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Slattery et al. Neuroimmunol Neuroinflammation 2018;5:11 I http://dx.doi.org/10.20517/2347-8659.2018.05 Page 9 of 15

150

Nitrite (% control) 100

50

0
0 2 20 50
AM404 (µmol/L)

0 µmol/L SR144528 0.5 µmol/L SR144528
0.02 µmol/L SR144528 2 µmol/L SR144528

Figure 7. Effects of AM404 alone or in combination with the selective CB2 receptor antagonist SR144528 on lipopolysaccharide (LPS)-
induced nitric oxide release. BV-2 cells were treated for 15 min with various concentrations of SR144528 (0.02, 0.5, 2 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 18.9 ± 1.4 mmol/L. The effect of treatments was assessed by the two-way analyses of variance (ANOVA), followed by
Tukey’s post hoc test. No significant differences were observed for stimulated cells incubated in the absence of SR144528 (ANOVA F = 1.56,
##
#
P = 0.21). P < 0.05 and P < 0.01, significantly different from stimulated cells incubated in the absence of AM404 (ANOVA F = 22.23, P
< 0.0001). ANOVA interaction F = 0.45, P = 0.90
Subsequently, we studied whether p-aminophenol and AM404 suppress NO release from activated
microglia through inhibition of COX-1 and COX-2. Involvement of COX was investigated because
previous studies have demonstrated that AM404 modulates NO release as well as inhibits purified COX
isoforms 1 and 2 [23,27] . Furthermore, BV-2 cells express both isoforms of COX and these enzymes participate
[59]
in inflammatory processes involving the AM404 precursor, arachidonic acid . BV-2 cells were exposed
for 15 min to various concentrations (2, 20, 50 µmol/L) of the nonselective COX inhibitor indomethacin
prior to treatment with p-aminophenol or AM404 [Figures 8 and 9]. Previous reports have indicated
[60]
that indomethacin is effective at inhibiting NO production by LPS-stimulated microglia in vitro . We
hypothesized that if p-aminophenol or AM404 lost their ability to inhibit NO release by stimulated
microglia in the presence of indomethacin, it could be concluded that they exert their effect through
COX inhibition. Consistent with the previous studies, indomethacin on its own significantly reduced
[60]
NO production by stimulated BV-2 cells . Both p-aminophenol and AM404 were able to attenuate NO
secretion by stimulated microglia in the presence of indomethacin at all concentrations used. Notably,
p-aminophenol and AM404 caused a greater reduction in NO secretion than indomethacin. While we
cannot completely exclude COX inhibition as one of the mechanisms of p-aminophenol or AM404 inhibitory
activity, it appears not to be the primary mechanism by which these compounds reduce microglial activation.
It is important to note that, unlike other inhibitors used, indomethacin at 50 µmol/L was significantly toxic
to BV-2 cells according to the LDH and MTT cell viability assays (data not shown), which could also be
partially responsible for its inhibitory effect on NO production at this high concentration.

After determining that p-aminophenol and AM404 attenuate microglial activation independently of
COX inhibition, we explored the effects of these compounds on TRPV1 signaling. TRPV1 has been
previously identified as a molecular target of AM404. Signaling through this receptor may contribute to
[23]
the antiallodynic and antihyperalgesic effects of AM404, which are mediated by the NO pathway . As
such, we hypothesized that p-aminophenol and AM404 may reduce NO secretion from stimulated BV-2
[61]
microglia by interacting with TRPV1 . BV-2 cells were treated for 15 min with various concentrations