Figure 1.
Effect of MVC treatment on MMP-2 and MMP-9 levels in astrocyte culture supernatants.
Primary astrocytes (1×105 cells/ml), incubated in serum-free DMEM, were treated with MVC at the indicated concentrations in the presence of PMA (100 nM) (A) or LPS (10 µg/ml) (B). Negative and positive control supernatants were obtained from unstimulated and untreated astrocytes in serum-free DMEM (control) and cells stimulated with LPS or PMA, respectively. After 24 h of incubation equal amounts of cell culture supernatants were subjected to gelatine zymography as described in the Methods section. Representative gels are reported in (A) and (B). MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kDa, respectively, using pre-stained molecular weight markers (Bio-Rad). Histograms represent results, expressed as mean±SD, after scanning densitometry and computerized analysis of gels, from at least three independent experiments with different cell populations. Asterisk represents values statistically different from positive control (PMA-treated astrocytes) (One-way Anova followed by Student-Newman-Keuls post hoc test; * = p<0.05).
Figure 2.
Effect of MVC treatment on MMP-2 and MMP-9 levels in microglial culture supernatants.
Primary microglial cells (1×105 cells/ml), incubated in serum-free DMEM, were treated with MVC at the indicated concentrations in the presence of PMA (100 nM) or LPS (10 µg/ml). Negative and positive control supernatants were obtained from unstimulated and untreated microglia in serum-free DMEM (control) and cells stimulated with LPS or PMA, respectively. After 24 h of incubation equal amounts of cell culture supernatants were subjected to gelatine zymography as described in the Methods section. Representative gels in A and B show MMP-2 and MMP-9, as identified by their apparent molecular mass of 67 and 92 kDa, respectively, using pre-stained molecular weight markers (Bio Rad). Histograms represent results, expressed as mean±SD, after scanning densitometry and computerized analysis of gels, from at least three independent experiments with different cell populations. No statistically significant inhibition of MMP-2 and MMP-9 in both PMA- and LPS-activated cells was observed. (One-way Anova followed by Student-Newman-Keuls post hoc test).
Figure 3.
Effect of MVC on astrocyte morphological changes and cell viability.
Primary astrocytes (3×104), were activated with PMA 100 nM and simultaneously treated with different concentrations of MVC for 24 h at 37°C, 5%CO2 in serum-free DMEM (Control). (A) Photomicrographs show representative results of three independent experiments. Cell morphology was observed under phase-contrast microscope (Magnification 50×). (B) Cell viability was examined by MTT assay. The results are expressed as percentage of surviving cells over control cells. Data are presented as mean ± SD for three separate experiments with independent cell populations.
Figure 4.
Zymographic analysis of MMP-2 and MMP-9 activity after in vitro incubation with MVC.
Purified standard MMP-2 (0.35 ng) and MMP-9 (0.88 ng) were subjected to gelatin zymography as described in the text. After the electrophoresis, the gel was cut into lanes and each lane was incubated in the absence (control) or in the presence with MVC at the reported concentrations during the development of the zymograms. 1,10 phenantroline (4 mM) (PA) was used as a positive control. A: Staining and destaining of the gels revealed that inhibition of both MMP-2 and MMP-9 activity was observed only in the lane incubated in the presence of PA, but not in the lanes developed in the presence of MVC. B: Percentage of MMP-9 and MMP-2 inhibition of repeated analyses calculated in comparison to control.
Figure 5.
Inhibition of MMP-9 mRNA expression in PMA-activated astrocytes by MVC.
Primary astrocytes (1×105 cells/ml), incubated in serum-free DMEM (Control), were activated with 100 nM PMA (positive control) and simoultaneusly treated with MVC at the indicated concentrations. The isolated RNA samples were analyzed by RT-PCR, using the primer pairs specific for MMP-2, MMP-9 and GAPDH. The products were run on a 1,5% agarose gel containing ethidium bromide. The bands were visualized under UV. Representative results are shown in A. Quantitation of the above experiment and two others after scanning densitometry are shown in B. Positive control MMP-2 and MMP-9 mRNA were set at 100%, and the treatments with MVC represented as percent of positive control (mean± SD). Asterisk indicates statistically significant decrease in comparison to positive control. (One-way Anova followed by Student-Newman-Keuls post hoc test; * = p<0,05).