Skin inflammation induced by topical applications of DNFB
in mice.
DNFB, a known chemical contact sensitizer, is widely used to induce skin
inflammation model. We started recapitulating the mouse chronic model of
contact dermatitis by topical applications of DNFB. As shown in Fig. 1a,
DNFB was topically applied to mouse dorsal skin for three times starting
day 1 (100 μl of 0.5 % DNFB), day 5 (50 μl of 0.2 % DNFB) and day 6
(50 μl of 0.2 % DNFB). Obvious inflammation of dorsal skin started to
develop at day 2 after first application of topical DNFB (Fig. 1b) and
aggravated with increase of skin keratinization following subsequent
applications of DNFB in time-dependent manner (Fig. 1b). H&E staining
of skin tissue sections further revealed a significant increase of skin
thickness and infiltration of inflammatory cells (Fig. 1c). These
results indicate that topical applications of DNFB can cause contact
dermatitis in mice.
Selective activation
of TRPA1 channels by chemical DNFB in
calcium fluorescent
assay and patch-clamp recordings
To test the effect of DNFB (Fig. 2a) on TRPA1 and other thermo-TRPs, we
started using the calcium fluorescent imaging of HEK293 cells expressing
several TRP channels in FlexStation3 microplate reader assay. Adding
different concentrations (3-300 µM) of DNFB caused a dose-dependent
increase of intracellular Ca2+ level, as compared with
TRPA1 agonist AITC (300 µM) as positive control (Fig. 2a). There was a
lack of detectable signals from TRPV1 (Fig. 2b), TRPV3 (Fig. 2c) and
TRPV4 (Fig. 2d) channels in response to DNFB in the same range of
concentration (3-300 µM). These results suggest that DNFB selectively
activates TRPA1 over the other tested members of TRP channels.
To confirm the activation of TRPA1 channels by DNFB, we recorded the
whole-cell currents of TRPA1, TRPV1, TRPV3 and TRPV4 channels expressed
in HEK293 cells in the presence of 5 µM DNFB. As shown in Fig. 3,
DNFB-mediated activation of TRPA1 current was blocked by the channel
blocker ruthenium red (RR) at 20 µM (Fig. 3a). In contrast, TRPV1 (Fig.
3b), TRPV3 (Fig. 3c) and TRPV4 (Fig. 3d) channels were not responsive to
DNFB (5 µM), although these channels were activated by their agonists
such as 1 µM capsaicin for TRPV1, 50 µM 2-APB for TRPV3 and 0.1µM GSK101
for TRPV4. Consistent with the data from calcium fluorescent assay,
these results confirmed that DNFB is a selective agonist of TRPA1.
To determine the potency of DNFB on TRPA1 activation, we made the
whole-cell recordings of TRPA1 currents in the presence of different
concentration of DNFB (0.1-100 µM) and observed a dose-dependent
activation of TRPA1 currents with an EC50 value of 2.36
± 0.26 µM (Fig. 3e left panel and f). As a control, TRPA1 agonist AITC
(3-3000 µM) also elicited a dose-dependent activation of the channel
current with an EC50 value of 17.77 ± 5.02 µM (Fig. 3e,
right panel and f), consistent with a previous report (Cavanaugh et al.,
2008). These results indicate that DNFB activates TRPA1 currents in
dose-dependent manner with approximately 7.5-fold potency better than
AITC.