Curcumin Alleviates oxLDL Induced MMP-9 and EMMPRIN Expression through the Inhibition of NF-κB and MAPK Pathways in Macrophages

Abstract

Rupture of vulnerable atherosclerotic plaques is the leading cause of acute myocardial infarction (AMI) and unstable angina pectoris (UA). However, it still lacks an effective therapy to stabilize the vulnerable atherosclerotic plaques. Numerous reports have shown that upregulation of MMP-9 (matrix metalloproteinase-9) and EMMPRIN (extracellular matrix metalloproteinase inducer) in macrophages is involved in the progression and development of vulnerable plaques. Here we evaluated the impact of curcumin on the expression of MMP-9 and EMMPRIN in macrophages. Macrophages were pretreated with curcumin or specific inhibitors (p38 MAPK inhibitor, NF-κB p65 inhibitor) for 1 h, then cells were cultured with oxLDL for indicated time. Real-time PCR and Western blot analysis were used to evaluate the expression of mRNA and proteins. Translocation of NF-κB p65 was detected by using laser confocal microscopy. Here we showed that curcumin attenuated the MMP-9 and EMMPRIN expression in oxLDL stimulated macrophages. Further studies revealed that curcumin inhibited oxLDL induced NF-κB activation and p38 MAPK phosphorylation. These findings illustrated that curcumin can inhibit the expression of EMMPRIN and MMP-9 in oxLDL stimulated macrophages through down regulation of NF-κB and p38 MAPK signaling pathways, which might be the molecular mechanism for the anti-atherosclerotic effect of curcumin.

Keywords: EMMPRIN; MMP-9; atherosclerosis; curcumin; macrophage; oxLDL.

Figure 1

Curcumin inhibits MMP-9 and EMMPRIN in oxLDL-stimulated macrophages. (A–D) Curcumin suppresses EMMPRIN and MMP-9 expression in oxLDL-stimulated macrophages at dose-dependent manner (6.25, 25, and 50 μM). (E,F) Curcumin reduced MMP-9 and EMMPRIN expression at time-dependent manner (3, 6, and 12 h). Real-time PCR and Western blot were used to measure the mRNA and protein level. ^*P < 0.05 vs. oxLDL group, ^**P < 0.01 vs. CTL group (Control group). CTL group suggest the cells incubated in a medium with DMSO. 6.25, 25, and 50 μM group suggest that indicated concentrations of curcumin were used to pretreated macrophages for 1 h, and then they were stimulated with 50 ug/L oxLDL for another 12 h. 3, 6, and 12 h indicates that macrophage were pretreated with curcumin (25 μM) for different time intervals (3, 6, and 12 h).

Figure 2

Curcumin suppresses the phosphorylation of IκB-α (p-IκB-α) and nuclear translocation of p65 in oxLDL-stimulated macrophages. Macrophages were pretreated with vehicle or curcumin (25 μM) for 1 h, followed by oxLDL (50 uFg/L) for 1, 3, and 6 h. The key protein levels, p-IκB-α in the cytosolic part and NF-κB p65 subunit, were assessed by Western blot analysis. (A,B) Effect of curcumin on phosphorylation of IκB-α in oxLDL-stimulated macrophages at different time points. (C,D) Effect of curcumin on p65 expression (p65 translocation) at different time points. (E,F) Macrophages were pretreated with vehicle, curcumin or p65-specific inhibitor (BAY-11-7082, 20 μM). p65-specific inhibitor significantly decreased MMP-9 and EMMPRIN expression. ^*P < 0.05 when compared with the control group.

Figure 3

Confocal laser scanning microscope observe the effect of curcumin on the translocation of NF-κB p65 in oxLDL-treated macrophages. The Cy3-conjugated secondary antibody was used to analyze the localization of NF-κB p65. Cell nucleus were stained by DAPI. The fluorescence images were observed by confocal laser scanning microscope, Red marked NF-κB p65, light blue represents nuclei with DAPI. Images were merged and a purple fluorescence indicate the areas of co-localization (three independent experiments). THP-1 induced macrophages were pretreated with vehicle or curcumin (25 μM) for 1 h, and exposed to oxLDL (50 ug/L) for 1, 3, and 6 h. It was found a significant translocation of p65 to the cell nucleus after cells were stimulated with oxLDL for 3 h. In treatment groups, cells were pretreated with curcumin (25 μM) for indicated times, NF-κB p65 was retained significantly in the cytoplasm.

Figure 4

Curcumin inhibits p38 and JNK phosphorylation. (A) Macrophages were pretreated with vehicle or curcumin (25 μM) for 1 h, and then treated with 50 μg/ml oxLDL for 10, 20, and 60 min, and assayed by Western blot using indicated antibodies. (B–D) Protein quantification was carried out by densitometric analysis. Normalized proteins of JNK, p-JNK, ERK, p-ERK, p38, and p-p38 were normalized based on the internal control β-actin. (E,F) Macrophages were pretreated with vehicle, curcumin or specific p38 inhibitor (SB203580, 10 μM). The addition of a specific p38 inhibitor efficiently blocks oxLDL-stimulated MMP-9 and EMMPRIN expression. ^*P < 0.05 when compared with the control group, and (NS) P > 0.05 when compared with the control group.