2.10. Determination of Total COX, COX-1 and COX-2 Activity

Total COX, COX-1, and COX-2 activity in the colonic tissue were established with help of COX Fluorescent Activity Assay Kit (Cayman Chemicals, Ann Arbor, MI, USA), which utilizes the peroxidase component of COXs. The reaction between hydroperoxy endoperoxide (PGG₂) and ADPH (10-acetyl-3,70 dihydroxyphenoxazine) produces highly fluorescent compound: resorufin. Its fluorescence can be easily analyzed with an excitation wavelength between 530–540 nm and an emission wavelength between 585–595 nm. The kit included isozyme-specific inhibitors for distinguishing COX-2 activity from COX-1 activity. Tissue samples were homogenized in cold 50 mM Tris-HCl buffer, pH = 7.5 containing protease inhibitor- Pierce™ Protease and Phosphatase Inhibitor (ThermoScientific, Waltham, MA, USA). Centrifugation of homogenates at 10,000× g for 15 min at 4 °C was subsequently performed. Collected supernatants (put on ice) were assayed following the assay protocol. Fluorescence was measured with an excitation wavelength of 530 nm and an emission wavelength of 590 nm (Victor X Microplate Reader, PerkinElmer, USA). All measurements were performed in duplicate. Total COX activity was demonstrated in units per milligram of wet tissue (1 U equals the amount of enzyme that will cause the formation of 1 nmol of fluorophore per minute at 22 °C). Fluorophore concentrations of the samples were calculated from standard curve using resorufin standard.

For measurements of COX-1 and COX-2, DuP-697- a potent and time-dependent inhibitor of COX-2 was used. The assay followed the same procedure as described above. Total COX Activities of DuP- 697 treated samples were subtracted from the Total COX Activity of their corresponding samples and then divided by the Total COX Activity of the samples and multiplied by 100 to give the percent inhibition. The amount of inhibition corresponded to the amount of COX-2 in the sample. Through subtracting the COX-2 from Total COX we calculated the amount of COX-1, and then presented them as percentage.