Figure 5.

Knockout of Nrf2 abolished the protective effects of betulin on inflammatory and oxidant reactions in LPS-stimulated macrophages. (a) Normal (WT) and Nrf2 knockout (Nrf2^−/−) RAW264.7 cells were cultured with betulin (36 μM) for 6 h to detect Nrf2 expression. (b) WT and Nrf2^−/− cells were cultured with or without betulin (36 μM) for 1 h and then challenged with LPS (500 ng/ml) for 18 h; then, the cells were incubated with 50 μM of DCFH-DA for 40 min, and DCF fluorescence intensities were detected by flow cytometry. (c–f), WT cells and Nrf2^−/− cells were cultured with or without betulin (36 μM) for 1 h and then challenged with LPS (500 ng/ml) for 18 h; then, the expression of proinflammatory genes (iNOS and COX-2) and antioxidant enzymes (HO-1 and NQO1) were detected by quantitative real-time PCR. All of the data shown represent the average of three independent experiments. *P≤0.05, **P≤0.01 versus the LPS group; NS, no significant difference