Figure 1

ROS is a fundamental element during BK-induced MMP-9 expression in RBA-1 cells. (A) Cells were pretreated without or with N-acetylcysteine (NAC, 10 mM) for 1 h before exposure to 10 nM BK for 24 h. The conditioned media were collected for zymographic analysis of MMP-9 expression and activity. (B) Cells were pretreated with or without NAC for 1 h before exposure to BK for 16 h. The total RNA was collected and analyzed by RT-PCR. (C) Cells were incubated with the DCF-DA (5 μM) for 45 min, followed by stimulation with 10 nM BK for 5 min in presence or absence of NAC. The fluorescence intensity of cells was determined. (D) Cells were transiently cotransfected with 0.9 μg of pGL-MMP9-Luc and 0.1 μg of pGal coding for β-galactosidase for overnight. Cells were treated with BK for 16 h in the absence or presence of NAC (10 mM) for 1 h before being harvested for measuring the luciferase and β-galactosidase activities. (E) Immunofluorescence staining for MMP-9 or GFAP in serial sections of the brain cortex tissue from vehicle-treated rat (Sham), BK-injected rat (BK), or NAC-pretreated rat (NAC/BK). The arrow indicates GFAP-positive cells overlapping with MMP-9 expression. DAPI was used to stain the nucleus. Image of fluorescence microscope, 400×. Similarly, the MMP-9 protein and mRNA of brain cortex tissues (Sham, BK, and NAC/BK) were analyzed by Western blot, zymography, and real-time PCR. (F) Rat primary astrocytes were isolated and cultured, and pretreated with or without NAC (1 or 10 mM) before exposure to BK for 24 h. The conditioned media and cell lysates were collected and analyzed by zymography and Western blotting. Data are expressed as the mean ± SEM (n = 3). ^*P < 0.05; ^#P < 0.01, as compared with the respective values of cells stimulated with BK only.