Chapter 2 Experimentation I…
2.6 Conclusion
In this study, curcumin effectively inhibited the TNF-α-induced migration of HASMCs as compared with the control group. The ROS production, MMP-9 secretion and expression, nuclear translocation of NF-κB p50 and p65 were reduced by curcumin pretreatment. These results led us to conclude that curcumin could restrict the migration of HASMCs by suppressing MMP-9 through down-regulation of NF-κB.
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Fig. 2-1. Cytotoxic effect of curcumin on human aortic smooth muscle cells (HASMCs) with MTT test. HASMCs were treated with increasing concentrations (10~75 μmol/l) of curcumin for 24 h in 10 % FBS-F12K. Statistical analyses of MTT were performed using One-way ANOVA followed by Dunnett’s test; n=3. *p<0.05 compared with control.
* *
*
0 20 40 60 80 100 120
0 10 20 30 50 75
Curcumin (μmol/l)
% of control
(A)
Fig. 2-2. Effect of curcumin on the MMP-9 activity of TNF-α-induced human aortic smooth muscle cells (HASMCs). HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 h, and induced by TNF-α (100 ng/ml) for additional 23 h. The activation of MMP-9 was assessed by gelatin zymography (A). Densitometric analysis was conducted with image analysis system software to quantify gelatin zymography data (B). Values are mean±SD, n=3. a-cMeans with different letters are significantly different compared at p<0.05. Abbreviation: C (control without TNF-α or curcumin), TNF (TNF-α), Cu10 (curcumin 10 μmol/l), Cu20 (curcumin 20 μmol/l).
MMP-9
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Fig. 2-3. Curcumin inhibits the protein expression of MMP-9 in human aortic smooth muscle cells (HASMCs). HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 h, and induced by TNF-α (100 ng/ml) for additional 23 h. The expression of MMP-9 was assessed by western blot analysis. Representative western blot showing MMP-9 protein levels in cell lysates (top) and β-actin (bottom) (A). Densitometric analysis was conducted with image analysis system software to quantify western blot data (B). Values are mean±SD, n=3. a-cMeans with different letters are significantly different compared at p<0.05. Abbreviation: C (control without TNF-α or curcumin), TNF (TNF-α), Cu10 (curcumin 10 μmol/l), Cu20 (curcumin 20 μmol/l).
MMP-9
C TNF TNF TNF
＋ ＋
Cu10 Cu20
Fig. 2-4. Effect of curcumin on TNF-α-induced activation of NF-κB p50 in human aortic smooth muscle cells (HASMCs). HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 h and induced by TNF-α (100 ng/ml) for 23 h. Nuclear extracts were prepared and analyzed for activation of NF-κB family. Five micrograms of nuclear protein was used in each experiment. Values are mean±SD, n=3. a-dMeans with different letters are significantly different compared at p<0.05. Abbreviation: C (control without TNF-α or curcumin), TNF (TNF-α), Cu10 (curcumin 10 μmol/l), Cu20 (curcumin 20 μmol/l).
46
C TNF TNF TNF
＋ ＋
Cu10 Cu20
Fig. 2-5. Effect of curcumin on TNF-α-induced activation of NF-κB p65 in human aortic smooth muscle cells (HASMCs). HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 h and induced by TNF-α (100 ng/ml) for 23 h. Nuclear extracts were prepared and analyzed for activation of NF-κB family. Five micrograms of nuclear protein was used in each experiment. Values are mean±SD, n=3. a-dMeans with different letters are significantly different compared at p<0.05. Abbreviation: C (control without TNF-α or curcumin), TNF (TNF-α), Cu10 (curcumin 10 μmol/l), Cu20 (curcumin 20 μmol/l).
(A)
(a) (b)
(c) (d)
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(B)
C TNF TNF TNF
＋ ＋
Cu10 Cu20
Fig. 2-6. Effect of curcumin on migrarion of human aortic smooth muscle cells (HASMCs) induced by TNF-α treatment. (A) Microphotographs of migrated cells without TNF-α or curcumin (a), with TNF-α (100 ng/ml) (b), with TNF-α (100 ng/ml) and 10 μmol/l of curcumin (c), and with TNF-α (100 ng/ml) and 20 μmol/l of curcumin (d) were captured. (B) HASMCs (1.5×105 cells/300 μl) were resuspended in conditioned medium collected from TNF-α treated cells for 23 h, and added to the upper components of migration chamber in the presence of 10 and 20 μmol/l curcumin.
Values are mean±SD, n=3. a-cMeans with different letters are significantly different compared at p<0.05. Abbreviation: C (control without TNF-α or curcumin), TNF (TNF-α), Cu10 (curcumin 10 μmol/l), Cu20 (curcumin 20 μmol/l).
b
(A)
(a) (b)
(c) (d)
50
(B)
C TNF TNF TNF
＋ ＋
Cu10 Cu20
Fig. 2-7. Effect of curcumin on TNF-α-induced ROS production in human aortic smooth muscle cells (HASMCs). (A) Microphotographs of ROS production in HASMCs without TNF-α or curcumin (a), with TNF-α (100 ng/ml) (b), with TNF-α (100 ng/ml) and 10 μmol/l of curcumin (c), and with TNF-α (100 ng/ml) and 20 μmol/l of curcumin (d) were captured. (B) HASMCs were pretreated with 10 and 20 μmol/l curcumin for 1 h and induced by TNF-α (100 ng/ml) for 23 h. Values are mean±SD, n=3. a-cMeans with different letters are significantly different compared at p<0.05.
Abbreviation: C (control without TNF-α or curcumin), TNF (TNF-α), Cu10 (curcumin 10 μmol/l), Cu20 (curcumin 20 μmol/l).
c
Chapter 3
鼠尾草酸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
Carnosic acid inhibits MMP-9 activity and migration of
TNF-α-induced human aortic smooth muscle cells
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3.1 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).
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 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).
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 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).
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.
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3.2 Materials and Methods
3.2.1 Materials
3.2.1.1 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
3.2.1.2 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
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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
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3.3 Methods
3.3.1 DPPH scavenging assay
Free radical scavenging effect was determined using the free radical generator DPPH (2, 2-diphenyl-1-picrylhydrazyl) by a similar method to Yamaguchi et al, 1998.
Briefly, the reaction mixture contained 500 μl of CA concentrations (0-200 μmol/l) and 500 μl of DPPH (0.5 mmol/l in methanolic solution). The DPPH radical scavenging activity was evaluated by measuring the decrease of DPPH radical detected at 517 nm and by determining the difference in the peak area between control and reaction mixture. Inhibition % was calculated via Eq.
Inhibition % = (B1-B0/B0) × 100
(where B0: is the absorbance of control. B1: is the absorbance of reaction mixture.)
The decoloration was plotted against the sample extract concentration in order to calculate the IC50 values (inhibitory concentration 50 μmol/l), which is the amount of sample necessary to decrease the absorbance of DPPH by 50 %.
3.3.2 Trolox equivalent antioxidant capacity (TEAC) assay
The assay was carried out using a spectrophotometer by the improved ABTS+ method as described by Re et al, 1999 with slight modification. Briefly, ABTS+ radical cation
was generated by a reaction of 7 mmol/l 2,2’-azinobis(3-ethyl-benzothiazoline-6- sulfonicacid-diammoniumsalt) (ABTS) and 2.45 mmol/l potassium persulfate. The reaction mixture was allowed to stand in the dark for 16 h at room temperature and used within 2 days. The ABTS+ solution was diluted with ethanol to an absorbance of 0.700±0.050 at 734 nm. All samples were diluted appropriately to provide 20-80 % inhibition of control absorbance. Fifty microliters of the diluted sample were mixed with 1.9 ml of diluted ABTS+ solution. The assay with the mixture was carried out in triplicate, the mixture was allowed to stand for 6 min at room temperature and the absorbance was immediately recorded at 734 nm (Li et al, 2007). Trolox solution (final concentration 0~15 μmol/l) was used as a reference standard. The results were expressed as μmol/l Trolox of CA.
3.3.3 Isolation of low density lipoproteins
Blood was collected from healthy donors after a 12 h overnight fasting using EDTA as anticoagulant. Plasma was obtained after low-speed centrifugation of the blood, adjusted to a density of 1.21 g/ml with KBr and a discontinuous density gradient was made by overlaying the plasma solution with a 10 mmol/l PBS and 1 mmol/l EDTA pH 7.4. LDL were isolated after ultracentrifugation as described previously (Vieira et al, 1996). The LDL fraction was exhaustively dialyzed against PBS buffer pH 7.4 without EDTA in the dark, filtered through a 0.45 μm filter, stored at 4 °C under nitrogen and used in 24-72 h.
3.3.4 Inhibition of LDL Oxidation
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LDL were oxidised using the classical copper-induced LDL auto-oxidation.
Incubations were carried out at 37 °C for measuring the diene conjugated formation (Vieira et al, 1996). Increasing concentrations of CA (0~10 μmol/l) dissolved in DMSO were present in the incubation media. Briefly, 0.9 mg/ml of LDL total cholesterol was incubated in PBS in the presence of CuSO4 (50 μmol/l). After incubation,150 µL EDTA (2 mmol/l) was added. A 100 µL portionof the mixture was then transferred to vials containin0.9 ml of 2-propanol. The precipitates were removed by centrifugation. The concentration of conjugated diene in the supernatant was determined by absorption at 234 nm.
3.3.5 Cell culture
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 hours.
3.3.6 Cell viability assay (MTT assay)
The cytotoxic effect of CA 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 CA 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.
3.3.7 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
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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.
3.3.8 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 for made a standard curve and the protein concentration of sample was determined by standard curve.
3.3.9 Western blot analysis
HASMCs were treated with various concentrations of CA 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.
3.3.10 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 sec at 14000×g in a microcentrifuge pre-cooled at 4 °C (Dschietzig et al, 2001). Then resuspend nuclear pellet in 50 μl complete lysis buffer
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containing 10 mmol/l DTT, lysis buffer AM2, and protease inhibitor cocktail by pipetting up and down. The suspension was incubates 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.
3.3.11 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 were subtracted from all measurements.
3.3.12 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 CA and TNF-α-treated cells for 23 h, 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 h 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.
3.3.13 Measurement of intracellular ROS
HASMCs were pretreated with 10 and 20 μmol/l CA for 1 hour and induced by
66
TNF-α (100 ng/ml) for 23 h. Then were incubated with 10 μmol/l 2,7-dichlorofluorescein (DCF) diacetate (DCFH-DA) for 30 min, 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.
3.3.14 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.
3.4 Results
3.4.1 The antioxidant activity of CA
Free radical scavenging effect was determined using the free radical generator DPPH (2, 2-diphenyl-1-picrylhydrazyl) and the IC50 of DPPH assay is 35.92±1.65 μmol/l (Table 3-1). In inhibition of LDL oxidation assay, LDL was oxidatied using the classical copper-induced LDL auto-oxidation and the IC50 of inhibition of LDL oxidation is 5.63±0.19 μmol/l (Table 3-1). The TEAC assay is based on the reduction of the ABTS (2, 2′-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)) radical cation by antioxidants and TEAC value of CA is 5.75±0.49 μmol/l (Table 3-1). A value of 1 TEAC in a sample is defined as a concentration equivalent to 1 μmol/l Trolox, a water-soluble analog of α-tocopherol.
3.4.2 Cytotoxicity of CA on HASMCs.
The cytotoxity of CA on HASMCs were evaluated using MTT assay. The HASMCs (1×104 cells/well) were incubated for 24 hours in cultures in 96-well with various concentrations of CA (0, 10, 20, 30, 50, and 75 μmol/l). Dose-dependent cytotoxic effect of CA against HASMCs was shown in Fig. 3-1 (100 %, 94.9±1.2 %, 92±0.5 %, 84.9±0.6 %, 79.7±1.6 %, and 67.7±2.6 %, respectively.). According to the MTT assay, we chose 10 and 20 μmol/l of CA to do all the following experiments.
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3.4.3 CA prevents TNF-α-induced activation of MMP-9 in HASMCs.
The inhibitory effect of CA on TNF-α-induced MMP-9 activation were analysed by gelatin zymography. HASMCs were pretreated with 10 and 20 μmol/l CA for 1 h, and then induced by TNF-α (100 ng/ml) for additional 23 h. As shown in Fig. 3-2, MMP-9 secretion was markedly induced by TNF-α, and suppressed by CA. The 20 μmol/l CA treatment is more effective on activation of MMP-9 than 10 μmol/l CA.
3.4.4 CA suppresses TNF-α-induced MMP-9 expression in HASMCs.
The effect of MMP-9 expression by CA in VSMCs was assessed by Western blot. HASMCs were pretreated with 10 and 20 μmol/l CA 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 CA (Fig. 3-3).
3.4.5 CA suppresses nuclear translocation of NF-κB p50 and p65 in TNF-α-induced HASMCs.
To determine whether the inhibitory effect of CA 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. 3-4) and p65 (Fig. 3-5). Pretreatment
of HASMCs with 10 and 20 μmol/l CA prior to TNF-α stimulation did significantly prevent the nuclear translocation of p50 and p65. As shown in Fig. 3-4, the 20 μmol/l CA treatment is more effective on decreased nuclear translocation of NF-κB p50 than 10 μmol/l CA. In Fig. 3-5, the 20 μmol/l CA treatment is more effective on decreased nuclear translocation of NF-κB p65 than 10 μmol/l CA.
3.4.6 CA suppresses TNF-α-induced HASMCs migration.
HASMCs (1.5×105 cells/300 μl) were resuspended in pretreated with 10 and 20 μmol/l CA for 1 h, and induced by TNF-α (100 ng/ml) for 23 h. As shown in Fig. 3-6, the migration of HASMCs was increased by treatment with TNF-α when compared with TNF-α-untreated control cells. The stimulatory effect of TNF-α was significantly reduced by CA. The 20 μmol/l CA treatment is more effective on decreased HASMCs migration than 10 μmol/l CA.
3.4.7 CA 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
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