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3.1 Prolegomenon
Atherosclerosis, a progressive pathological disorder leading to cardiovascular and cerebrovascular diseases, is still the leading cause of mortality and morbidity in industrialized countries, in spite of improved pharmacological and lifestyle approaches (Ross, 1993). The adhesion of circulating leukocytes to the vascular endothelium is a critical early event in the development of atherosclerosis (Joris et al., 1983; Faggiotto et al., 1984). This process depends on the interaction between cell adhesion molecules
expressed on the surface of endothelial cells and their cognate ligands on leukocytes (Price & Loscalzo, 1999). Previous studies have indicated that NF-κB/Rel transcription factors may play an important role in the development of the atherosclerosis (Collins, 1993; Qwarnstrom et al., 1994). The activation of NF-κB in endothelial cells is associated with the activation of genes responsible of an increased transcription of adhesion molecules, cytokines and chemokines (True et al., 2000; Valen et al., 2001;
Thornburg et al., 2003; Hatada et al., 2003). Plant polyphenols are large group of naturally-occuring antioxidants and epidemiologic studies have suggested that higher polyphenol intake from fruits and vegetables is associated with decreased risk for cardiovascular disease (Ilja & Peter, 2005). Previous studies showed that polyphenolic compounds such as dietary flavonoids or red wine polyphenols could prevent atherosclerosis by inhibiting adhesion molecules expression in endothelial cell (Murase et al., 1999; Silvina & Balz, 2006). Carnosic acid, an antioxidant polyphenolderived from Sage (Salvia oddicinalis) and Rosemary (Rosmarinus officinalis), is a lipophilic antioxidant that scavenges singlet oxygen, hydroxyl radicals,and lipid peroxyl radicals, thus preventing lipid peroxidationand disruption of biological membranes (Aruoma et al.,
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1992; Haraguchiet al., 1995). Previous study also demonstrated that carnosic acid can inhibit plasma triglyceride elevation in olive oil-loaded mice and reduce the gain of body weight and the accumulation of epididymal fat weight in high fat diet-fed mice (Kiyofumi et al., 2004). Therefore, we designed to examine the effect of carnosic acid on monocyte adhesion to cultured human endothelial cells and the expression of adhesion molecules (VCAM-1, ICAM-1 and E-selectin) and to elucidate its possible mechanism.
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3.2.2 Chemicals
ABTS (2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) Sigma, MO, USA BSA (Bovine serum albmin) Sigma, MO, USA Bradford reagent Bio-Rad, CA, USA Carnosic acid Sigma, MO, USA CHCl3 (Chloroform) BDH, Poole, England CuSO4 (Cupric sulfate) Sigma, MO, USA DCFH-DA (2’, 7’-dichlorofluorescin diacetate) Molecular Probe, Oregon, USA DEPC (Diethyl pyrocarbonate) Sigma, MO, USA DMSO (Dimethyl sulfoxide) Sigma, MO, USA DPPH (2, 2-diphenyl-1-picrylhydrazyl) Sigma, MO, USA DTT (1, 4-Dithio-D,L-thereitol) Bio-Rad, CA, USA ECG (Endothelial cell growth supplement) Sigma, MO, USA EDTA-Disodium (Ethyienediamine Teraacetate Disodium Salt) Bio-Rad, CA, USA FBS (Fetal Bovine serum) Gibco, NY, USA
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KCl (potassium chloridem) SHOWA, Tokyo, Japan KH2PO4 (potassium dihydrogenphosphate) SHOWA, Tokyo, Japan L-Glutamine (200 mmol/l) Gibco, NY, USA MDA (malonaldehyde bis-(dimethyl acetal)) Alderich, WI, USA MTT (3-[4, 5-Dimethylthiazol-2-yl]-2, 5-diphenyl-terazoliumbromide)
Sigma, MO, USA
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2-propanol Sigma, MO, USA 95 % Ethanol Echo Chemical, Taichung, Taiwan Mouse anti-human β-actin Abcam, Cambridge, UK Mouse anti-human ICAM-1 Abcam, Cambridge, UK Mouse anti-human VCAM-1 Abcam, Cambridge, UK Mouse anti-human E-selectin Abcam, Cambridge, UK Sheep anti-mouse IgG antibody Abcam, Cambridge, UK
3.2.3 Cell Culture
Human umbilical vein endothelial cells (HUVECs) were isolated from human umbilical cords using collagenase type II (Jaffe, 1973), and cultured on 0.1 % gelatin-coated culture dishes in medium M199 (Sigma, MO, USA) supplemented with 10
% FBS, 1 % Antibiotic -Antimycotic, Glutamine (2 mmol/l), Heparin (10 U/ml), Hepes (10 mmol/l), Endothelial cell growth supplement (ECG) (12.5 μg/ml) at 37℃ in a humidified atmosphere of 5 % CO2 and 95 % air. After 3 days, the medium was replaced by fresh medium and subcultured at 1: 4 ratio one time per week. All experiments were performed with HUVEC from passages two to five.
3.2.4 Cell viability assay (MTT test)
The viability of the cells was assessed by MTT (3-[4,5-dimethylthiazol-2-yl] -2,5 -diphenyl-tetrazolium bromide) assay (Mosmann, 1983), which is based on the reduction of MTT by the mitochondrial dehydrogenase of intact cells to a blue formazan product.
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Briefly, 1 × 104 cells/well were dispensed within 96-well culture plates and incubated with various concentrations of carnosic acid (which was dissolved in dimethyl sulphoxide) for 24 h. Four hours before the end of the treatment 10 μl MTT (5 mg/ml) was added to each well. At the end of the treatment the incubation medium was removed and the formazan crystals were dissolved in 100 μl of solution of DMSO. MTT reduction was quantified by measuring the light absorbance with a ELISA plate reader (μQUANT, Bio-Tek, USA) at 590 nm. The reduction in optical density caused by carnosic acid was used as a measurement of cell viability, normalized to cells incubated in medium with DMSO only, which were considered to be 100 % viable.
3.2.5 Adhesion of U937 cells to endothelial cells
Adhesion was evaluated using the human leukemia pro-monocytic U937 cells which were labeled with calcein AM (10 nmol/l; Molecular Probe; Invitrogen) (Yu et al., 2007). HUVEC (2 ×105) were distributed into 6-well plates before the assay and allowed to reach confluence. Then the growth medium was supplemented with 10 and 20 μmol/l carnosic acid for 18 h, followed by incubation 10 ng/ml IL-1 β for 6 h in the continued presence of carnosic acid . U937 cells were grown in RPMI 1640 medium (Gibco, New York, USA) containing 10 % FBS and subcultured at a 1:5 ratio three times per week.
U937 cells were incubated with 10 nmol/l calcein AM in RPMI 1640 medium for 30 min at 37℃, then washed with PBS to remove free dye and resuspended in M-199 median containing 10 % FBS. Labeled U937 cells (2×105) were added to each HUVEC-containing well and incubation continued for 30 min. Non-adherent cells were removed by two gentle washes with PBS, then the number of bound U937 cells was
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determined by a fluorescence plate reader at an excitation wavelength of 485 nm and emission at 530 nm; HUVEC cell monnolayers with DMSO only were served as the blank.
3.2.6 Bradford assay
The Bradford assay (Bradford, 1976), a colorimetric protein assay, is based on an absorbance shift in the dye Coomassie when bound to arginine and hydrophobic amino acid residues present in protein. The anionic (bound) form of the dye is blue and has an absorption spectrum maximum historically held to be at 595 nm. The cationic (unbound) forms are green and red. The increase of absorbance at 595 nm is proportional to the amount of bound dye, and thus to the amount (concentration) of protein present in the sample. Standard solutions contain a range of 0 to 25 micrograms protein (BSA) in 800 μl H2O, followed by adding 200 μl dye reagent and incubate 5 min. l μl of sample solution add into 799 μl H2O, followed by adding 200 μl dye reagent and incubated for 5 min. The absorbance was read at 595 nm. The results made a standard curve and the protein concentration of sample was determined by standard curve.
3.2.7 Western blot
For Western blotting, 3×106 cells were seeded in 10 cm dishes and treated 10 and 20 μmol/l carnosic acid for 18 h, followed by incubation 10 ng/ml IL-1 β for 6 h in the continued presence of carnosic acid, afterwards cells were scraped with a rubber policeman in PBS and centrifuged at 1,200 rpm for 10 min. Cells were lysed for 1 h at 4
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℃ with lysis buffer (0.32 mol/l Sucrose, 10 mmol/l Tris, 5 mmol/l EDTA, 2 mmol/l DTT, 1 % Triton X-100, and 1 mmol/l PMSF) and centrifuged at 12,000 g for 30 min at 4 ℃.
The protein concentration of cell extracts was determined with a Bradford-based assay (Bradford, 1976). Cell extracts were loaded per lane, resolved by 10 % SDS -PAGE and transferred at room temperature by blotting to polyvinylidene difloride (PVDF) membrane (Shishodia et al., 2003). Nonsecific binding was blocked by soacking the membrane in PBS-Tween 20 buffer containing 50 g/L fat-free milk and separately incubated for 1 h at room temperature with mouse anti-human-VCAM-1, ICAM-1, and E-selectin antibodies. Subsequently, the membrane was incubated with a sheep anti-mouse IgG antibody. The protein levels was determined with the enhanced chemiluminescence (Upstate, USA) and High performance chemiluminescence film (Amersham biosciences, USA). Incubation with mouse anti-human β-actin antibody was also performed as an internal control. Results were quantified with scanning densitometer using an image analysis system with software.
3.2.8 Nuclear extract preparation
Nuclear protein extracts were prepared using a nuclear extract kit (TransAM nuclear extract kit, CA, USA) from HUVECs to assay the NF-κB activity. Nuclear protein were prepared as described previously (Dschietzig et al., 2001). 3×106 cells were seeded in 10 cm dishes and treated 10 and 20 μmol/l carnosic acid for 18 h, followed by incubating 10 ng/ml IL-1 β for 6 h in the continued presence of carnosic acid, afterwards cells were scraped with a rubber policeman in PBS, collected and centrifuged at 1,200 rpm for 10 min. Cells were lysed in hypotonic buffer and centrifuge suspension for 30
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seconds at 14,000×g in a microcentrifuge pre-cooled at 4 °C. Then resuspend nuclear pellet in 50 μl complete lysis buffer containing 10 mmol/l DTT, lysis buffer AM1, and protease inhibitor cocktail by pipetting up and down. The suspension was incubated for 30 min on ice, and centrifuged for 10 min at 14,000×g in a microcentrifuge pre-cooled at 4 °C. Transfer supernatant and stored at -80 °C. The protein concentration was determined with a Bradford-based assay (Bradford, 1976).
3.2.9 Measurement of NF-κB activation
For analysis of NF-κB activation with the TransAM NF-κB Family kit (TransAM, Active Motif, CA, USA) (Yu et al., 2007). The oligonucleotide containing the NF-κB consensus binding site (5′-GGGACTTTCC-3′) specific for the active form of NF-κB was immobilized to a 96-well plate and the well was filled with 10 μg of nuclear extract. After 1 hour incubation and three washings, the primary antibody against the active form of NF- B recognizing an epitope on p65, p50 that is accessible only when NF- B is activated and bound to its target DNA was added for 1 hour. After washing, the secondary antibody conjugated to horseradish peroxidase was added to achieve a sensitive readout by spectrophotometry at 450 nm and the blank was subtracted from all measurements.
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3.2.10 Measurement of reactive oxygen species
ROS generation in cells was assessed using the probe 2,7-dichlorofluorescein (DCF) (Molecular Probes Europe BV, Leiden, The Netherlands) according to the method reported by Wang and Joseph (1999). Confluent HUVECs (1×104 cells/well) in 96-well plates were pretreated with 10 and 20 μmol/l carnosic acid for 18 h, followed by incubation of 10 ng/ml IL-1 β for 6 h in the continued presence of carnosic acid. After the removal of curcumin or carnosic acid from wells, cells were incubated with 10 μmol/L DCFH-DA for 30 minutes. The fluorescence intensity (relative fluorescence units) was measured at 485-nm excitation and 530-nm emission using a fluorescence microplate reader.
3.2.11 Statistical analysis
Results are shown as mean ± S.D. Statistical analyses of MTT were performed using One-way ANOVA followed by Dunnett’s test and others were performed using One-way ANOVA followed by Duncan's Multiple Range Test. A value of P < 0.05 was considered statistically significant.
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3.3 Results
3.3.1 Cell viability of carnosic acid for HUVECs
Cell viability was determined by the MTT test. After 24h incubation with 5, 10 and 20, 40 and 60μmol/l carnosic acid, cell viability was 105.1 ± 2.5, 106.3 ±5.4, 99.1 ± 5.2, 88.2 ± 3.3 and 71.5 ± 3.8 % of control levels, respectively, the two highest concentrations causing a significant reduction in cell viability (Fig 3-1). Therefore, according to the MTT test we chose 10 and 20 μmol/l carnosic acid to do all the following experiments.
3.3.2 Carnosic acid inhibited the adhesion of U937cells to IL-1 β -stimulated HUVECs
In order to determine the effect of carnosic acid on the adhesion of U937 cells to endothelial cells, HUVECs were treated with 10 and 20 μmol/l carnosic acid for 18 h, followed by incubation of 10 ng/ml IL-1 β for 6 h and the percentage of cell adhesion was evaluated by the quantification of calcein AM (Fig. 3-2 (B)). The adhesion of U937 cells to HUVECs also was photographed (Fig. 3-2 (A)). The control group showed that minimal binding of HUVECs to U937 cells, but adhesion significantly increased when the HUVECs were treated with IL-1 β (Fig 3-2 A & B). Pretreatment with 10 and 20 μmol/l carnosic acid could significantly reduce the number of U937 cells adhering to IL-1 β-stimulated HUVECs. The concentration of 20 μmol/l carnosic acid was more effective on cell adhesion than 10 μmol/l carnosic acid (Fig 3-2 A & B). These results indicated
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that carnosic acid could inhibit monocytes adhesion to endothelial cells.
3.3.3 Carnosic acid inhibits IL-1 β-induced cell protein expression of VCAM-1, ICAM-1and E-selectin in HUVECs
To determine if the inhibition of cell adhesion by carnosic acid was due to inhibit the expression of adhesion molecules, HUVECs were pretreated for 18 h with 10 and 20 μmol/l carnosic acid before the addition of 10 ng/ml IL-1 β. The expression of VCAM-1, ICAM-1 and E-selectin was increased after IL-1 β stimulation (Fig 3-3, 3-4, 3-5).
Pretreatment of HUVECs with 10 μmol/l carnosic acid significantly inhibited the expression of VCAM-1 and E-selectin, but not ICAM-1 (Fig 3-3, 3-4, 3-5). Pretreatment of HUVECs with 20 μmol/l carnosic could significantly inhibit the expression of ICAM-1, VCAM-1 and E-selectin. Therefore, 20 μmol/l carnosic could was more effective than 10 μmol/l carnosic acid on the inhibition of cell adhesion molecular expression (Fig 3-3, 3-4, 3-5).