The objective of this research

Chapter 1 Literature review

1.7 The objective of this research

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). It is a chronic inflammatory disease driven by risk factors that cause oxidative and inflammatory mechanisms. Oxidative stress may lead to many cellular events, such as inactivation of NO, oxidative modifications of DNA and proteins, lipid oxidation, enhanced mitogenicity and apoptosis of vascular cells, and increased expression and activation of redox-sensitive genes, such as the receptor for oxidized LDL, adhesion molecules, chemotaxis factors, proinflammatory cytokines, regulators of cell cycle progression, and matrix metalloproteinases (Wassmann et al, 2004).

Previous studies also indicated that proinflammatory cytokines, such as tumor necrosis factor (TNF) and interleukin-1 (IL-1), play an important role in the pathogenesis of atherosclerosis (Zhu et al, 1999; Rahman at al, 1998).

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 activity may contribute to the pathogenesis of atherosclerosis by facilitating migration of vascular smooth muscle cells (VSMCs) (Jones et al, 2003). MMPs (MMP-9 and MMP-2) production and SMCs migration may play key roles in the pathogenesis of neointima formation and atherosclerosis. The activity of the 92 kDa (MMP-9) but not the 72 kDa (MMP-2) gelatinase is induced by IL-1α, TNF-α and phorbol esters, in a variety of cell types (Birkedal-Hansen et al, 1993; Fabunmi et al, 1996).

retained in the cytoplasm in an inactive state through interaction with IκB. NF-κB is rapidly activated in response to a variety of inflammatory and other stimuli that lead to degradation of IκB (Martin et al, 2000). Upon activation of NF-κB, a large number of genes are induced including various inflammatory cytokines, adhesion molecules, and MMPs (Baeuerle, 1991; Grilli et al, 1993; Martin et al, 2000).

Curcumin, which is consumed daily by millions of people, is a polyphenol derived from the plant Curcuma longa. In general, curcumin has been associated with a large number of biological and cellular activities, including antioxidative, anti-inflammatory, anticarcinogenic, and hypocholesterolemic properties (Gafner et al, 2004; Shishodia et al, 2005; Aharma et al, 2005; Aggarwal and Shishodia, 2006;

Maheshwari et al, 2006). In this study, we investigated the inhibitory effect of curcumin on TNF-α-induced human aortic smooth muscle cells (HASMCs) migration and MMP-9 activity.

Carnosic acid (CA) is the primary phenolic compound in rosemary and salvia.

Previous study indicated that CA has a typical O-diphenol structure and most diphenol compounds show potent chain-breaking antioxidant activity in food systems (Shahidi et al, 1992). This molecule has antimicrobial activity (Oluwatuyi et al, 2004; Moreno et al, 2006), is able to inhibit lipid absorption in humans (Ninomiya et al, 2004) and is a free radical scavenger, due to its phenolic skeleton (Masuda et al, 2001, 2002; del Bano et al, 2003). In this study, we investigated the inhibitory effect of CA on TNF-α-induced HASMCs migration and MMP-9 activity.

In the present study, two polyphenolic compounds, Curcumin and CA, were examined for their effects on TNF-α-induced cell migration in HASMCs and also elucidate its possible mechanism.

Fig. 1. The structure of the normal blood wall. (Libby, 2002)

Smooth muscle cells

Adventitia

Fig. 2. The function of VSMCs during different stages of atherosclerosis. (Dzau et al, 2002)

Fig. 3. The regulation of MMPs. (Dollery et al, 1995)

(Creemers et al, 2001)

Fig. 4. The signal transduction pathways of NF-κB activation. (Martin et al, 2000)

Fig. 5. The structure of curcumin.

Fig. 6. The structure of carnosic acid.

Chapter 2 薑黃素抑制細胞激素誘發人類大主動脈平滑肌細胞的遷移和基質金屬蛋白酶的活化

Curcumin inhibits MMP-9 activity and migration of

TNF-α-induced human aortic smooth muscle cells

2.1 Introduction

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). It is a chronic inflammatory disease driven by risk factors that cause oxidative and inflammatory mechanisms. Oxidative stress may lead to many cellular events, such as inactivation of NO, oxidative modifications of DNA and proteins, lipid oxidation, enhanced mitogenicity and apoptosis of vascular cells, and increased expression and activation of redox-sensitive genes, such as the receptor for oxidized LDL, adhesion molecules, chemotaxis factors, proinflammatory cytokines, regulators of cell cycle progression, and matrix metalloproteinases (Wassmann et al, 2004).

variety of cell types (Birkedal-Hansen et al, 1993; Fabunmi et al, 1996).

Transcription factor NF-κB and its target genes are involved in the pathogenesis of atherosclerosis (Kutuk and Basaga, 2003). NF-κB subunits form homo- and heterodimers, the most prominent one is p50/p65 heterodimers. The dimmer is retained in the cytoplasm in an inactive state through interaction with IκB. NF-κB is rapidly activated in response to a variety of inflammatory and other stimuli that lead to degradation of IκB (Martin et al, 2000). Upon activation of NF-κB, a large number of genes are induced including various inflammatory cytokines, adhesion molecules, and MMPs (Baeuerle, 1991; Grilli et al, 1993; Martin et al, 2000).

Maheshwari et al, 2006). In this study, we investigated the inhibitory effect of curcumin on TNF-α-induced human aortic smooth muscle cells (HASMCs) migration and MMP-9 activity.

2.2 Materials and Methods

2.2.1 Materials

2.2.1.1 Instruments

CO2 incubator NUAIRE, MN, USA Laminar flow NUAIRE, MN, USA Microscope Nikon, Japan

pH meter HANNA, RI, USA Stirrer/Hotplate Corning, Taiwan Waterbath tank TKS, Taiwan Haemocytometer Boeco, Germany Eppendorf centrifugator Hamburg, Germany Pipetman Gilson, France Spectrophotometer HITACHI, Japan

Spectrophotometer Beckman Coulter, CA, USA MicroPlate fluorescence reader Bio-Tek, VT, USA

Shaking incubator Orbital, VA, USA ELISA plate reader Bio-Tek, VT, USA Electrophoresis tank Bio-Rad, CA, USA Transfer system Bio-Rad, CA, USA

Electrophoresis chamber Bio-Rad, CA, USA Power supply Hoefer, CA, USA

2.2.1.2 Chemicals

40 % Acrylamide Amresco, OH, USA

2, 7-dichlorofluorescein diacetate (DCFH-DA) Molecular Probe, OR, USA 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)

Sigma, MO, USA Enhanced chemiluminescence (ECL) Upstate, CA, USA

Ethanol 景明化工, Taichung, Taiwan

Hepes Gibco, NY, USA

Invasion assay kit Chemicon, CA, USA

Isopropanol Sigma, MO, USA

Methanol Tedia, OH, USA

Nuclear extract kit TransAM, CA, USA

NF-κB kit TransAM, CA, USA

Penicillin-Streptomycin Gibco, NY, USA

Recombinant human TNF-α Cytolab, Rehovot, Israel Rabbit anti-human matrix metallproteinases-9 Abcam, Cambridge, UK

Secondary antibodies

Sheep anti-mouse IgG antibody Abcam, Cambridge, UK Goat anti-rabbit IgG antibody Abcam, Cambridge, UK

2.3 Methods

2.3.1 Cell culture

Human aortic smooth muscle cells (HASMCs) were purchased from Food Industry Research and Development Institute, 新竹, Taiwan (CCRC 60293). They were maintained in Ham’s F12K containing 10 % fetal bovine serum, 2 mmol/l L-glutamine, 1.5 g/l sodium bicarbonate, 10 mmol/l HEPES, 10 mmol/l TES, 0.05 mg/ml ascorbic acid, 0.01 mg/ml transferrin, 0.01 mg/ml insulin, 10 ng/ml sodium selenite, 0.03 mg/ml ECGs. All experiments were performed with HASMCs from passages 21 to 31, which were grown to 80-90 % confluence and made quiescent by serum starvation (0.1 % FBS) for at least 24 h.

2.3.2 Cell viability assay (MTT assay)

The cytotoxic effect of curcumin on HASMCs was investigated using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay (Chen et al, 2002). The principle of this assay is that mitochondria dehydrogenase in viable cells reduces MTT to a blue formazan. Briefly, the cells were grown in 96-well culture plates at a density of 1×10⁴ cells per well in F-12K culture medium and incubated with various concentrations of curcumin for 24 hours. 10 μl MTT (5 mg/ml) were then added to each well and incubation continued at 37 °C for an additional 4 hours. The medium was then carefully removed, so as not to disturb the formazan crystals which had formed. Dimethyl sulphoxide (100 μl), which solubilizes formazan crystals, was

added to each well and absorbance of the solubilized blue formazan was measure the optical density at 590 nm using µQuant Microplate Spectrophotometer (Bio-Tek, VT, USA). All determinations were performed according to three individual experiments.

The data were shown mean±SD as percentage of control.

2.3.3 Gelatin zymography for MMP-9

MMP-9 activity in conditioned medium of cultured HASMCs was analyzed by substrate-gel electrophoresis (zymography) using SDS-PAGE (10 %) containing 0.1 % gelatin. Substrate gel zymography of the activity of MMP-9 was performed with a Mini-Protein II apparatus from Bio-Rad, according to a method described previously (Demeule et al, 2000). Cells were grown to sub-confluence and were rinsed with phosphate-buffered saline (PBS) and then incubated in serum-free medium for 24 h.

Equal volumes of samples of conditioned cell culture medium were mixed with sample buffer containing 62.5 mmol/l Tris-HCl (pH 6.8), 10 % glycerol, 2 % SDS, and 0.00625 % (w/v) bromophenol blue, loaded onto the gel and separated by electrophoresis. Thereafter, gels were washed 3 times for 30 minutes at room temperature in buffer (50 mmol/l Tris-HCl, pH 8.0, 5 mmol/l CaCl2, 0.02 % NaN3, and 2.5 % Triton X-100) and incubated for 18 h at 37 °C with the same buffer except Triton X-100. Gels were stained with Coomasssie Brillant Blue R-2500 (0.1 %) and destained in 5 % methanol and 7 % acetic acid. Gelatinolytic activity appeared as a clear band on a blue background.

2.3.4 Bradford protein 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.

2.3.5 Western blot analysis

HASMCs were treated with various concentrations of curcumin in the presence of 100 ng/ml TNF-α. Cellular lysates were prepared in a lysis buffer containing 10 mmol/l Tris/HCl (pH 8), 0.32 mol/l sucrose, 5 mmol/l Ethyienediamine Teraacetate Disodium Salt (EDTA), 1 % Triton X-100, 2 mmol/l 1, 4-Dithio-D,L-thereitol (DTT), 1 mmol/l PMSF. The cells were disrupted and extracted at 4 °C for 30 min. After centrifugation at 13,000 rpm for 15 min, the supernatant was obtained as the cell lysate.

Protein concentrations were measured using the bradford assay. Total protein (20 μg) were subjected to SDS-PAGE (10 %) and blotted on PVDF membranes (Shishodia et al, 2003). Nonspecific binding was blocked by soaking the membrane in PBS-Tween

20 (PBST) buffer containing 50 g/l nonfat milk. The membrane was incubated with monoclonal mouse anti-human β-actin (1:1000) and polyclonal rabbit anti-human MMP-9 (1:1000). Subsequently, the membrane was incubated with sheep anti-mouse IgG antibody (1:5000) and goat anti-rabbit IgG antibody (1:5000). The protein levels were determined using the enhanced chemiluminescence detection reagents (Upstate, CA, USA) and high performance chemiluminescence film (Amersham, IL, 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.

2.3.6 Preparation of nuclear extract

Nuclear protein extracts of HASMCs were prepared using a nuclear extract kit (TransAM nuclear extract kit, CA, USA). Cells were lysed in hypotonic buffer and centrifuge suspension for 30 seconds at 14,000×g in a microcentrifuge pre-cooled at 4

°C (Dschietzig et al, 2001). Then resuspend nuclear pellet in 50 μl complete lysis buffer containing 10 mmol/l DTT, lysis buffer AM2, 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. Protein concentrations were measured using the bradford protein assay.

2.3.7 ELISA-Based Nuclear Factor-κB Assay

Additionally to gel-shift assays, an ELISA-based kit was used for quantitative detection of NF-κB activity (TransAM NF-κB kit, CA, USA). For each sample, 20 μl of nuclear extracts (5 μg protein) were used according to the instructions of the manufacturer (Yu et al, 2007). Nuclear extracts were incubated with the oligonucleotide-coated wells for 60 min. Where indicated a competitor for NF-κB binding (NF-κB wild-type consensus oligonucleotide) was added in molar excess prior to the probe. The wells were then washed and incubated with the primary antibodies for p50 and p65 for 60 min. After incubation with a horseradish peroxidase-conjugated secondary antibody, a substrate was added to produce blue colour and then for quantitation by µQuant Microplate Spectrophotometer (Bio-tek, VT, USA). The absorbance was read at 590 nm and the blank was subtracted from all measurements.

2.3.8 Cell migration assay

VSMCs invasion through the extracellular matrix was determined by using a commercial cell invasion assay kit (Chemicon, CA, USA). HASMCs (1.5×10⁵ cells/300 μl) were resuspended in conditioned medium collected from pretreatment with curcumin and TNF-α-treated cells for 23 hours, and added to the upper components of migration chamber (Bedoui et al, 2005). Five hundred microliters of same conditioned medium were added to the lower compartment of migration chamber.

Cells without TNF-α-treated conditioned medium served as control. The migration chambers were incubated at 37 °C for 24 hours in 5 % CO2. After incubation, the inserts were removed from the wells, and the cells on the upper side of the filter were removed using cotton swabs. The filters were fixed, and stained according to the

manufacturer’s instructions. The cells that invaded and were located on the underside of the inserts. Then transfer 100 μl of the dye mixture to a 96-well plate, and measure the optical density at 560 nm.

2.3.9 Measurement of intracellular ROS

HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 hour and induced by TNF-α (100 ng/ml) for 23 hours. Then were incubated with 10 μmol/l 2,7-dichlorofluorescein (DCF) diacetate (DCFH-DA) for 30 minutes, which is converted to DCF by intracellular esterase (Kim et al, 2006). The latter was then oxidized by ROS to the highly fluorescent DCF. The fluorescence of each dish was immediately analyzed at excitation wavelength of 485 nm and emission wavelength of 528 nm by FLx800 microplate fluorescence reader (Bio-tek, VT, USA). All measurements were at least triplicated.

2.3.10 Statistical analysis

Results are shown as mean±SD. 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.

2.4 Results

2.4.1 Cytotoxicity of curcumin on HASMCs.

The cytotoxity of curcumin on HASMCs were evaluated using MTT assay. The HASMCs (1×10⁴ cells/well) were incubated for 24 h in cultures in 96-well with various concentrations of curcumin (0, 10, 20, 30, 50, and 75 μmol/l). Dose-dependent cytotoxic effect of curcumin against HASMCs was shown in Fig. 2-1 (100 %, 92±0.5

%, 91.2±0.8 %, 84.8±4.2 %, 81.8±6.5 %, and 67±13 %, respectively.). According to the MTT assay, we chose 10 and 20 μmol/l of curcumin to do all the following experiments.

2.4.2 Curcumin prevents TNF-α-induced activation of MMP-9 in HASMCs.

The inhibitory effect of curcumin on TNF-α-induced MMP-9 activation were analysed by gelatin zymography. HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 h, and then induced by TNF-α (100 ng/ml) for additional 23 h. As shown in Fig. 2-2, MMP-9 secretion was markedly induced by TNF-α, and suppressed by curcumin. The 20 μmol/l curcumin treatment is more effective on activation of MMP-9 than 10 μmol/l curcumin.

2.4.3 Curcumin suppresses TNF-α-induced MMP-9 expression in

HASMCs.

The effect of MMP-9 expression by curcumin in HASMCs was assessed by Western blot. HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 h, and induced by TNF-α (100 ng/ml) for 23 h. MMP-9 expression was markedly induced by TNF-α, and suppressed by curcumin (Fig. 2-3). The 20 μmol/l curcumin treatment is more effective on protein expression of MMP-9 than 10 μmol/l curcumin.

2.4.4 Curcumin suppresses nuclear translocation of NF-κB p50 and p65 in TNF-α-induced HASMCs.

To determine whether the inhibitory effect of curcumin on the TNF-α-induced expression of MMP-9 is medicated via NF-κB, we measured the nuclear translocation of p50 and p65 of the NF-κB family. Treatment of TNF-α (100 ng/ml) for 23 h enhanced the nuclear translocation of p50 (Fig. 2-4) and p65 (Fig. 2-5). Pretreatment of HASMCs with 10 and 20 μmol/l curcumin prior to TNF-α stimulation did significantly prevent the nuclear translocation of p50 and p65. As shown in Fig. 2-4, the 20 μmol/l curcumin treatment is more effective on nuclear translocation of NF-κB p50 than 10 μmol/l curcumin. In Fig. 2-5, the 20 μmol/l curcumin treatment is more effective on decreased nuclear translocation of NF-κB p65 than 10 μmol/l curcumin.

2.4.5 Curcumin suppresses TNF-α-induced HASMCs migration.

HASMCs (1.5×10⁵ cells/300 μl) were pretreated with 10 and 20 μmol/l curcumin for 1 hour, and induced by TNF-α (100 ng/ml) for 23 h. As shown in Fig.

2-6, the migration of HASMCs was increased by TNF-α stimulation. The stimulatory

effect of TNF-α was significantly reduced by curcumin pretreatment. The 20 μmol/l curcumin treatment is more effective on decreased HASMCs migration than 10 μmol/l curcumin.

2.4.6 Curcumin suppresses TNF-α-induced ROS generation.

To characterize the events underlying TNF-α-induced migration, we examined the generation of ROS after TNF-α treatment in HASMCs. HASMCs were exposed to TNF-α (100 ng/ml) for 23 h, and DCF fluorescence produced was measured (Fig. 2-7).

The production of ROS was induced by TNF-α and decreased by curcumin. The 20 μmol/l curcumin treatment is more effective on reduced ROS generation than 10 μmol/l curcumin.

2.5 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

the experiments (Fig. 2-1).

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

在文檔中薑黃素和鼠尾草酸抑制細胞激素誘發之人類大主動脈平滑肌細胞的遷移和基質金屬蛋白酶的活化; Curcumin and Carnosic acid inhibit MMP-9 activity and migration of cytokine-induced human aortic smooth muscle cells (頁 18-0)