Figure 1.
The AA Metabolic Network in Human PMNs
AA is metabolized through three main pathways: the 5-LOX pathway (red line), the 15-LOX pathway (blue line), and the COX-2 pathway (green line). PGE2, LTA4, and LTB4 are the major inflammatory mediators produced in the COX-2 and 5-LOX pathways. A total of 24 feedback loops, which are involved in the network, make important contributions to the regulation of inflammatory mediators.
Figure 2.
Comparison of the Experimental Data and Computational Results
PMNs (20 × 106 cells/ml) were incubated at 37 °C with 10 μM A23187 (a calcium ionophore) (A) and 10 μM A23187 + 30 μM AA (B), respectively. The calculated results (line without star: red for LTB4 and blue for ω-LTB4) fit well to experimental data [22] (line with star: red for LTB4 and blue for ω-LTB4), considering experimental error.
Figure 3.
Flux Analysis on the Three Main Pathways: The 5-LOX Pathway, the 15-LOX Pathway, and the COX-2 Pathway
Four states were studied: the disease state with no addition of inhibitors (A), the inhibited state with addition of COX-2 inhibitor (B), addition of 5-LOX inhibitor (C), or addition of both COX-2 and 5-LOX inhibitors (D). Red line, 5-LOX pathway; blue line, 15-LOX pathway; and green line, COX-2 pathway.
Figure 4.
Effects of Exogenous AA on the Network Balance
The 1-h output of ω-LTB4 (20-OH-LTB4 + 20-COOH-LTB4) (A) and 15-HETE (B) were calculated to evaluate changes in the 5-LOX pathway and the 15-LOX pathway. To validate the simulation, experiments were performed: PMNs (1.66 × 107 cells/ml) were incubated at 37 °C with 10 μM A23187 (a calcium ionophore) and arachidonic acid of different concentration for 1h. The output of ω-LTB4 (C) and 15-HETE (D) were measured. The y-axis represents the production with exogenous AA normalized against that without exogenous AA.
Figure 5.
The Production of 15-HETE in the Presence of Inhibitors
PMNs (1.66 × 107 cells/ml) were preincubated with inhibitors for 10 min at 37 °C and then were stimulated with 10 μM A23187 (a calcium ionophore) for 1 h at 37 °C. MK886, an inhibitor of 5-LOX, was used to block the production of LTs. CAY10404, an inhibitor of COX-2, was used to block the production of PGs. The y-axis represents the production of 15-HETE with exogenous AA normalized against that without exogenous AA.
Figure 6.
The Effect of the Mixture and the Dual-Functional COX-2/5-LOX Inhibitors
(A) The effect of MR on the efficacy of the mixture of COX-2/5-LOX inhibitors was simulated.
(B) The effect of DR on the efficacy of the dual functional COX-2/5-LOX inhibitor was simulated.
(C) Comparison of the therapeutic effects of these two kinds of inhibitors. The inhibition intensity on the production of LTs and PGs was calculated to evaluate the efficacy of inhibitors, where I (Mix) is the inhibition intensity of the mixture and I (Dual) is the inhibition intensity of the dual functional inhibitor. ER is the relative activity of COX-2 to 5-LOX, which is 0.02 in the current model. CIt is the total concentration of inhibitors while CEt is the total concentration of COX-2 and 5-LOX. DR is the relative inhibition constant to different enzymes (DR = Ki5-LOX / KiCOX-2), and MR is the mixing ratio of the COX-2 inhibitor and the 5-LOX inhibitor (MR = [COX-2] / [5-LOX]). The efficacy of these two types of inhibitors was compared by the value of difference between I(Dual) and I(Mix).