In vitro activity: Adelmidrol is a diethanolamide derivative of azelaic acid that exerts potent anti-inflammatory effects that are partly dependent on PPARγ. Adelmidrol reduces NF-κB translocation, and COX-2 expression.

Kinase Assay: The levels of Ikb-α, nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), phosphoextracellular signal-regulated kinase (p-ERK), Bcl-2, Bax, lamin a/c, and β-actin were calculated, as previously described, in cytosolic and nuclear fractions from colon tissue collected at the end of the experiment with minor modifications. Colon tissue from each mouse was suspended in extraction buffer A containing 0.2 mM phenylmethylsulfonyl fluoride, 0.15 mM pepstatin A, 20 mM leupeptin, 1 mM sodium orthovanadate, homogenized at the maximum setting for 2 minutes, and centrifuged at 12,000g × rpm for 4 minuteS at 4°C. Supernatants represented the cytosolic fraction. The pellets, containing enriched nuclei, were resuspended in buffer B containing 1% Triton X-100, 150 mM NaCl, 10 mM Tris–HCl pH 7.4, 1 mM EGTA, 1 mM EDTA, 0.2 mM phenylmethylsulfonyl fluoride, 20 mM leupeptin, 0.2 mM sodium orthovanadate. After centrifugation 10 minutes at 12,000 rpm at 4°C, the supernatants containing the nuclear protein were stored at –80°C for further analysis. The filters were blocked with 1× PBS, 5% (w/v) nonfat dried milk for 40 minutes at room temperature, and successively probed with anti-Ikb-α (1/500 in PBS, v/v, Santa Cruz Biotechnology), anti-NF-κB (1/500 in PBS, v/v, Santa Cruz Biotechnology), anti-COX-2 (1/500 in PBS, v/v, Cayman), anti-p-ERK (1/500 in PBS, v/v, Santa Cruz Biotechnology), anti-Bax (SantaCruz Biotechnology 1/500 in PBS, v/v), anti-Bcl-2 (1/500 in PBS, v/v, Santa Cruz Biotechnology), and anti-lamin a/c (1/500 in PBS, v/v, Santa Cruz Biotechnology) in 1× PBS, 5% (w/v) nonfat dried milk, 0.1% Tween-20 (PMT) at 4°C overnight. Membranes were incubated with peroxidase-conjugated bovine anti-mouse IgG secondary antibody or peroxidase-conjugated goat anti-rabbit IgG (1:2000, Jackson Immuno Research, West Grove, PA) for 1 hour at room temperature. To establish that blots were loaded with equivalent amounts of protein lysates, they were similarly incubated with antibody against β-actin (1/1000 in PBS, v/, Santa Cruz Biotechnology v). Relative expression of the protein bands for Ikb-α (37 kDa), NF-κB (65 kDa), COX-2 (72 kDa), p-ERK (46 kDa), Bcl-2 (29 kDa), Bax (23 kDa), lamin a/c (65 kDa), and β-actin (42 kDa) were detected with the enhanced chemiluminescence detection system according to the manufacturer’s instructions (SuperSignal West Pico Chemiluminescent Substrate, Thermo Fisher Scientific, Waltham, MA). Expression of protein bands was calculated by densitometry with Bio-Rad ChemiDoc XRS + software (Hercules, CA) and standardized to β-actin levels. Images of blot signals (8-bit/600-dpi resolution) were imported to an analysis program (Image Quant TL, v2003). Commercially available molecular weight markers (10–250 kDa) were used to establish molecular weight positions.

Myeloperoxidase Assay: Neutrophil infiltration in the colon was examined by determining tissue myeloperoxidase (MPO) activity using a spectrophotometric assay with tetramethylbenzidine as substrate, according to a previously published method. After DNBS injection, colon tissues were collected and weighed. Every piece of tissue was homogenized in a mixture containing 0.5% hexadecyltrimethyl ammonium bromide dissolved in 10 mM potassium phosphate buffer, pH 7, and centrifuged for 30 minutes at 20,000g at 4°C. An aliquot of the supernatant was then allowed to react with a solution of 1.6 mM tetramethylbenzidine and 0.1 mM H2O2. The degree of change in absorbance was measured spectrophotometrically at 650 nm. MPO activity was described as the quantity of enzyme degrading 1 mmol of peroxide per minute at 37°C and expressed in U/g wet tissue.