Matrix metalloproteinase 9 (MMP-9), a member of MMPs family, is involved in many physiological processes, including cardiovascular diseases (CVD). Tumor necrosis factor-α (TNF-α) is considered a cytokine with pleiotropic biologic capabilities, and led to the process of CVD when TNF-α is abnormally released and stimulates MMP-9 expression and activation. Curcumin and genistein have anti-inflammatory and antioxidant properties, and both are the potential molecules for the prevention and treatment of pathological cardiac hypertrophy and heart failure.
In this study, we investigated the molecular mechanism of curcumin and genistein treatment on the TNF-α-mediated MMP-9 expression. The experimental result confirms that TNF-α could upregulate MMP-9 expression in rat heart myoblast cells H9c2, and this TNF-α-induced MMP-9 expression could be repressed by MEK1/2 specific inhibitor. It is interesting that the curcumin and genistein treatment could inhibit TNF-α-induced MMP-9 expression. To evaluate the MMP-9 regulation at transcriptional level, a DNA fragment of 2.2 kb (-2168/ + 1) of human MMP-9 gene was cloned and constructed into a vector of luciferase reporter gene. The 2.2 kb sequences were identified having three candidate NF-κB binding sites, NF-κB I (-1418/-1409), NF-κB II (-626/-617) and NF-κB III (-353/-345). A series of reporter vectors with the mutated NF-κB sites of MMP-9 promoter sequences were constructed and transfected into H9c2. These constructs were used to investigate the regulating role of NF-κB in the curcumin and genistein suppression on the TNF-α-induced MMP-9 by promoter activity assay.
Our results show that NF-κB II binding site (-626/-617) within the promoter region of MMP-9 gene plays a key role of MMP-9 expression by TNF-α stimulation. Moreover, the inhibitory effect of curcumin and genistein on TNF-α-induced MMP-9 expression correlates with the suppression of MMP-9 promoter activity, and it is associated with NF-κB. The expression of MEK1/2 in the TNF-α-treated H9c2 was significantly decreased when the cells were pretreated with curcumin or genistein. Taken together, we suggest that curcumin and genistein down-regulate TNF-α-induced MMP-9 expression through the inhibition of MEK/ERK signaling pathway and associated with NF-κB transcriptional factor in cardiac myoblast cells H9c2.