Figure 1.
Mechanism of inflammatory response via NF-κB activation against bacterial invasion.
Inflammation proceeds mainly via two different routes, which are initiated by TLRs-PAMPs binding (Infection process) or bradykinin-BR interactions (Inflammation process). The former invokes inflammation by producing cytokines as a result of the response. NF-κB activation also increases the densities of other mediators such as TLRs and BRs.
Figure 2.
Testing the effective dose and treatment timing, relative to bacterial stimulation, of sodium salicylate as an anti-inflammatory substance.
Experimental data were obtained by measuring the signals in colorimetric mode, which represented the TLR1 density expressed on the cell surfaces (A549). When stimulated with the P. aeruginosa lysate, expression inhibited by sodium salicylate was most effective at a dose of 50 mM and with pre-incubation of the drug, i.e., pre-incubation in B. Data are shown in mean ± SD (n = 3) and comparisons to the control (i.e., stimulated cells without anti-inflammatory substance treatment) are marked as *** very highly significant (P<0.001), ** highly significant (P<0.01), or * significant (P<0.05). No significance (ns) was indicated otherwise.
Figure 3.
Application of the semi-continuous analytical approach for TLR regulation to test the anti-inflammatory substance effect.
Optimal conditions for the TLR regulation scheme were determined (A) and TLR expression was observed in response to two cyclic repeated bacterial stimulations (B, No treatment). Sodium salicylate was the positive inhibitor (see Figure 2) and an effective dose (50 mM) was used either by co-incubation with the stimulus agent (Co-addition) or by pre-incubation (Pre-incubation). Both treatments revealed inhibition of the TLR response to serial stimulations, suggesting a model for anti-inflammatory substance screening. Each measurement was repeated twice under identical conditions and comparisons to the control (No treatment) were marked as stated in Figure 2 legend.
Figure 4.
Schematic representation of NF-κB inhibition by CAPE and dose response of the cells to the inhibitor.
The inhibition site in the NF-κB activation pathway for CAPE is very different from that of sodium salicylate (A). CAPE effectively suppressed up-regulation of TLR1 density when stimulated with the bacterial lysate, and the degree was proportional to the concentration used (B). This compared with the results of no response to acetaminophen as the negative control. Data were expressed as mean ± SD (n = 4) and each significance was also indicated.
Figure 5.
Simulation of anti-inflammatory agent screening based on standard controls in the repetitive inhibition response curve.
According to the testing scheme (Figure 3A), the stimulation-and-restoration cycle was repetitively maintained three times without chemical treatment (no inhibition) or separately with sodium salicylate treatment (positive inhibition). When two different substances were employed in the inhibition testing, CAPE (90 µM) showed positive anti-inflammatory response (A) when compared with the two control curves, whereas acetaminophen (10 mM) revealed a negative inhibition response (B). All experiments were carried out in duplicate and comparisons to the control (No inhibition) were marked as mentioned in Figure 2 legend.
Figure 6.
Simulation of the drug effect persistency testing using sodium salicylate.
Pre-incubation with sodium salicylate during each cycle caused consistent anti-inflammatory responses to the three repeated bacterial stimulations (“1,2,3-inhibition” as a positive control) compared to that without inhibition (Non-inhibition as negative control). However, when the treatment was skipped at either the second (“1,3-inhibition”) or third cycle (“1,2-inhibition”), the inhibitory effect was not shown at the corresponding stage. This may indicate that drug efficacy persisted for 1 day. Each measurement was repeated twice under the same conditions and the significance was indicated.