Figure 6.

Pristimerin attenuates ROS generation, proMMP-9 expression, and cell migration induced by LPS in RBA-1 cells. (A) The cells were incubated with various concentrations of pristimerin (1, 0.5, 0.3, 0.1 μM) for 24 h and then cell viability was measured by an XTT assay kit. (B) Cells were pretreated with or without pristimerin (0.5 µM) for 1 h and then incubated with LPS (2 μg/mL) for 10 min. The fluorescence intensity of DCFH-DA or DHE staining was detected by a fluorescence microscope. The figure represents one of three individual experiments. Scale bar represents 50 µm. (C) Cells were pretreated with or without pristimerin (0.1, 0.3, and 0.5 µM) for 1 h and then exposed to LPS (2 μg/mL) for the indicated time intervals (12 and 24 h). The MMP-9 level was determined by gelatin zymography. The GAPDH level of cell lysates was assayed by Western blot. (D, E) Cells were pretreated with or without pristimerin (0.1, 0.3, and 0.5 μΜ for mRNA expression; 0.5 μΜ for promoter activity) for 1 h and then incubated with LPS (2 μg/mL) of 4 h for mRNA expression or 6 h for promoter activity. The mRNA expression (D) and promoter activity (E) of MMP-9 were determined by real-time PCR and promoter assay, respectively. (F) Cells were pretreated with or without pristimerin (0.5 μM) for 1 h and then incubated with LPS (2 μg/mL) for 48 h. The number of cell migration was determined (magnification = 40×). Data are expressed as mean ± SEM of three independent experiments. # p < 0.01 as compared with the cells exposed to vehicle or LPS, as indicated.

Abbreviations: XTT, 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide; PTM, pristimerin.