Effect of Rosemarinus officinalis L. on MMP-9, MCP-1 levels, and cell migration in RAW 264.7 and smooth muscle cells

Abstract

Atherosclerosis is a chronic and progressive inflammatory disease. Novel anti-inflammatory therapies may have promise as treatment strategies for cardiovascular risk reduction. Rosemary (Rosemarinus officinalis L.) has been used in folk medicine to treat headaches, epilepsy, poor circulation, and many other ailments. It was found that rosemary could act as a stimulant and mild analgesic and could reduce inflammation. However, the mechanisms underlying the anti-inflammatory and antiatherosclerotic effects of rosemary need more study. This study investigated effects of the rosemary components, carnosic acid (CA), and carnosol (CAR), on cell migration. Monocyte chemoattractant protein-1 (MCP-1) and matrix metalloproteinase-9 (MMP-9) were determined by Western blot and gelatin zymography, respectively, in RAW 264.7 macrophages and vascular smooth muscle cells (VSMCs). VSMC migration was assessed by a Matrigel migration assay. Active compounds of rosemary extracts were also analyzed using a reversed-phase high-performance liquid chromatography. MMP-9 and MCP-1 activities were markedly diminished with methanol extract (RM), n-hexane fraction (RH), and CA in RAW 264.7 cells. RM, RH, CA, and CAR suppressed tumor necrosis factor-alpha-induced VSMC migration by inhibiting MMP-9 expression. Chromatograms of RM- and RH-containing CA and CAR revealed higher CA contents of RM (9.4%, 93.85 μg/mg dry wt.) and, especially, RH (18.4%, 184.00 μg/mg dry wt.), which were appreciably elevated compared with the similar CAR content in RM and RH (3.7%, 37.30 μg/mg dry wt.; and 2.5%, 25.05 μg/mg dry wt., respectively). Rosemary, especially its CA component, has potential antiatherosclerosis effects related to cell migration.

FIG. 1.

Effect of RM and RH on LPS-induced MMP-9 secretion and MCP-1 expression in RAW 264.7 cells. Cells were pretreated with various concentrations of RM or RH for 1 h and induced by LPS (100 ng/mL) for an additional 23 h. The supernatants of cell culture were used for gelatin zymography (A, B). Total cell lysates were examined for MCP-1 protein expression (C, D) by Western blot. β-actin was used as an internal control. Data represent the mean±SEM of three independent experiments (n=3). *P<.05, **P<.01, ***P<.001 when compared with the groups treated with LPS (100 ng/mL) alone. CA, carnosic acid; LPS, lipopolysaccharide; MCP, monocyte chemotactic protein; MMP, matrix metalloproteinase; RH, n-hexane fraction; RM, methanol extract; SEM, standard error of the mean.

FIG. 2.

Effect of RM and RH on TNF-α-induced cell migration by inhibiting the MMP-9 activity in VSMC. Cells were treated with the indicated concentrations of RM and RH with the TNF-α (100 ng/mL) for 24 h. The supernatant of VSMC culture was prepared and used for gelatin zymography (black background). The pellets were used for Western blot (white background). β-actin was used as an internal control (A, B). A Matrigel migration assay was carried out with RM and RH with the TNF-α (100 ng/mL). After 12 h incubation, cells on the bottom side of the filter were fixed, stained, and counted (400×) (C). Data represent the mean±SEM of three independent experiments (n=3). *P<.05, **P<.01 when compared with the groups treated with LPS (100 ng/mL) alone. TNF, tumor necrosis factor; VSMC, vascular smooth muscle cell.

FIG. 3.

Effect of CA and CAR on LPS-induced MMP-9 secretion and MCP-1 expression in RAW 264.7 cells. Cells were pretreated with various concentrations of RM or RH for 1 h and induced by LPS (100 ng/mL) for an additional 23 h. The supernatants of cell culture were used for gelatin zymography (A, B). Total cell lysates were examined for MCP-1 protein expression (C, D) by Western blot. β-actin was used as an internal control. Data represent the mean±SEM of three independent experiments (n=3). **P<.01 when compared with the groups treated with LPS (100 ng/mL) alone. CAR, carnosol.

FIG. 4.

Effect of CA and CAR on TNF-α-induced cell migration by inhibiting the MMP-9 activity in VSMC. Cells were treated with the indicated concentrations of RM and RH with the TNF-α (100 ng/mL) for 24 h. The supernatant of VSMC culture was prepared and used for gelatin zymography (black background). The pellets were used for Western blot (white background). β-actin was used as an internal control (A, B). A Matrigel migration assay was carried out with RM and RH with the TNF-α (100 ng/mL). After 12 h incubation, cells on the bottom side of the filter were fixed, stained, and counted (400×) (C). Data represent the mean±SEM of three independent experiments (n=3). *P<.05, **P<.01 when compared with the groups treated with LPS (100 ng/mL) alone.