Figure 4 YDZG promote Nrf2 translocation from the cytoplasm into the nucleus and inhibited ROS generation. (A) RAW264.7 cells pretreated with YDZG (20, 40, 80 μM) for 1 h were stimulated with LPS (1 μg/ml) for 8 h. The ROS level was determined by a ROS kit (Sigma MAK143) according to the manufacturer’s instructions (n=6). (B) RAW264.7 cells treated with for YDZG (20, 40, 80 μM) for 1 h were incubated with LPS for 8 h. Cells were stained with DCFH2-DA (1μM) for 30 min. The fluorescence intensity was determined by flow cytometry (n=5). (C) RAW264.7 cells treated with for YDZG (20, 40, 80 μM) for 1 h were incubated with LPS for 8 h. Cells were stained with MitoSOX Red mitochondrial superoxide indicator (10 μM) for 30 min. The fluorescence intensity was determined by flow cytometry (n=5). (D) RAW264.7 cells treated with for YDZG for 2 h were co-cultured with LPS for 2 h. The protein expression of Nrf2 in cytoplasm and nucleus were detected by Western blotting (n=5). (E) ROS level in cells was stained with DCFH2-DA (10 μM). The images were captured by fluorescence microscopy (scale bar = 100 μm). (F) RAW264.7 cells were pretreated with YDZG (80μM) for 1 h before stimulated with LPS for another 2 h. Nrf2 translocation was determined using the immunofluorescence assay described in the Methods section (n = 3). Primary antibody anti-Nrf2 (1:100) and secondary antibody goat anti-Rabbit Alexa Fluor 488 (1:200) were employed (scale bar = 10 μm). Data are reported as mean ± SEM; *P < 0.05 compared with the LPS alone group.