Discussion

In this study, we investigated the effect of curcumin on HASMCs migration and MMP-9 activation induced by TNF-α. Curcumin lowered the secretion and protein expression of MMP-9 by gelatin zymography and Western blot assays. It also decreased nuclear translocation of nuclear factor-κB (NF-κB) P50 and P65. In addition, the migration assay showed that curcumin effectively inhibited the TNF-α-induced migration of HASMCs as compared with the control group. In our in vitro study, we also found that curcumin could scavenge DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals, alkoxyl radical (RO ), and peroxyl radical (ROO ). It is approximately 2-3-folds more potent than Trolox in antioxidative ability (Appendix 1). It also could suppress TNF-α-induced intracellular ROS production.

Curcumin, which is consumed daily by millions of people, is a polyphenol derived from the plant Curcuma longa (Fang et al, 2005). It exhibits a variety of pharmacological effects including anti-tumor, anti-inflammtory, anti-infectious activities and is currently in clinical trials for AIDS patients (Mazumder et al,1995;

Ruby et al, 1995; Surh, 2002). Commercial curcumin usually isolated from the rhizome of Curcuma longa Linn. which contain approximately 77 % of curcumin (Ahsan et al, 1994). Previous study indicated that the serum concentration was 1.77±1.87 μmol/l after 8 g curcumin intake in human (Cheng et al, 2001), therefore, curcumin was absorbable in digestive tract in human. In the present study, we found that 20 μmol/l of curcumin did not have any significant effect on the cytotoxity of HASMCs from MTT test. Therefore, we chose 10 and 20 μmol/l curcumin to do all

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the experiments (Fig. 2-1).

The migration of SMCs from the tunica media to the subendothelial region is a key event in the development and progression of many vascular diseases including atherosclerosis and post-angioplasty restenosis (Maeda et al, 2002). MMPs (MMP-2 and MMP-9) activities may contribute to the pathogenesis of atherosclerosis by facilitating migration of VSMCs (Jones et al, 2003). Although MMP-2 and MMP-9 have similar substrate specificities, the regulation of their expression are different.

MMP-2 is constitutively expressed by several cell types, including SMCs, and its expression is not induced by cytokines or growth factors. In contrast, MMP-9 can be induced by TNF-α in SMCs (Cho et al, 2000; Galis et al, 1994). Therefore, we have investigated the effect of curcumin on the migration of HASMCs and activation of MMP-9. The results indicated that the migration of HASMCs was significantly induced by TNF-α, and suppressed by curcumin (Fig. 2-6). This inhibition against TNF-α-induced migration of HASMCs is consistent with the inhibition of activation and expression of MMP-9 (Fig. 2-2 and Fig. 2-3). A similar result was seen when HASMCs were pretreated with other polyphenolic compound, such as tea flavonoid epigallocatechin-3-gallate (20 μmol/l), quercetin (40 μmol/l), and other flavonoids (Kim et al, 2005; Moon et al, 2003).

The NF-κB family controls the expression of genes involved in the inflammation and immune response (Baeuerle, 1991). In the cytoplasm, inactive NF-κB exists as a heterodimeric complex of subunits p50 and p65 that binds to a cytoplasmic protein, IκB (Baeuerle and Henkel, 1994). Upon activation, IκB is rapidly degraded, and the p50/p65 heterodimer is translocated from the cytoplasm into the nucleus where the dimmer interacts with regulatory κB elements in promoters and

enhancers, thereby controlling gene transcription (Baeuerle and Baltimore, 1988).

NF-κB is activated by a multitude of stimuli, including various inflammatory cytokines and reactive oxygen intermediates (Baeuerle and Baltimore, 1998; Grilli et al, 1993; Muller et al, 1997), which are activated in atherosclerotic lesions (Brand et al,

1996). In the present study, curcumin reduced cytokine-induced nuclear translocation of p50 (Fig. 2-4) and p65 (Fig. 2-5) in HASMCs.

Much further work, which is beyond the scope of the present study, is necessary to elucidate the mechanisms underlying the synergistic regulation of MMP secretion by cytokines. We focused instead on defining the role played by the NF-κB transcription factor in the regulation of MMPs in HASMCs. A functional NF-κB site occurs in the proximal stimulatory region of the MMP-9 promoter (Sato and Seiki, 1993; Fini et al, 1994) and deletion of this site reduces upregulation of reporter gene constructs in response to cytokines. Until now, however it has been unclear what contribution NF-κB plays in the upregulation of the endogenous MMP-9 gene.

Previous study has demonstrated that transient overexpression of IκBα in vascular SMCs only partially impaired upregulation of MMP-9, suggesting that NF-κB simply plays a permissive role in the upregulation of MMP-9 (Bond et al, 2001). In the present study, curcumin reduced cytokine-induced expression of MMP-9 and prevented the nuclear translocation of p50 (Fig. 2-4) and p65 (Fig. 2-5) in HASMCs.

We suggested that inhibitory mechanisms of curcumin might interrupt a signaling cascade involving MMP-9 transcription-mediated activation of NF-κB.

Several studies have indicated that ROS are implicated in the activation of NF-κB (Muller et al, 1997). The current study shows that the ROS production stimulated by TNF-α was decreased by curcumin pretreatment in HASMCs (Fig. 2-7).

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Based on this result, we proposed that the inhibitory effect of curcumin on MMP-9 expression and NF-κB activation may be due to its antioxidant and anti-inflammatory properties. In our in vitro study, we also found that curcumin could scavenge DPPH (2, 2-diphenyl-1-picrylhydrazyl) radicals, alkoxyl radical (RO ), and peroxyl radical (ROO ) (Table 2-1). It is approximately 2-3-folds more potent than Trolox in antioxidative ability (Appendix 1). Since atherosclerosis is a chronic inflammatory disease associated with increased oxidative stress in the VSMCs, it would be conceivable that the anti-atherogenic effects of curcumin might due to its antioxidative and anti-inflammatory properties. The inhibition of cytokine-induced MMP-9 expression has been described for other substances with anti-inflammatory properties such as tea flavonoid epigallocatechin-3-gallate, quercetin, and other flavonoids (Kim et al, 2005; Moon et al, 2003).