Heart disease and cardiovascular disease are the top ten factors of death in Taiwan (Department of Health, Executive Yuan, R.O.C. (TAIWAN)).
Atherosclerosis is considered to be a form of chronic inflammation and a disorder of lipid metabolism (Madamanchi, Vendrov et al. 2005), among the many genetic and environmental risk factors that have been identified by epidemiologic studies elevated levels of serum cholesterol, low levels of HDL, diabetes mellitus, metabolic syndrome, and insulin resistance are probably unique in being sufficient to drive the development of atherosclerosis in human and experimental animals, even in the absence of other known risk factors (Glass and Witztum 2001). High levels of circulating saturated fatty acids are associated with diabetes, obesity and hyperlipidemia. In heart, the accumulation of saturated fatty acids has been proposed to play a role in the development of heart failure and diabetic cardiomyopathy (Zhou, Grayburn et al. 2000; Chiu, Kovacs et al.
2001; Grynberg 2005) as well as ischemia–reperfusion (Grynberg 2005).
Palmitic acid, a 16-carbon saturated fatty acid (CH3(CH2)14COOH), found in animals and plants, which is a major circulating saturated fatty acid. Meat, dairy products, palm oil, and several other plant oils, including soybean, peanut oils contain very small amounts of palmitic acid.
Ex
F
whi
9
ischemia–reperfusion (Zhou, Grayburn et al. 2000; Chiu, Kovacs et al.
2001). There are multiple pathways can be involved in the acute and chronic cellular effects of NEFA (non‐esterified fatty acid) excess, such as reactive oxygen species production, mitochondrial permeability transition pore opening, IκB kinase and NF-κB activation, finally leading to cell dysfunction, apoptosis or necrosis (Weinberg 2006). Apoptosis of cardiomyocytes was shown recently to play a central role in the development of heart failure (Olivetti, Abbi et al. 1997). Saturated fatty acid–induced apoptosis has been demonstrated in neonatal rat cardiomyocytes (de Vries, Vork et al. 1997; Hickson-Bick, Buja et al.
2000; Sparagna, Hickson-Bick et al. 2000).
4. Palmitic acid-induced oxidative stress in cardiomyocytes
Oxidative stress is caused by reactive oxygen species (ROS), also termed oxygen free radicals, are molecules containing unpaired electrons, which is derived from many cellular enzyme systems within the cardiovascular system (Fearon and Faux 2009). ROS are involved in inflammation, endothelial dysfunction, cell proliferation, migration and activation, extracellular matrix deposition, fibrosis, angiogenesis, and cardiovascular remodeling, important processes contributing to cardiovascular and renal remodeling in hypertension, atherosclerosis, diabetes, cardiac failure, and myocardial ischemia-reperfusion injury (Pawlak, Naumnik et al. 2004; San Martin, Du et al. 2007). The major enzymes responsible for ROS generation in the vasculature include NAD(P)H oxidase, xanthine oxidase, mitochondrial autooxidations,
10
lipoxygenase, and uncoupled NOS. NADPH oxidase was initially identified in phagocytes and it exists as a multisubunit enzyme complex consisting of membrane subunits (p22phox and gp91phox) which are the major components responsible for enzyme stability and activity, and at least four cytosolic subunits (p47phox, p67phox, p40phox and Rac1 or 2) which translocate to the membrane when enzyme was activated (Ray and Shah 2005). The sequential reduction of oxygen through the addition of electrons leads to the formation of a number of ROS including:
superoxide; hydrogen peroxide; hydroxyl radical; hydroxyl ion; and nitric oxide (App.4).
APP.4 Electron structures of common reactive oxygen species. The · designates an unpaired electron.
Antioxidant defense systems may be generally classified into indirect enzymatic antioxidant enzymes and into small molecular weight molecules which directly scavenge free radicals and related reactants. The antioxidant enzymes are regulated by multiple factors, it’s represent a first line of defense against these toxic reactants by metabolizing them to
11
innocuous byproducts. The first enzymatic reaction in the reduction pathway of oxygen occurs during the dismutation of two molecules of O2-.
when they are converted to hydrogen peroxide (H2O2) and diatomic oxygen. The enzyme at this step is one of two isoforms of superoxide dismutase (SOD); CuZnSOD is present in the cytosol while MnSOD is located in the mitochondrial matrix. Two enzymes participate in the removal of H2O2 from the cellular environment, peroxidases and catalase (Rodriguez, Mayo et al. 2004). APP. 5 showed exogenous and endogenous stimuli leading to ROS generation and activation of stress-sensitive gene expression (Evans, Goldfine et al. 2002).
APP. 5 Exogenous and endogenous stimuli leading to ROS generation and activation of stress-sensitive gene expression.
An imbalanced production of ROS plays a role in the pathogenesis of a number of human diseases such as ischemia/reperfusion injury, atherosclerosis, cancer, and allergy. Oxidative stress induced by free fatty acids (FFAs) plays a key role in the development of cardiovascular diseases in metabolic syndrome (Madamanchi and Runge 2007). Elevated
12
FFA can cause oxidative stress due to increased mitochondrial uncoupling (Wojtczak and Schonfeld 1993; Carlsson, Borg et al. 1999) and β-oxidation (Rao and Reddy 2001; Yamagishi, Edelstein et al. 2001), leading to the increased production of ROS and the activation of stress-sensitive signaling pathways.
5. Sinaling pathway in palmitic acid-induced cardiomyocyte dysfunction
ROS are not only toxic consequences of cellular metabolism but also participants in many intracellular signaling pathways leading to changes in gene transcription and protein synthesis (Griendling, Sorescu et al.
2000). Exposure to a high fat diet induces cardiac contractile dysfunction, which is associated with a permanent relocation of CD36 to the plasma membrane (Ouwens, Diamant et al. 2007). Furthermore, relocation of CD36 appears to be a general phenomenon in insulin resistant hearts (Carley, Atkinson et al. 2007), and precedes cardiac contractile dysfunction (Ouwens, Diamant et al. 2007). In the diabetic heart, mitochondrial oxidative stress induces apoptosis by release of cytochrome c and upregulation of caspase-3 and caspase-9 (Cai, Li et al.
2002; Li, Zhang et al. 2009). In APP.6, high plasma FA concentrations lead to an increase in cytoplasmic FA levels. This stimulates β-oxidation and also stimulates CD36 relocation to the plasma membrane (via PPAR or via increased basal Akt phosphorylation), which leads to an even further increase in cellular FA uptake. The increased intracellular FA concentration causes mitochondrial overload, leading to dysfunction and
oxy
A
15
identification of molecules, which regulate the activation of the NF-κB heterodimer, RelA(p65) and p50 has enhanced our understanding of the molecular mechanisms controlling inflammation. Signaling systems induced by a variety of stimuli activate two serine kinases, termed IκB kinase (IKK)α and IKKβ (or IKK1; IKK2), which target the inhibitors of κB (IκB) (Nakano, Nakajima et al. 2006). Regulation of cell death and survival is also controlled in part by another signaling cascade activated by the mitogen-activated protein kinase(MAPK), which is induced following cellular stress or cytokine signaling (Davis 2000; Kyriakis and Avruch 2001). In mammals, the MAPK cascades are composed of three distinct signaling modules, the c-Jun N-terminal kinase (JNK) cascade, the p38MAPK cascade, and the extracellular signal-regulated kinase (ERK) cascade. Conversely, the c-Jun N-terminal kinase (JNK) promotes apoptosis when activated for prolonged periods (Nakano, Nakajima et al.
2006). Prolonged activation has been shown to be caused by exposure to ROS directly as well as by inactivating JNK inhibitors such as MAP Kinase phosphatases (Nakano, Nakajima et al. 2006). Suppression of TNF-α-induced ROS accumulation seems to be the mechanism by which NF-κB downregulates JNK activation.
6. High density lipoprotein
Lipoproteins consist of lipids and proteins called apoproteins (apo).
They are classified according to size, density, and lipid and apoprotein composition: chylomicron (CHY), very low-density lipoprotein (VLDL), intermediate density lipoproteins (IDL), low-density lipoproteins (LDL), and high-density lipoproteins (HDL) (Forti and Diament 2006). HDL are
a fr
17
Castelli et al. 1977; Castelli, Garrison et al. 1986; Assmann, Schulte et al.
1996; Sharrett, Ballantyne et al. 2001). HDL promotes the mobilization and clearance of excess cholesterol via the series of reactions collectively termed ‘‘reverse cholesterol transport’’ (Toth 2009). Another mechanism cited is that HDL possesses such as antioxidant capabilities, anti-inflammatory, anti-thrombotic, and anti-apoptotic activity (Chapman, Assmann et al. 2004).
The effects of dietary fats on the risk of coronary artery disease have traditionally been estimated from their effects on serum total cholesterol (Keys, Anderson et al. 1957; Hegsted, McGandy et al. 1965). A direct atherogenic effect of TG-rich particles, particularly intermediate-density lipoprotein, and remnant particles has been presumed. In a more recent analysis, adjustment for established coronary risk factors, especially HDL cholesterol, substantially attenuated the magnitude of risk associated with high TG levels (Sarwar, Danesh et al. 2007). Therefore, the aim of this study was to explore the mechanisms underlying HDL protects against palmitic acid-induced oxidative stress in cardiomyocytes. We investigated the ROS-mediated NF-κB activation and subsequent inflammatory and apoptotic signaling pathways.
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Materials
40%Acrylamide/Bis solution 29:1 (SERVA, Germany) Annexin V-FITC Apoptosis Detection Kit (BioVion, USA) Ammonium persulfate/APS (USB, USA)
beta-mercaptoethanol (Pharmacia Biotech, Sweden) Bovine serum albumin/BSA (Sigma, USA)
Bromophenol Blue (Sigma, USA)
Cosmic Calf Serum/CCS (Hyclone, USA) DAPI (Sigma, USA)
DCF-AM (2’,7’-dichlorofluorescein acetoxymethyl ester; Molecular Probes, Eugene, OR)
Dihydroethidium(DHE)
DMEM (Dulbeccco’s Modified Eagle’s Medium; Sigma, USA ) DMSO (dimethyl sulfoxide; Sigma, USA)
DTT (1,4-Dithio-D, L-threitol; GERBU, Germany)
Dual-Luciferase®Reporter Assay system (Promega, USA )
ECL kit (enhanced chemiluminescent detection system;Millipore, MA, USA )
Ethylenediaminetetraacetic acid/EDTA (Sigma, USA) FBS (Fetal bovine serum; GIBCO, USA)
Glucose (USB, USA) Glycine (Sigma, USA) Glycerol (Amresco, USA)
Mem-PER®Eukaryotic Membrane Protein Extraction Reagent Kit (PIERCE, 89826)
19
ME-PER®Nuclear and Cytoplasmic Extraction Reagent Kit (Thermo, 78833)
Methanol 20L (慕容科技有限公司/Taiwan)
Mitochondria Isolation Kit for Cultured cells (Thermo, 89874)
MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-terazolium bromide;
Sigma, USA)
Neonatal Rat Cardiomyocyte Isolation Kit (Cellutron Life Technology, MD, USA)
Paraformaldehyde (Sigma, USA) PBS (GIBCO, New Zealand) Penicillin (Sigma, USA)
Protease inhibitor cocktail tablets (Roche, Germany) Protein maker (Fermentas )
PVDF membrane pore size 0.45m (Millipore, USA) Sodium dodecyl sulfate /SDS (Sigma, USA)
Sodium chloride/NaCl (Sigma, USA)
Sodium bicarbonate/NaHCO3 (Sigma, USA) TEMED (Sigma, USA)
Tris(USB, USA) Tris-base (USB, USA) Tris-HCl (USB, USA)
Triton X-100(TEDIA, USA)
TUNEL (Terminal deoxynucleotide dUTP-biotin nick-end labeling;
Roche, Mannheim, Germany) Tween 20(Pharmacia, Sweden)
20
Trypsin-EDTA (GIBCO, USA) 脫脂奶粉(安佳, New Zealand)
Antibody
1st Ab Brand
p-Akt (Ser473) Cell Signaling
β-actin (C4) Santa Cruz
Bax (P-19) Santa Cruz
BCL2 BD
Caspase-3 (H-277) Santa Cruz
Cox-2 Cell signaling
Cox IV (Va) Invitrogen
p-ERK 1/2 (E-4) Santa Cruz
Flotinllin Cell Signaling
IKKα/β (H-470) Santa Cruz
IκBα (H-4) Santa Cruz
p-IKKα/β (Ser176) Santa Cruz
p-IκB-α (Ser32) (14D4) Cell signaling
p-SAPK/JNK (Thr183/Tyr185) Cell Signaling MMP-3
NFκB p65 (A) Santa Cruz
Nitrotyrosine (HM11) Biomol
NOX-2/gp91 phox abcam
p-NF-κB p65 (Ser536) (93H1) Cell Signaling
p22-phox (FL-195) Santa Cruz
21
p47-phox upstate
PCNA (FL-261) Santa Cruz
p-p38 (D-8) Santa Cruz
Rac1 abcam
SOD-1 (C-17) Santa Cruz
SOD-2 (MnS-1) Santa Cruz
anti-mouse IgGhorseradish peroxidase conjugated Santa Cruz anti-rabbit IgGhorseradish peroxidase conjugated Santa Cruz anti-gout IgGhorseradish peroxidase conjugated Santa Cruz
22
Methods
1. Cell culture
H9c2 cell lines were obtained from American Type Culture Collection (ATCC), cultured in Dulbecco’s modified essential medium (DMEM) supplemented with 10% Cosmic CalfR serum (CCS), 2mM glutamine, 100units/ml penicillin, 100μg/ml streptomycin, and 1mM pyruvate in humidified air (5% CO2) at 37 oC. During the treatment, pretreated with HDL for 2 hours and then stimulated with palmitic acid (PA) for 24 hours. The specificity of the inhibit ROS and mitochondria complex Ⅰ inhibitor by adding N-acetly cysteine (NAC) (500μM).
2. Lipoprotein separation
Human plasma was obtained from the Taichung Blood Bank (Taichung, Taiwan) and HDL was isolated using sequential ultracentrifugation (=1.019-1.063 g/ml) in KBr solution containing 30 mM EDTA, stored at 4℃ in sterile, dark environment and used within 4 days as previously described. HDL was separated from EDTA and from diffusible low molecular mass compounds by gel filtration on PD-10 Sephadex G-25 Mgel (Pharmacia) in 0.01 mol/l phosphate-buffered saline (136.9 mmol/l NaCl, 2.68 mmol/l KCl, 4 mmol/l Na2HPO4, 1.76 mmol/l KH2PO4) at pH 7.4. Protein concentration was determined by Bradford Protein Assay.
23
3. MTT assay
MTT, [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium-bromide].
The H9c2 cells were inoculated into 24-well plate. After HDL and palmatic acid treatments, the medium was removed and MTT solution (0.5mg/ml) was added to each well which containing cells, subsequently incubated the plate in a 5% CO2 incubator at 37ºC for 1 hour. MTT solution was replaced by isopropanol to dissolve blue formazan crystals, and absorbance was measured at 570 nm by using a microplate reader.
4. DAPI staining and TUNEL assay
After various treatments, H9c2 cells grown on 6 mm plate were fixed with 4% paraformaldehyde solution for 30 min at room temperature.
After a rinse with PBS, cells were treated with permeation solution (0.1% Triton X-100 in 0.1% sodium citrate) for 2 min at 4ºC.
Following wash with PBS, samples were first incubated with Terminal Deoxynucleotide Transferase-mediated dUTP Nick End Labeling (TUNEL) reagent containing terminal deoxynucleotidyl transferase and fluorescent isothiocyanate-dUTP. The cells were also stained with 1µg/ml DAPI for 30 min to detect cell nucleus by UV light microscopic observations (blue). Samples were analyzed in a drop of PBS under a fluorescence and UV light microscope, respectively.
Apoptotic cells were assessed by fluorescence microscope or in a flowcytometer.
24
5. Reactive oxygen species and mitochondrial superoxide production
Intracellular ROS generation was monitored by flow cytometry using peroxide-sensitive fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA, Molecular Probes), dihydroethidium (DHE) and MitoSOX™ as a probe for the presence of H2O2 or superoxide.
DCFH-DA is converted by intracellular esterases to DCFH, which is oxidized into the highly fluorescent dichlorofluorescein (DCF) in the presence of a proper oxidant, and then analyzed by flow cytometry.
Dihydroethidium (DHE), by virtue of its ability to freely permeate cell membranes is used extensively to monitor superoxide production. It had long been postulated that DHE upon reaction with superoxide anions forms a red fluorescent product (ethidium) which intercalates with DNA. DHE is perhaps the most specific and least problematic dye; as it detects essentially superoxide radicals, is retained well by cells, and may even tolerate mild fixation. MitoSOX™ Red mitochondrial superoxide indicator is a novel fluorogenic dye for highly selective detection of superoxide in the mitochondria of live cells, which is rapidly and selectively targeted to the mitochondria.
Once in the mitochondria, MitoSOX™ Red reagent is oxidized by superoxide and exhibits red fluorescence. MitoSOX™ is readily oxidized by superoxide but not by other ROS- or reactive nitrogen species (RNS)–generating systems, and oxidation of the probe is prevented by superoxide dismutase. The oxidation product becomes highly fluorescent upon binding to nucleic acids.
25
6. Immunoblotting
Culture H9c2 cells were scraped and washed once with PBS, then cell suspension was spun doen, and lysed in RIPA buffer (HEPES 20mM, MgCl2 1.5mM, EDTA 2mM, EGTA 5mM, dithiothreitol 0.1mM, phenylmethylsulfonyl fluoride 0.1mM, pH 7.5), and spun down 12,000 rpm for 20 min, the supernatant was collected in new eppendorf tube. Proteins (30 μg) were separated by electrophoresis on SDS-polyacrylamide gel. After the protein had been transferred to polyvinylidene difluoride membrane, the blots was incubated with blocking buffer (1X PBS and 5% nonfat dry milk) for 1 hour at room temperature and then probed with primary antibodies (1:1000 dilutions) overnight at 4°C , followed by incubation with horseradish peroxidase-conjugated secondary antibody (1:5000) for 1 hour. To control equal loading of total protein in all lanes, blots were stained with mouse anti-β-actin antibody at a 1:50000 dilution. The bound immunoproteins were detected by an ECL kit.
7. Measurement of mitochondria membrane potential
The lipophilic cationic probe fluorochrome 5,5',6,6'-tetrachloro1,1',3,3'-tetraethylbenzimidazolocarbocyanine
iodide (JC-1) was used to explore the effect HDL on the mitochondria membrane potential (△Ψm). JC-1 exists either as a green fluorescent monomer at depolarized membrane potential or as a red fluorescent J-aggregate at hyperpolarized membrane potential. JC-1 exhibits potential-dependent accumulation in mitochondria, as indicated by the fluorescence emission shift from 530 to 590 nm. After treating cell
26
with palmitic acid (0.5mM) for 24 hours in the presence or absence various concentrations of HDL, cell (5X104 cell/24-well plates) were rinsed with DMEM, and JC-1 (5μM) was loaded. After 20 min of incubation at 37 ℃ , cell were examined under a fluorescent microscope. Determination of the △Ψm was carried out using a FACScan flow cytometer.
8. Isolation of cytosolic fraction for cytochrome c analysis
After treating cells with palmatic acud in the presence and absence of natural products, the cells were collected and lysed with lysis buffer (20mmol/L HEPES/ NaOH, pH 7.5, 250 mmol/L sucrose, 10 mmol/L KCl, 1.5 mmol/L MgCl2, 2mmol/L EDTA, 5 mmol/L EGTA, 1 mmol/L DTT, protease inhibitor cocktail) for 20 min on ice. The samples were homogenized 30 strockes by glass Dounce and pestle.
The homogenates were then centrifuged at 500x g to remove unbroken cells and nuclei. Supernatant were centrifuged at 17000x g for 30 min to isolate mitochondria fraction. Supernatant was cytoslic extraction and pellet was mitochondria fraction lysed by RIPA buffer. Cytosol and mitochondria protein were resolved by SDS-polyacryamide gel electrophoresis.
9. Nuclear protein extraction
Cells grown to 80% confluency and subjected to various treatments were subsequently washed with ice-cold PBS and it was prepared for nuclear protein extraction. Cells grown on 10-cm dish were gently
27
scraped with 3 ml ice-cold PBS and it were centrifuged at 1,000x g for 10 min at 4°C. After carefully aspirating the supernatant, cells were resuspended with 200μl ice-cold BUFFER-I (10 mM Hepes (pH 8.0), 1.5 mM MgCl2, 10 mM KCl, 1 mM dithiothreitol, and proteinase inhibitor cocktail and incubated for 15 min on ice to allow cells to swell, followed by adding 20μl IGEPAL-CA630. After vigorously vortexing for 10 s and centrifuging at 16,000 g for 5 min at 4°C, the supernatant (cytoplasmic fraction) were carefully aspirated and the pellet were resuspended with ice-cold BUFFER-II (20 mM Hepes (pH 8.0), 1.5 mM MgCl2, 25% glycerol, 420 mM NaCl, 0.2 mM EDTA, 1 mM dithiothreitol and proteinase inhibitor cocktail and vigorously vortex. After vortexing, the suspension was placed on ice for 30 min before centrifuging at 16,000x g for 15 min at 4°C. The supernatants (nuclear extracts) were stored aliquots at -80°C. Protein concentration of the supernatants was determined by the colorimetric assay.
10. Transfection luciferase or siRNA assay
Transient transfections were carried out by the proprietary cationic polymer reagent (Fermentas) (TurboFect™ in vitro Transfection Reagent) following the manufacturer's instruction. In some experiments 2×104 cells were plated onto 24-well plates and grown overnight. Vectors, including the reporter vectors, and the internal Renilla luciferase control vector (0.1 μg), and other protein expression vectors were cotransfected as indicated in the figure legends. All assays for firefly and Renilla luciferase activity were performed using one reaction plate sequentially. Briefly, at 24 h post-transfection and
28
stimulation, the cells were washed with phosphatebuffered saline and lysed with Passive Lysis Buffer. After a freeze/thaw cycle, samples were mixed with Luciferase Assay Reagent II, and the firefly luminescence was measured with a Luminometer. Next, samples were mixed with the Stop & Glo reagent, and the Renilla luciferase activity was measured as an internal control and to normalize the luciferase activity values. Double-stranded siRNA sequences targeting JNK, NF-κB mRNAs were obtainedfrom Santa Cruz Biotechnology. The non-specific siRNA (scramble) consisted of a nontargeting. Cells were cultured in 60-mmwell plates in medium. Transfection of siRNA was carried out with transfection reagent. Specific silencing was confirmed by immunoblotting with cellular extracts after transfection.
11. Annexin V-FITC/PI Staining
H9c2 cells seeded at a density of 2 × 105 cells/well in 6-well plates were exposed to hypoxia for 24 h. Apoptotic cells were monitored by FACSCanto flow cytometry using the Annexin V-FITC Apoptosis Detection Kit. Total cells and supernatants were collected, washed and incubated for 15 min with 1 × binding buffer containing annexin V-conjugated fluorescein isothiocyanate (FITC) and propidium iodide (PI). Annexin V positive cells were considered as early apoptotic cells.
Cells with annexin V and PI positive were considered as late apoptotic and/or necrotic cells whereas viable cells were unstained.
12. Cardiomyocyte Culture
Neonatal cardiomyocytes were isolated and cultured using the
29
commercial Neonatal Cardiomyocyte Isolation System Kit according to manufacturer’s directions. Briefly, hearts from one- to two-day-old Sprague-Dawley rats were removed, the ventricles were pooled, and the ventricular cells were dispersed by digestion solution at 37 °C.
Ventricular cardiomyocytes were isolated and cultured in DMEM containing 10% fetal bovine serum, 100 μg/ml penicillin, 100 μg/ml streptomycin, and 2 mM glutamine. After 3-4 days, cells were incubated in serum-free essential medium overnight before treatment with indicated agents.
13. Statistical analysis
Statistical differences were assessed by one way-ANOVA. P < 0.05 was considered statistically significant. Data were expressed as the mean
± SEM.
30
Results
Palmitic acid (PA)-induced apoptosis, and cells death.
To clarify palmitic acid induced cytotoxicity in cardiacmyocyte, H9c2 were treated with different concentrations of PA for 24 and 48 h. The result of MTT showed that after treatment with various concentrations of PA for indicated time peroid significantly decreased the cell viability in a dose-dependent manner (Fig.1A). The cell viability is lower then 50% in concentration of PA on 0.5mM treated with H9c2 cells ,therefore 0.5 mM was used for the following experiments.We also used TUNEL analysis for observing cells undergoing apoptosis. After incubation with PA for 24 h, we observed a significant increase apoptosis bodies (Fig.1B).
Palmitic acid increased generation of mitochondrial reactive oxygen species (ROS).
Previous investigation demonstrated that free fatty acid (FFA) induced-oxidative stress plays an important key role in development of cardiovascular disease in metabolic syndrome (Madamanchi and Runge 2007). We therefore, examined the cellular ROS levels after treatment with 0.5 mM PA for 24 h by fluorometric assay using DCF-AM and DHE.
As shown in Fig 2A and 2B, an approximately three-fold and two-fold increase of ROS and superoxide was observed in cells incubated with PA compared with untreated cells. NADPH oxidase and mitochondrion are known major sources of superoxide (Land 2012), so we measured the expression levels of NADPH oxidase subunits by using Western blot (Fig.2D) and generation of superoxide in mitochondira by using
31
MitoSOX™ Red (Fig.2C). In Fig 2C, an approximately three-flod increase of mitochondrial superoxide was observed in cells incubated with 0.5 mM PA for 24 h compared with normal condition. However, the protein levels of gp91phox, p47phox, Rac-1 protein in H9c2 cells in a time
MitoSOX™ Red (Fig.2C). In Fig 2C, an approximately three-flod increase of mitochondrial superoxide was observed in cells incubated with 0.5 mM PA for 24 h compared with normal condition. However, the protein levels of gp91phox, p47phox, Rac-1 protein in H9c2 cells in a time