4.3.6. Cotton Pellet-Induced Granuloma

Mice (*n* = 6) previously anesthetized with ketamine (80 mg/kg) and xylazine (20 mg/kg) had four cotton pellets (0.01 g each) implanted through a small dorsal incision. Twenty-four hours later, the animals were treated orally with vehicle or NTK (10 mg/kg) for ten consecutive days. On day 11, the animals were euthanized, and the pellets, as well as the surrounding tissue, were removed, dried at 37 ◦C for 24 h, and weighed. The results were expressed as the di fference between the final weight and the initial weight [28].

For total protein quantification, the pellets were placed in test tubes and homogenized with 1 mL of 0.9% saline. The concentration of total proteins was determined using a specific kit (Labtest, Lagoa Santa, Brazil) that is based on the reaction with copper ions in an alkaline medium, creating a violet color complex whose absorbance is proportional to the concentration of proteins in the sample. The readings were performed at 550 nm using a spectrophotometer.

#### *4.4. In Silico Analysis of COX-2 Inhibition*

Docking simulations were carried out for ligand-bound protein complexes, obtained from the Protein Data Bank (PDB, ID. 1PXX and 3RZE). The complexed structures were adjusted using a protein preparation tool provided by the Chimera package, the 3D structures of ligands were obtained using the corina ® 3D structure generator and minimization of energy was achieved using the UCSF Chimera structure build module. The binding region was defined by a 10 Å × 10 Å × 10 Å box set at the centroid of the co-crystallized ligand in the crystal complex to explore a large region of the enzyme structure. The docking analysis was carried out using the UCSF Chimera and AutoDock Vina software based on the Iterated local search global optimizer. Proteins and ligands were maintained flexible during the docking process. The selection of flexible residues from proteins was based on the active site at 4.0 Å from the co-crystallized ligands. The most favorable binding free energy was represented by clustering the positional RMSD results with not more than 1.0 Å. The final docked complexes were analyzed using Discovery Studio 3.1 visualizer [29].
