiluminescent substrate was added and the luminescence was detected by using a luminescence analyzer (Fluoroskan Ascent FL, Thermo Fisher Scientific Inc, Waltham, MA) (Hsu et al. 2013).
Complement system products C3a and C5a in pouch lavage
C3a and C5a levels in lavage were measured by using commercially ELISA kits (Uscn, USA). Briefly, standards or samples are then added to the microtiter plate wells coated with C3a or C5a biotin-conjugated polyclonal antibody. Then, avidin conjugated to horseradish peroxidase and TMB was added. The enzyme-substrate reaction is terminated by the addition of a sulphuric acid and the absorbance was measured spectrophotometrically at 450 nm.
Statistical analysis
Data are means ± standard deviation (SD). Student’s t-test was used to make pairwise comparisons between the treatments in time course study. Group comparisons were done using one-way analysis of variance (ANOVA) and then Tukey’s honestly significant difference (HSD) post-hoc test. Significance was set at P < 0.05.