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).