Triple Inhibitory Activity of Cliona celata Against TNF-α-Induced Matrix Metalloproteinase-9 Production Via Downregulated NF-κB and AP-1, Enzyme Activity, and Migration Potential

Abstract

Extracellular matrix-degrading protease, matrix metalloproteinase-9 (MMP-9), is known to be involved in vascular smooth muscle cell (SMC)'s aberrant proliferation and movement in atherosclerotic lesions. During screening of the MMP-9-inhibitory compounds from marine animal resources, we have found that the ethyl acetate extract from Cliona celata (ECC) effectively inhibits the SMC-derived MMP-9 enzyme activity and gene expression. In addition, the ECC effectively repressed the migration potential of the tumor necrosis factor-α (TNF-α)-stimulated human aortic smooth muscle cell (HASMC). As assessed by Western blot analysis, the produced MMP-9 protein levels in the TNF-α-induced HASMC were significantly decreased by the concomitant treatment of ECC at the 50- to 300-μg/mL concentration ranges. In addition, in the RT-PCR experiment, the expressed MMP-9 mRNA levels in the TNF-α-induced HASMC were seemingly decreased by ECC treatment at the same concentration ranges (50-300μg/mL). For the action mechanism(s) of ECC to the phenotype changes in HASMC, we have further evaluated the ECC's pharmacological activities on the signal molecules which are importantly linked in the MMP-9 expression and cell migration potential of HASMC. We have found that extracellular signal-regulated kinase 1 and 2 (ERK1/2) phosphorylation as a target point is suppressed by the ECC treatment in the TNF-α-treated HASMC. Using electrophoretic mobility shift assay, the nuclear extracts purified from ECC-treated HASMCs were shown to decrease the binding potentials on the labeled nuclear factor-kappaB (NF-κB) and activator protein 1 probes. NF-κB p65 and phosphorylated c-Jun contents were also decreased in the purified nuclear extracts from the ECC-treated HASMC, as confirmed by Western blot analysis. Finally, it was shown that the ECC-treated HASMCs were less migrated when compared to the TNF-α-treated cells, as confirmed by HASMC migration assays using the 8-μm pore transwell membranes. From these results, it was proposed that ECC has a potentially applicable anti-atherosclerotic activity.