Evaluating Pharmacological Inhibitors on ROS and Profibrotic Proteins

Treatment with NOX-4 inhibitor GKT 137831 was performed at three different concentrations (10, 20, and 30 µM) to explore the effect on ROS production according to the literature (Sedeek et al., 2013a (link)). GKT 137831 was then used at 30 µM for protein expression analysis. Similarly, we tested the effects of PD98059, a potent and selective inhibitor of MAP kinase kinases (MAPKK), MEK1 and MEK2 (Alessi et al., 1995 (link)) at two concentrations (30 and 50 µM) (Gonzalez et al., 2017 (link)), to explore the effects on ROS production and induction of profibrotic proteins mediated by hrPR. NS-398 was used at 10⁻⁵ mol/l (Ferguson et al., 1999 (link)) to determine COX-2 inhibition effect on ROS and profibrotic protein expression. CD cells show high expression of EP4 receptors (Gonzalez et al., 2013 (link); Wang et al., 2016 (link)). We used L-161982 (Cayman Chemical), a potent and selective EP4 receptor antagonist that demonstrates selective binding to human EP4 receptors with a Ki value of 0.024 M. We used a fourfold higher concentration (100 nM) (Takayama et al., 2002 (link)). All pharmacological inhibitors were added 30 min before incubations with hrPR. M-1 CD cells were harvested after 6 h. Controls were performed with vehicle (DMSO, 0.06% vol/vol).