Figure 4

LPS-induced Tnfa expression in macrophages decreases after bezafibrate treatment. (a) RAW 264.7 cells were cultured 24 h with or without 10 ng/ml LPS in the presence or absence of indicated concentrations of bezafibrate. Relative Tnfa expression was then analyzed by realtime PCR. Data represent mean Tnfa expression relative to Gapdh ± SD (each n = 3, *P < 0.05; **P < 0.01; ***P < 0.001 by Student’s t-test). (b) Nine-week-old C57BL/6 mice were treated with bezafibrate or vehicle once daily for eight days. Staple fixation was performed on left hind limbs on day two of drug treatment. On day nine, mice were sacrificed and gastrocnemius muscles were harvested. Specimens were stained with Alexa Fluor 488-conjugated rat anti-mouse F4/80 and rabbit anti-mouse TNFα, followed by Alexa546-conjugated goat anti-Rabbit IgG. Nuclei were stained with DAPI. Sections were observed under a fluorescence microscope and all magnifications are 400x. Scale bar, 100 µm. In merged samples, images shown in the rightmost column are higher magnifications of squared areas in the adjacent image. In the latter, scale bars indicate 50 µm. (c) Shown is the ratio of the number of F4/80/TNFα double-positive cells and the number of DAPI-positive nuclei measured at five randomly selected locations. Data represent mean ratios relative to numbers observed in the vehicle (control) group ± SD (n = 5, *P < 0.05; **P < 0.01; ***P < 0.001 by one-way ANOVA and Tukey post hoc test).