Lichens have been known to possess multiple biological activities, including anti-proliferative and anti-inflammatory activities. Vascular cell adhesion molecule-1 (VCAM-1) may play a role in the development of atherosclerosis. Hence, VCAM-1 is a possible therapeutic target in the treatment of the inflammatory disease. However, the effect of lobaric acid on VCAM-1 has not yet been investigated and characterized. For this study, we examined the effect of lobaric acid on the inhibition of VCAM-1 in tumor necrosis factor-alpha (TNF-α)-stimulated mouse vascular smooth muscle cells. Western blot and ELISA showed that the increased expression of VCAM-1 by TNF-α was significantly suppressed by the pre-treatment of lobaric acid (0.1?10 μg/ml) for 2 h. Lobaric acid abrogated TNF-α-induced NF-κB activity through preventing the degradation of IκB and phosphorylation of extracellular signal-regulated kinases (ERK), c-Jun N-terminal kinases (JNK), and p38 mitogen activated protein (MAP) kinase. Lobaric acid also inhibited the expression of TNF-α receptor 1 (TNF-R1). Overall, our results suggest that lobaric acid inhibited VCAM-1 expression through the inhibition of p38, ERK, JNK and NF-κB signaling pathways, and downregulation of TNF-R1 expression. Therefore, it is implicated that lobaric acid may suppress inflammation by altering the physiology of the atherosclerotic lesion.
Atherosclerosis is a chronic inflammatory disease, characterized by the accumulation of lipids and fibrous elements in the large arteries. Vascular smooth muscle cells (VSMCs) plays a major role in early phase of atherosclerosis (Lusis, 2000; Owens
Lobaric acid, an ingredient of the lichen
In the present study, we therefore examine the influence of lobaric acid on the expression of adhesion molecule to cultured mouse vascular smooth muscle cells. The results suggest that lobaric acid inhibits TNF-α-induced VCAM-1 expression through the inhibition of TNF-α receptor expression, and MAPK and NF-κB signaling pathways.
Unless otherwise indicated, all the chemicals used in this study were purchased from Sigma Chemical Co. (St Louis, MO, USA). Anti-VCAM-1 antibody was purchased from R&D Systems, USA. Lipofectamine Plus, DMEM medium, and fetal bovine serum were purchased from Life Technologies, Inc. (Carlsbad, CA, USA). The reporter plasmid pGL3-NF-κB used in the luciferase assay system was obtained from Promega (Madison, WI, USA), and pCMV-β-gal was obtained from Lonza (Walkersville, MD, USA). 3-amino-1,2,4-triazole was purchased from Calbiochem (La Jolla, CA, USA). Antibodies against IκB-α, p65, JNKs, phospho-JNK (p-JNK), ERK, phos-pho-ERK (p-ERK), phospho-38, p38, lamin A, TNF-α receptor 1 and β-actin were purchased from Abcam Inc, USA. Lobaric acid was acquired from Korea Polar Research Institute., Korea.
The extraction of lobaric acid was performed using a modification of the technique described previously (Ingolfsdottir
The vascular smooth muscle cell line (MOVAS-1) that has been utilized in various vascular studies (Charlmers
MOVAS-1 cells were seeded at a concentration of 5×104 cells/well in 96-well tissue culture plates (Nunc, Denmark) and incubated with different concentrations of lobaric acid (0.01, 0.1, 1, 10, 100 μg/ml) for 24 h. After treatment, cell proliferation was assessed by incubating the cells with 25 μg/ml of MTT (Sigma, St. Louis, MO, USA) for another 4 h. Then, the MTT-formazan produced by viable cells was dissolved in dimethyl sulfoxide (DMSO) and a Molecular Device microplate reader (Menlo Park, CA, USA) was used to measure absorbance at 560 nm. The blank control only contained cell culture medium and the absorbance of untreated cultures was set at 100%. At least three independent experiments were performed.
The cell surface expression of the adhesion molecules on the muscle cell monolayers was quantified by ELISA using a modification of the methods described previously (Mo
Cells (5×105 cells/well) were plated into each well of a 6-well plate. The cells were transiently co-transfected with the plasmids, pGL3-NF-κB and pCMV-β-gal, using LipofectAMINE Plus according to the manufacturer’s protocol. Briefly, a transfection mixture containing 0.5 μg pGL3-NF-κB and 0.2 μg pCMV-β-gal was mixed with the Lipofectamine plus reagent and added to the cells. After 4 h, the cells were pretreated with lobaric acid for 2 h followed by the addition of TNF-α for 4 h, and then lysed with 200 μl of lysis buffer (24 mM Tris-HCl (pH 7.8), 2 mM dithiothreitol, 2 mM EDTA, 10% glycerol, and 1% Triton X-100). Ten microliters of cell lysates were used for the luciferase activity assay. The luciferase and β-galactosidase activities were determined. The values shown represent an average of three independent transfections, which were normalized with β-galactosidase activity. Each transfection was carried out in triplicate and experiments were repeated three times.
Western blot analysis was performed by a modification of the technique described previously (Cho
The total RNA was extracted using a single-step guanidinium thiocyanate-phenol-chloroform method. The yield and purity of the RNA were confirmed by measuring the ratio of the absorbances at 260 and 280 nm. Quantitative RT-PCR was performed using VCAM-1-specific primers to identify cDNA. cDNA was amplified in 20 μl of PCR (8 μl of cDNA solution in water, 1 μl of forward primer and reverse primer, and 10 μl of PowerSYBR Green PCR Master Mix) in a quantitative Real-time PCR System, and fluorescence was monitored at each cycle. The VCAM-1 RT-PCR primers were 5′-CTCAGGTG-GCTGCACAAGTT-3′ (forward primer) and 5′-AGAGCTCAA-CACAAGCGTGG-3′(reverse primer). The GAPDH RT-PCR primers were 5′-TGCATCCTGCACCAA-3′ (forward primer) and 5′-TCCACGATGCCATTG-3′ (reverse primer).
MOVAS cells, grown on 22-mm diameter glass coverslips were pretreated with lobaric acid (10 μg/ml) for 2 h and incubated with fresh growth medium containing TNF-α (10 ng/ml) for 4 h. Cells were washed in PBS and fixed with 4% paraformaldehyde for 15 min at room temperature. The fixed cells were permeabilized with 0.5% Triton-X 100 in PBS for 10 min and blocked with a 5% bovine serum albumin in PBS. The anti-NF-κB p65 antibody was diluted 1:1000 and incubated overnight at 4°C. The cells were then incubated with Alexa 594-conjugated anti-rabbit antibody (red channel) for 1 h in 1% BSA/0.05% Triton X-100/PBS. Cells were washed twice with permeabilization buffer, incubated for 5 min in a Hoechst 33342-containing PBS solution, and washed with PBS. Coverslips were mounted to glass slides using ProLong Gold antifade agent and photographed with a confocal microscope (LSM 510 META; Carl Zeiss).
Results are represented as means ± S.E.M. All experiments were performed at least three times. For comparisons between two groups, the Student’s
We examined the anti-proliferative effect of lobaric acid on mouse vascular smooth muscle cell line, MOVAS-1 cells, by exposing them to lobaric acid for 24 h. Cell proliferation was determined by MTT assay. When MOVAS cells were exposed to lobaric acid (0.01?100 μg/ml), cell growth was inhibited at a concentration of 100 μg/ml (Fig. 2). Thus, the concentration selection for the present experiments was based on the cell proliferation results. In subsequent experiments, cells were treated with lobaric acid at concentrations of 0.1, 1, and 10 μg/ml.
This experiment was conducted to determine the effect of lobaric acid on the expression of TNF-α-induced adhesion molecules. VSMCs were pretreated with or without various concentrations of lobaric acid for 2 h, followed by treatment with TNF-α (10 ng/ml) for 8 h. As detected by ELISA, pretreatment with lobaric acid significantly suppressed cell surface expression of TNF-α-induced VCAM-1 in a concentration-dependent fashion (Fig. 3A). Additionally, the expression of total cellular adhesion molecule on VSMCs in response to TNF-α stimulation and lobaric acid treatment was examined by Western blot analysis. The results showed a similar pattern of inhibition by lobaric acid at total cellular protein level (Fig. 3B). Thus, these results strongly suggest that lobaric acid is effective in blocking the expression of VCAM-1 induced by TNF-α.
Next, we determined if lobaric acid interferes with the expression of TNF-α-induced adhesion molecules at the transcriptional level. To examine gene transcription, total cellular RNA was isolated from VSMCs and analyzed by real time-PCR. VSMCs were pretreated with various concentrations of lobaric acid for 2 h, followed by treatment with TNF-α for 4 h (Fig. 3C). Lobaric acid concentration-dependently attenuated VCAM-1 mRNA expression, suggesting that the effect of lobaric acid on TNF-α-induced VCAM-1 mRNA expression occurs at the level of RNA.
Because NF-κB activation in the inflammatory response may trigger upregulation of adhesion molecule, we examined the effect of lobaric acid on NF-κB transcriptional activation using Luciferase reporter assays. Cells were pretreated with various concentrations of lobaric acid for 2 h before stimulation with TNF-α for 4 h. Stimulation of the cells with TNF-α resulted in an approximately 2-fold increase in luciferase activity, and this increase was considerably suppressed by lobaric acid at 10 μg/ml (Fig. 4A). We also examined the effect of lobaric acid on the expression of p65 NF-κB protein (Fig. 4B). As shown in Fig. 4B, pre-incubation of VSMCs with lobaric acid decreased the nuclear translocation of p65 NF-κB. These data indicate that lobaric acid inhibits TNF-α-induced nuclear translocation of NF-κB.
To examine whether lobaric acid affects TNF-α-induced degradation of IκBα, the expression of IκBα protein was determined by Western blot assay (Fig. 4C). Stimulation with TNF-α significantly degraded IκBα at 45 min as compared to untreated control cells, but TNF-α-induced cells pretreated with lobaric acid failed to degrade IκBα. Consistent with the protein expression, a significant inhibitory effect of lobaric acid on the TNF-α-induced NF-κB p65 nuclear translocation determined by immunofluorescence assay was observed (Fig. 4D). These results further demonstrate that lobaric acid inhibits TNF-α-induced NF-κB activation. Collectively, these results suggest that lobaric acid inhibits TNF-α-induced VCAM-1 expression by blocking the activation of NF-κB.
Many studies have demonstrated that MAPK plays a role in the induction of adhesion molecules by TNF-α (Ho
Since it has been known that TNF-α signaling through TNF-R1 could contributes to arterial inflammation and induction of VCAM-1 expression (Sawa
It has been reported that lichens synthesize and accumulate photoprotective compounds against UV radiation-induced damage in the photobiont (Hidalgo
Cytokines in the atherosclerosis could contribute to the expression of adhesion molecules (Huo and Ley, 2001; Lee
Cytokines mediate the actions through interactions with high affinity surface receptors. Hence, interfering with ligand-receptor binding represents potential therapeutic approach. Various biological effects of TNF-α are mediated through two receptors, TNF-R1 and TNF-R2 (Armitage, 1994). TNF-R1 is the major signaling receptor in most cells and a critical mediator for upregulation of cellular adhesion molecules involved in atherosclerosis (Zhang L
Activated MAPKs have been known to stimulate various transcription factors such as NF-κB, which has been shown to control inflammatory responses. In addition, activation of the transcription factor NF-κB is required for the transcriptional activation of cell adhesion molecules by TNF-α (Angel and Karin, 1991; Beg
In summary, the results of the present study demonstrate that lobaric acid is capable of inhibiting the expression of VCAM-1 in VSMCs. This action results from suppression of MAPK pathways and NF-κB activation by blocking the expression of TNF-R1 (Fig. 7). The present data might account, at least in part, for the anti-inflammatory activities of lobaric acid. In addition, lobaric acid has been reported to have the anti-inflammatory effect by inhibiting the 5-lipoxygenase (Ogmundsd?ttir
This research was supported by the grant of the Ministry of Oceans and Fisheries’ R&D project (PM13030) and the Korea Polar Research Institute (KOPRI) project (PE13040).