L 161 982

HUVECs were obtained from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. Cells were cultured in RPMI 1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% foetal bovine serum (FBS, HyClone), streptomycin (100 mg/ml; Sigma‐Aldrich, Saint Louis, Missouri, USA) and penicillin (100 units/ml; Sigma‐Aldrich) and maintained at 37 °C under a humidified atmosphere containing 5% CO₂. The cells were seeded into 6‐ or 96‐well plates, stimulated by various drugs for different desired time intervals and then collected for further analysis. The drugs used to stimulate the cells were as follows: SC‐560 (COX‐1 inhibitor); NS‐398 (COX‐2 inhibitor); SC51322 (EP1 antagonist); AH6809 (EP2 antagonist); L798106 (EP3 antagonist); L‐161982 (EP4 antagonist); PD98059 (ERK inhibitor); SB203580 and SB202190 (p38 MAPK inhibitors); SP600125 (JNK inhibitor); Y27632 (ROCK inhibitor); cicaprost, PGE₂, and AA (Cayman Chemical, Ann Harbor, MI, USA); TCDD (Cambridge Isotope Laboratories, Tewksbury, MA, USA).

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).

AlCl₃·6H₂O (Sinopharm Chemical Reagent Co., Ltd., China) and maltol (Aladdin, USA) were of analytical grade. Maltol solution(60 mM) was prepared by adding 0.3784g maltol into 50 ml of autoclaved PBS and 0.1207g AlCl₃·6H₂O was added into 25 ml of autoclaved PBS as AlCl₃ solution(20 mM). Aluminum-maltolate solution (10 mM) was prepared by adding 25ml maltol solution and 25 ml AlCl₃ solution. After filtered through 0.22 mm millipore filter, 60 mM maltol solution and 10 mM aluminum-maltolate solution was stored at 4°C until used [24 (link), 78 (link)]. SC19220, AH6809 and L-161982 (Cayman, USA) were dissolved in the DMSO (Sigma, USA) to be 10 mM reserve liquid. 17-phenyl trinor Prostaglandin E2 ethyl amide and CAY10598 (Cayman, USA) were dissolved in ethyl alcohol to be 10 mM reserve liquid. Butaprost and Sulprostone (Cayman, USA) were dissolved in methyl acetate to be 10 mM reserve liquid [24 (link)].