Effect of treatment with the <i>T</i>. <i>aurantium</i> and <i>T</i>. <i>citrina</i> sponge extracts from wild and reared specimens on MMP-2 and MMP-9 expression in astrocytes.

<p>Primary astrocytes (1×10⁵ cells/ml) were activated with LPS and treated with the crude extracts from <i>T</i>. <i>aurantium</i> and <i>T</i>. <i>citrina</i> at the indicated concentrations. Untreated and unstimulated cells represent negative control (CTRL). The isolated RNA samples were analyzed by RT-PCR, using the primer pairs specific for MMP-2, MMP-9 and 18S. 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 the aqueous extracts represented as the percent of control (mean ±SD). Statistically significant inhibiton of MMP mRNA expression in comparison to positive control (LPS) is indicated by asterisks (one way ANOVA followed by Tukey test; * = p<0.05).</p

Effect of treatment with the different sponge extracts on MMP-2 and MMP-9 levels in astrocyte supernatants.

<p>Primary astrocytes (1×10⁵ cells/ml), were activated with LPS (10 μg/ml) and simultaneously treated with each sponge extract at the indicated concentrations. Untreated and unstimulated cells represent negative control (CTRL). Culture supernatants were harvested after 24 h of incubation at 37°C, 5% CO₂ and subjected to gelatin-zymography. 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 in C and D represent results, expressed as mean ± SD, after scanning densitometry and computerized analysis of gels from at least three independent experiments with different cell populations. Asterisks represent values statistically different from LPS-activated astrocytes (positive control) (One-way Anova followed by Tukey test; * = p<0.05; ** p = 0.001).</p

Inhibitory effect of the different sponge extracts on ERK 1/2 signalling pathway in astrocytes.

<p>Primary astrocytes (1×10⁵ cells/ml) were pre-treated for 2 h with the crude extracts from <i>T</i>. <i>aurantium</i> (10 μg/ml), <i>H</i>. <i>perlevis</i> (30 μg/ml), <i>T</i>. <i>citrina</i> (10 μg/ml), <i>I</i>. <i>variabilis</i> (60 μg/ml), <i>A</i>. <i>aerophoba</i> (10 μg/ml), <i>C</i>. <i>nucula</i> (10 μg/ml), and <i>S</i>. <i>spinosulus</i> (60 μg/ml) or with 10 μM of the ERK 1/2 inhibitor PD98059, then activated for 2 h with LPS (10 μg/ml). Untreated and unstimulated cells represent negative control (CTRL). Representative autoradiographic films of Western blotting analysis are reported in <b>A</b> and <b>B</b>. Histograms in <b>C</b> and <b>D</b> represent the results, after densitometric scanning of autoradiographic films, normalized as the ratio of phosphorylated to total ERK protein. Data represent means ± SD of three independent experiments. Asterisks represent values statistically different from positive control (LPS-activated astrocytes), which was set at 100% (one-way ANOVA followed by Tukey test; *p < 0.05).</p

Comparison between the protein profile of the aqueous extracts and the corresponding aqueous and organic phases from the sponges <i>C</i>. <i>nucula</i>, <i>T</i>. <i>aurantium</i> and <i>A</i>. <i>aerophoba</i> and their effect on the release of MMPs from astrocytes.

<p><b>A</b>: Thirty μg of total proteins of the aqueous extract (AE) and aqueous phase (AP) and a corresponding equal volume of organic phase (OP) were subjected to SDS-PAGE on 10% polyacrilamide electophoretic gels. Molecular weight markers (sp) (BioRad) are indicated. <b>B:</b> Representative zymographic gels of supernatants from astrocytes activated with LPS (10 μg/ml) and simultaneously treated with the crude extracts from <i>C</i>. <i>nucula</i> (10 μg/ml), <i>T</i>. <i>aurantium</i> (10 μg/ml), <i>A</i>. <i>aerophoba</i> (10 μg/ml) or with equal amounts of the corresponding aqueous and the organic phases. Untreated and unstimulated cells represent negative control (CTRL).</p

Effect of crude extracts on cell viability.

<p>Primary astrocytes (1×10⁵ cells/ml), were activated with LPS (10 μg/ml) and simultaneously treated with the sponge extract from <i>T</i>. <i>aurantium</i> (Ta), <i>T</i>. <i>citrina</i> (Tc), <i>H</i>. <i>perlevis</i> (Hp), <i>I</i>. <i>variabilis</i> (Iv), <i>C</i>. <i>nucula</i> (Cn), <i>A</i>. <i>aerophoba</i> (Aa) and <i>S</i>. <i>spinosulus</i> (Ss) at the indicated concentrations. After 24 h of incubation at 37°C, 5% CO₂ astrocytes were subjected to the MTT assay. The results are expressed as percentage of surviving cells over untreated cells. Data are presented as mean ± SD of three different experiments with independent cell populations. The horizontal dashed line, set at 60%, indicates the threshold of cell viability. Concentrations of the crude extracts that yielded cell viability values < 60% of control were considered as toxic doses.</p

In-gel inhibition of MMP-2 and MMP-9 activity by sponge extracts.

<p>MMP-2 and MMP-9 standard were applied to gelatin-zymography. After the electrophoretic run in SDS, gels were cut in lanes and each lane was washed with Triton X-100 and incubated overnight in the incubation medium (50 mMTris; 1% Triton-X-100; 10 mM CaCl₂; 0.2% NaN₃; pH 7.5) in the absence or in the presence of the different aqueous extracts at the highest non-cytotoxic concentrations. 1,10 PA was used as a positive control. <b>A</b>: Staining and destaining of the gels revealed that <i>T</i>. <i>citrina</i>, <i>H</i>. <i>perlevis</i>, <i>I</i>. <i>variabilis</i> and <i>S</i>. <i>spinosulus</i> but not <i>T</i>. <i>aurantium</i>, <i>C</i>. <i>nucula</i> and <i>A</i>. <i>aerophoba</i>, were able to inhibit both MMP-2 and MMP-9. <b>B</b>: Percentage of MMP-9 and MMP-2 inhibition of repeated analyses, calculated in comparison to negative control (CTRL) represented by the lane incubated in the absence of the sponge extracts. Asterisks represent values statistically different from control (One-way Anova followed by Tukey test; * = p<0.05).</p

Effect of treatment with the different sponge extracts on MMP-2 and MMP-9 mRNA expression in astrocytes.

<p>Primary astrocytes (1×10⁵ cells/ml) were activated with LPS (10 μg/ml) (positive control) and treated with each aqueous extract at the indicated concentrations. Untreated and unstimulated cells represent negative control (CTRL). After 24 h of incubation at 37°C, 5% CO₂, total RNA was isolated from cells and RNA samples were analyzed by RT-PCR, using the primer pairs specific for MMP-2, MMP-9 and 18S. The products were run on a 1.5% agarose gel containing ethidium bromide. The bands were visualized under UV. Representative results are shown in <b>A</b>. Quantitation of the above experiment and two others after scanning densitometry are shown in <b>B</b>. Positive control MMP-2 and MMP-9 mRNA were set at 100%, and the treatments with the aqueous extracts represented as the percent of control (mean ±SD). Statistically significant inhibition of MMP mRNA expression in comparison to positive control (LPS) is indicated by asterisks (one way ANOVA followed by Tukey test; * = p<0.05).</p