Effect of curcuminoids on 5-lipoxygenase product formation in activated neutrophils

Human polymorphonuclear leukocytes (PMNL) were pre-treated with DMSO or curcuminoids, 5-LOX product formation was initiated by arachidonic acid (AA, 20 µM) and A23187 (2.5 µM) and 5-LOX products (LTB4, its isomers et-LTB4, t-LTB4, and 5-H(p)ETE) were analyzed by RP-UV-HPLC. 
Raw data including the absolute values (ng) and normalized data (% of control) of all individual experiments was uploaded.
The methods and results were published in Rao et al., Biochem Pharmacol. 2022 Sep:203:115202. doi: 10.1016/j.bcp.2022.115202 
Freshly isolated human PMNL (6 × 106 cells/mL) were resuspended in PBS pH 7.4 with glucose (1 mg/mL) and CaCl2 (1 mM). Cells were first pre-treated with vehicle (DMSO), test compounds or zileuton (3 μM, Cayman Chemical) for 10 min and then co-incubated with AA (20 μM) and calcium ionophore (A23187, 2.5 μM, Merck) for another 10 min at 37 °C. The reaction was terminated with an equal volume of ice-cold methanol containing the internal standard PGB1 (2 ng, Cayman Chemical). Formed lipid mediators were extracted by solid phase extraction using Sep-Pak C18 35 cc Vac Cartridges (Waters), and major 5-LOX products (LTB4, its all-trans isomers and 5-H(p)ETE) were analyzed by RP-UV-HPLC as described for the determination of cell-free 5-LOX activity. The 5-LOX reference inhibitor zileuton (3 μM, Cayman Chemical) suppressed 5-LOX product formation by 50.0 ± 20.5%.