ATO inhibits the activation of TLR4/NF-κB signalling in
macrophages
Previous studies showed that TLR4 promotes the transcription of
inflammatory cytokines and causes plaque instability by activating
downstream signalling pathways (such as the NF-κB pathway) in the
inflammatory site of atherosclerosis (Taket al. , 2001; Hansson et
al. , 2015). Before activation, p65 binds to its inhibitor, IκB-α, in
the cytoplasm. After activation, IκB-α is phosphorylated and the
released p65 is transferred to the nucleus, where it binds to target DNA
and regulates the expression of inflammation-related genes. To determine
whether ATO inhibits TLR4/NF-κB signalling pathway activation, we
extracted the aortic tissue protein from ApoE-/- model
mice after ATO treatment. We observed that ATO significantly reduced the
TLR4 protein levels in the aorta (Figure 4A,D). Consistently, ATO (2.5
and 5.0 μM) significantly reduced the TLR4 protein levels in
ox-LDL-treated RAW264.7 cells (Figure 4B, E). Next, we examined the
intracellular localisation of p65 following treatment with or without
BAY-11, an IκB-α degradation inhibitor, and ATO. The results showed that
in RAW264.7 cells, ATO treatment (2.5 μM) for 4–6 hours significantly
inhibited LPS-induced p65 translocation into the nucleus, which
increased its accumulation in the cytosol (Figure 4C, F, G, I).
Interestingly, BAY-11 (5 μM) treatment for 40 min showed a similar trend
(Figure 5C, F, G,I), indicating that ATO may inhibit IκB-α degradation.
To further evaluate this observation, we examined IκB-α protein
expression in LPS-treated RAW264.7 cells in the presence or absence of
ATO. The results showed that compared with the control group, the
LPS-stimulated groups showed significantly reduced IκB-α levels.
However, treatment with ATO (2.5 μM) for 4–6 h significantly increased
the IκB-α levels in RAW264.7 cells (Figure 5H, J). Taken together, these
data suggest that ATO inhibits TLR4 activation and inhibits nuclear p65
translocation, which may occur through the inhibition of IκB-α
degradation(Figure 5K).