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Page 10 of 15 Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05

150

Nitrite (% control) 100

50

0
0 2 20 50
P-aminophenol (µmol/L)

0 µmol/L indomethacin 20 µmol/L indomethacin
2 µmol/L indomethacin 50 µmol/L indomethacin

Figure 8. Effects of p-aminophenol alone or in combination with the non-selective cyclooxygenase inhibitor indomethacin on
lipopolysaccharide (LPS)-induced nitric oxide release. BV-2 cells were treated for 15 min with various concentrations of indomethacin
(2, 20, 50 mmol/L), then incubated with different concentrations (2, 20, 50 mmol/L) of p-aminophenol for a further 15 min period,
before being stimulated with LPS. After 24 h, nitrite concentrations in the BV-2 cell-free supernatants were measured using the Griess
assay. Data from four independent experiments are normalized against nitrite concentration in samples stimulated in the absence of
inhibitors. Nitrite concentration in these samples was 27.1 ± 2.6 mmol/L. The effect of treatments was assessed by the two-way analyses
of variance (ANOVA), followed by Tukey’s post hoc test. **P < 0.01, significantly different from stimulated cells incubated in the absence
of indomethacin (ANOVA F = 8.05, P = 0.0002); P < 0.01, significantly different from stimulated cells incubated in the absence of
##
p-aminophenol (ANOVA F = 95.51, P < 0.0001). ANOVA interaction F = 1.34, P = 0.24

150

Nitrite (% control) 100

50

0
0 2 20 50
AM404 (µmol/L)

0 µmol/L indomethacin 20 µmol/L indomethacin
2 µmol/L indomethacin 50 µmol/L indomethacin

Figure 9. Effects of AM404 alone or in combination with the non-selective cyclooxygenase inhibitor indomethacin on lipopolysaccharide
(LPS)-induced nitric oxide release. BV-2 cells were treated for 15 min with various concentrations of indomethacin (2, 20, 50 mmol/L), then
incubated with different concentrations (2, 20, 50 mmol/L) of AM404 for a further 15 min period, before being stimulated with LPS.
After 24 h, nitrite concentrations in the BV-2 cell-free supernatants were measured using the Griess assay. Data from four independent
experiments are normalized against nitrite concentration in samples stimulated in the absence of inhibitors. Nitrite concentration in
these samples was 26.6 ± 0.7 mmol/L. The effect of treatments was assessed by the two-way analyses of variance (ANOVA), followed by
Tukey’s post hoc test. **P < 0.01, significantly different from stimulated cells incubated in the absence of indomethacin (ANOVA F = 102.3,
##
#
P < 0.0001); P < 0.05 and P < 0.01, significantly different from stimulated cells incubated in the absence of AM404 (ANOVA F = 179,
P < 0.0001). ANOVA interaction F = 1.79, P = 0.10
(0.02, 0.5, 2 µmol/L) of the selective TRPV1 antagonist BCTC to block signaling through this receptor
prior to the addition of p-aminophenol or AM404. If BCTC reduces the inhibitory activity of p-aminophenol
or AM404 on NO secretion from stimulated microglia, it could be concluded that these metabolites
of acetaminophen exert their effect through TRPV1 signaling. Figures 10 and 11 illustrate that both