Fig. 6

AOPPs induced inflammation in BV2 cells through Nox4-ROS-MAPKs-NF-κB signaling pathway. a‑d Effects of AOPPs-MSA on p-p38, p-JNK, and p-ERK at different concentrations after incubation for 24 h. The levels of p-p38, p-JNK, and p-ERK were quantified and normalized with their respective total p38, JNK, or ERK levels. Data are representative of at least 3 independent experiments and the values are presented as mean ± SEM. *P < 0.05 versus control group. e Nuclear translocation of p65/NF-κB subunit was carried out by immunocytochemistry method. BV2 cells were labeled with anti-p65 antibodies (red) and DAPI (blue). Representative images were obtained from three independent experiments. Scale bar, 10 μm. f‑h The expression levels of p-IκB-α, IκB-α, p- p65, p65, and NF-κB p65 in nuclear or cytoplasm were measured by western blotting analysis. GAPDH (cytoplasm) and histone H3 (nuclear) were used as internal controls. i The levels of TNF-α were significantly increased in BV2 cells incubated with AOPPs-MSA. j‑l BV2 cells were pre-incubated with apocynin (100 μM), DPI (10 μM), NAC (5 mM), a p38 inhibitor (SB203580, 10 μM), a JNK inhibitor (SP600125, 100 μM), and a specific NF-κB inhibitor (PDTC, 100 μM) before AOPPs-MSA (200 μg/mL) treatment. The protein levels of TNF-α, p-p38, p-JNK, and GAPDH were measured by western blot analysis. (M) NF-κB p65 antibody conjugated with FITC, and nuclei was labeled with DAPI. Scale bar, 10 μm. Data are representative of at least 3 independent experiments and the values are presented as mean ± SEM. *P < 0.05 versus control group. #P < 0.05 versus AOPPs-MSA group