Evidence-Based Complementary and Alternative Medicine

Evidence-Based Complementary and Alternative Medicine / 2013 / Article
Special Issue

Role of Complementary and Alternative Medicine in Cardiovascular Diseases

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Research Article | Open Access

Volume 2013 |Article ID 364519 | https://doi.org/10.1155/2013/364519

Yi-Wen Chen, Hsiu-Chuan Chou, Szu-Ting Lin, You-Hsuan Chen, Yu-Jung Chang, Linyi Chen, Hong-Lin Chan, "Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury", Evidence-Based Complementary and Alternative Medicine, vol. 2013, Article ID 364519, 16 pages, 2013. https://doi.org/10.1155/2013/364519

Cardioprotective Effects of Quercetin in Cardiomyocyte under Ischemia/Reperfusion Injury

Academic Editor: Peng Nam Yeoh
Received10 Aug 2012
Revised22 Nov 2012
Accepted07 Feb 2013
Published14 Mar 2013

Abstract

Quercetin, a polyphenolic compound existing in many vegetables, fruits, has antiinflammatory, antiproliferation, and antioxidant effect on mammalian cells. Quercetin was evaluated for protecting cardiomyocytes from ischemia/reperfusion injury, but its protective mechanism remains unclear in the current study. The cardioprotective effects of quercetin are achieved by reducing the activity of Src kinase, signal transducer and activator of transcription 3 (STAT3), caspase 9, Bax, intracellular reactive oxygen species production, and inflammatory factor and inducible MnSOD expression. Fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) can reveal the differentially expressed proteins of H9C2 cells treated with H2O2 or quercetin. Although 17 identified proteins were altered in H2O2-induced cells, these proteins such as alpha-soluble NSF attachment protein (α-SNAP), Ena/VASP-like protein (Evl), and isopentenyl-diphosphate delta-isomerase 1 (Idi-1) were reverted by pretreatment with quercetin, which correlates with kinase activation, DNA repair, lipid, and protein metabolism. Quercetin dephosphorylates Src kinase in H2O2-induced H9C2 cells and likely blocks the H2O2-induced inflammatory response through STAT3 kinase modulation. This probably contributes to prevent ischemia/reperfusion injury in cardiomyocytes.

1. Introduction

Because of their high incidence and mortality rate, cardiovascular diseases have recently become a primary health concern worldwide. Myocardial ischemia/reperfusion injury, which causes excess reactive oxygen species (ROS) production that can lead to cardiac hypertrophy or dysfunction, is the most acute cardiovascular disease [1, 2]. In 2010, Chou et al. showed that ROS may affect intercellular connections and cytoskeleton resulting in cell detachment, morphology change, or death. Src kinase also plays a key role in ROS-induced phosphorylation and cell damage in cardiomyocytes [2].

The ROS in this study includes hydrogen peroxide (H2O2), singlet oxygen (O), superoxide (O2−), and the hydroxyl radical (OH). Among these ROS species, H2O2 is the most stable and the most abundant in human cells. Although the optimal amount of ROS plays an important role in signal transduction, excess ROS causes cell damage [3]. H2O2 regulates signal transduction-related proteins by phosphorylating or modifying the active sites of proteins but also inhibits phosphatase activity [4].

Quercetin, a type of polyphenolic compound, has anti-inflammatory, antiproliferation, anti-histamine, and antioxidant effects. Quercetin exists in many types of vegetables and fruits. Several reports have shown that quercetin has protective effects on different types of cells, including myocytes, testis, renal cells, and liver cells in ischemia/reperfusion injury [5]. A study conducted in 1992 showed that quercetin reduces the oxidative stress caused by ischemia/reperfusion in cardiomyocytes by inhibiting the xanthine dehydrogenase/xanthine oxidase system [6]. Several reports have also indicated that quercetin and isorhamnetin can scavenge ROS and inhibit the activation of ERK or MAP kinase in ROS-induced cardiomyopathy [7, 8]. In cancer therapy, combining quercetin with doxorubicin augmented the effects of doxorubicin in highly invasive breast cancer cells [9] and can protect cardiomyocytes from doxorubicin-induced toxicity by chelating iron, inducing antioxidant activity, and inhibiting carbonyl reductase [10]. Although quercetin has been reported to play a role in protecting myocardial cells from ischemia/reperfusion injury, its protective mechanism remains unclear in current knowledge.

Ischemia/reperfusion injury in cardiomyocytes is the result of myocardial inflammation [11]. Muthian and Bright showed that quercetin blocks the IL-12-induced inflammatory response through a signal transducer and activator of transcription 3 (STAT3) activation in T lymphocytes [12]. However, previous research failed to show a direct relationship between quercetin and STAT3-activated inflammation in cardiomyocytes. STAT3 is a transcription factor that plays an important role in numerous cytokine signaling transductions including cell survival, proliferation, cell cycle progression, and cell growth. STAT3 has two important phosphorylated and activate sites: Tyr705 and Ser727. STAT3 activation was phosphorylated at tyrosine 705 induced by various factors, including cardiotrophin-1, IL-6, tumor necrosis factor-alpha (TNF-α), and interferon-gamma (IFN-γ) [5, 13]. pY705-STAT3 is also essential for the dimerization of STAT3 and the translocation of STAT3 into the nucleus. In addition, STAT3 has been observed to be phosphorylated at serine 727 under oxidative stress to enhance the transcription activity of STAT3 in previous cerebral ischemia preconditioning study [14]. Moreover, The JAK2/STAT3 signaling pathways participate in an oxidative stress-induced immune response [3, 15].

Two-dimensional gel electrophoresis (2-DE) is a common tool for analyzing thousands of proteins in different biological samples and is complementary to LC-MS results. However, varying quantification between gels remains the primary challenge in 2-DE. Therefore, 2D-DIGE reduces the variation between gels and gels, which codetected the sample abundances on the same gel by using differential fluorescent labeling [2].

This study investigates the potential protective role of quercetin in H2O2-induced H9C2 cell injury. We focus on the correlation between quercetin in cardiomyocytes and the cardioprotective role of Src kinase inhibition and inflammatory response of STAT3 using 2D-DIGE combined with MALDI-TOF MS and immunoblotting.

2. Materials and Methods

2.1. Chemicals and Reagents

Quercetin was purchased from Sigma-Aldrich (St. Louis, USA). The primary antibody phopho-FAK, Bax, caspase9, Bcl-2, GAPDH, and STIP1 were purchased from Genetex (Hsinchu, Taiwan). Horseradish peroxidase and fluorescence conjugated secondary antibodies against mouse and rabbit were purchased from Sigma-Aldrich (St. Louis, USA). Annexin V-FITC and a propidium iodide (PI) labeling kit were purchased from Invitrogen. We purchased 2,7-dichlorofluorescein diacetate (DCFH-DA) from Molecular Probes. The chemicals and reagents of 2D-DIGE were purchased from GE Healthcare (Uppsala, Sweden).

2.2. Cell Lines, Cell Culture, and Cell Treatment

The H9C2 rat cardiomyocyte cell line purchased from American Type Culture Collection (Manassas, VA, USA) was chosen as a cellular model for this study as this cell line retains the characteristics of isolated primary cardiomyocytes and has been used as a model in ischemia and reperfusion studies [2]. The H9C2 was cultured in Dulbecco’s modified Eagle medium (DMEM) (Invitrogen) containing 10% fetal bovine serum (FBS) at 37°C. Cells cultured in normal growth medium were treated with various concentrations of H2O2 for 20 min. H9C2 cells were pretreated with quercetin (Sigma) for 1 h followed by treatment with H2O2 for 20 min.

2.3. Immunoblotting

The methods of quantifying and separating cell lysates for immunoblotting were similar to our previous paper [2]. The primary antibodies used in this study included Src-phospho-Y416, phospho-FAK, phosphor-Y99, phospho-AKT, p38, Bax, caspase9, Bcl-2, GAPDH, CDK4, and STIP1.

2.4. Immunostaining and Fluorescence Microscopy

For completing immunofluorescence staining, H9C2 cells grown on coverslips (12 mm) were treated with 5 mM H2O2 for 20 min alone, 1 mM quercetin for 1 h prior to treatment with 5 mM H2O2 for 20 min, or left untreated [2]. The cell fixing, immunostaining, and fluorescence image analysis methods in this study were similar to our previous paper [2].

2.5. Wound Healing Assay

H9C2 cells (105 cells/well) were incubated in 24-well plate at 37°C for 12 h and then scraped with a 10 μL tip and treated with H2O2 for 20 min, pretreated with quercetin, or left untreated. H9C2 cells were incubated with medium containing 10% FBS, and a fluorescence microscope captured images at different incubation times (0 h, 6 h, 24 h, 30 h, and 42 h).

2.6. Adhesion Assays

H9C2 cells ( cells/well) were incubated in a 3 cm dish containing DMEM containing 10% FBS and treated with 1 mM quercetin for 1 h followed by 5 mM H2O2 for 20 min. After treatment, H9C2 cells were incubated with serum-free medium for 1 h and 4 h and then were counted. The cell culture environment and cell counting were similar to our previous study [2]. All conditions have been performed in duplicate-independent experiments.

2.7. Apoptosis Assay Using Flow Cytometry

H9C2 cells (106 cells) were labeled with annexin V-FITC and PI at room temperature for 15 min and treated with H2O2, pretreated with quercetin, or left untreated. The FITC and PI fluorescence signals were recorded by fluorescence-activated cell sorting FACS (Accuri 6) and analyzed using CFlow plus software [2].

2.8. Reactive Oxygen Species in Cells Were Detected Using DCFH-DA Assay

H9C2 cells (105 cells/well) were grown on a 24-well plate, treated with H2O2 for 20 min, pretreated with quercetin for 1 h, or left untreated. After washing, H9C2 cells were incubated with 10 μM of 2,7-dichlorofluorescin diacetate (DCFH-DA) at 37°C for 20 min. Fluorescence was recorded by Spectra Max Gemini EM (Molecular Device) at an excitation wavelength of 488 nm and an emission wavelength of 504 nm [16, 17].

2.9. 2D-DIGE and Gel Image Analysis in Gel Digestion and Protein Identification by MALDI-TOF MS

The experiments in this study used Cy-Dye labeling and comparative quantification methods to perform lysine-2D-DIGE analysis. Proteins were identified through MALDI-TOF MS with peptide mass fingerprinting (PMF) in our previous paper [2].

3. Results

3.1. Quercetin Pretreatment Suppresses Hydrogen Peroxide-Induced Tyrosine Phosphorylation in Cardiomyocytes

H2O2, an important signal mediator, induces large scale of protein phosphorylation and protein modification resulting in cellular physiology alteration including cell morphology, adhesion, and viability. Because heart ischemia/reperfusion injury stimulates H2O2 production, H9C2 cells were treated with varying H2O2 doses to find the optimal phosphotyrosine response. The optimal response represents the maximal ratio of phosphotyrosine intensity to H2O2 concentration by immunoblotting (Figure 1(a)). Results show that 5 mM H2O2 treatment led to a robust phosphotyrosine response, but the phosphotyrosine response decreased in 10 mM H2O2 cells. Quercetin may also play an important role in oxidative stress-damaged cells, and phosphotyrosine signals were detected with a range of quercetin followed by treatment with 5 mM H2O2 (Figure 1(b)). These results reveal that H9C2 pretreated with 1 mM quercetin and subsequently treated with 5 mM H2O2 induced a lesser phosphotyrosine response than that of H9C2 cells treated with 5 mM H2O2. Subsequent experiments were carried out based on these H2O2 and quercetin treatment concentrations.

3.2. Quercetin Inhibits Hydrogen Peroxide-Induced Changes in Cell Morphology and Loss of Cell Adhesion

H2O2 stimulates the activation of Src kinase that regulates cytoskeleton, cell adhesion, and cell motility. Previous report indicated that PP1, a Src kinase inhibitor, inhibits H2O2-induced Src kinase activation [2]. In this study, quercetin pretreatment reduces the tyrosine phosphorylation of Src kinase and FAK in H2O2-treated H9C2 cells (Figure 2(a)). The immunostained images represent the H9C2 proteins against specific antibodies, including cytoskeleton protein (F-actin and α-tubulin) and cell-cell interaction protein (ZO-2). Oxidative damage affects cytoskeleton proteins and ZO-2, effectively altering cell morphology (Figure 2(b)). Quercetin pretreatment improved changes in ROS-induced cell morphology.

In the wound healing assay, H9C2 cell images were captured at different time points (0 h, 6 h, 24 h, 30 h, and 42 h) using a microscope (Zeiss). Cells were untreated, H2O2 treated, and quercetin pretreated followed by hydrogen peroxide treatment (Figure 2(c)). After incubation, the closure areas of H2O2-treated H9C2 cells were larger than those of untreated and quercetin pretreatment followed by H2O2 treatment.

An adhesion assay was also performed to analyze the effects of quercetin on ROS-damaged cardiomyocytes. H9C2 cells untreated, treated with H2O2 alone, or pretreated with quercetin were followed by treatment with H2O2. Cells were then incubated in a serum-free medium for 1 h or 4 h. The adherent cells were counted after incubation. Results show that H2O2-treated cells had reduced adhesive ability; yet, this could be significantly improved by pretreatment with quercetin (Figure 2(d)). Thus, quercetin can stimulate cell migration and maintain cell adhesion in H2O2-damaged H9C2 cell.

3.3. Quercetin Inhibits Phosphorylation of STAT3, PI3K/Akt, and p38 Kinase and the Expression of COX-2 in H2O2-Induced H9C2 Cells

To determine whether quercetin affects cell signalings associated with inflammatory response and cell proliferation, we examined the activation of AKT, p38, and STAT3 and the expression of COX-2 and MnSOD in ROS-induced cardiomyocytes. Results show that excess ROS increased the phosphorylation of Akt, p38, and STAT3 (Tyr-705 and Ser-727) and the level of COX-2 but repressed the expression of MnSOD in H9C2 cells. Quercetin significantly reduces the phosphorylation of STAT3 and level of COX-2 and increases the expression of MnSOD in H2O2-treated cells (Figure 3). These results show that quercetin suppresses inflammation in H2O2-induced H9C2 cells.

3.4. Pretreatment with Quercetin Suppresses ROS Production in H2O2-Treated H9C2 Cells

DCF fluorescence revealed ROS production in H9C2 cells induced by oxidative damage. Excess ROS accumulated in H2O2-induced H9C2 cells, but quercetin significantly inhibited H2O2-induced ROS production in cardiomyocytes (Figure 4).

3.5. Quercetin Reduces Hydrogen Peroxide-Induced H9C2 Cell Apoptosis

Excess ROS production from ischemia/reperfusion-injured cardiomyocyte [18] alters redox homeostasis and induces cell apoptosis. During cell apoptosis, the asymmetric distribution of phospholipids of the plasma membrane gets lost and phosphatidylserine is translocated to the outer surface of the plasma membrane which has a high affinity to annexin V-FITC. PI can penetrate the cell nucleus when cells undergo apoptosis. Cell apoptosis was detected using FACS. The dot plots of annexin V and PI staining are analyzed using FACS, appearing in Figures 5(a), 5(b), and 5(c). The cell apoptosis rate increased from 5% to 12.5% upon H2O2 treatment, whereas the cell apoptosis rate decreased to 5.5% after H9C2 cells were pretreated with quercetin before H2O2 treatment. In addition, the PI staining signal of H2O2-treated H9C2 shifted forward, compared to that of untreated cells and cells pretreated with quercetin followed by H2O2 (Figure 5(d)). The levels of Bax, Caspase 9, and Bcl-2 were detected using immunoblotting for untreated H9C2 cells, treated H2O2, and pretreated quercetin followed by H2O2 treatment. ROS increased the expression level of apoptosis factors caspase 9 and Bax and reduced anti-apoptosis marker Bcl-2 expression (Figure 5(e)). According to the data, quercetin can protect and stabilize the total chromosome of DNA in H9C2 cells from oxidative damage by inhibiting cell apoptosis and chromosome attrition.

3.6. 2D-DIGE Analysis of Untreated and H2O2-Treated H9C2 Cells and Quercetin Pretreatment Followed by Treatment with H2O2

Three types of cell lysates were analyzed using 2D-DIGE. The results of 2D-DIGE analysis and DeCyder processing identified 1535 proteins spots, and 44 proteins showed differential expression ( 1.5-fold or −1.5-fold; ) among these 3 conditions (Figure 6). Table 1 shows that 44 proteins were identified using MALDI-TOF MS and 17 protein spots of the 44 identified protein spots that displayed H2O2-dependent alteration could be reversed by pretreatment with quercetin (Table 1, boldface numbers). For example, the alpha-soluble NSF attachment protein (α-SNAP) (No.990) was upregulated (9.85-fold) in H2O2-treated cells, whereas quercetin reduced the overexpression of H2O2-treated α-SNAP (7.69-fold). Protein spot number 1405, which was identified as profilin-1, was downregulated in H2O2 treatment only (−3.01-fold) but showed no significant expression after quercetin pretreatment followed by H2O2 treatment (1.12-fold). These results suggest that the protective mechanisms of quercetin significantly reduced H2O2-induced damage in cardiomyocytes. Figure 7 shows the 2D-gel images, 3D images, and protein abundances from untreated, H2O2 treated, and quercetin pretreated followed by H2O2 cells.


Spot no.Swissprot no.Protein namePred. MWPred. PICov. %MASCOT scoreNo. of peptides
match/
supplied
H2O2/CtrlQuercetin + H2O2/CtrlPeptide sequenceFunction

1420P6332440S ribosomal protein S12148586.8242%55/516/30−1.25−2.77QAHLCVLASNCDEPMYVK,
TALIHDGLAR
Protein synthesis
691Q9JLJ34-Trimethylaminobutyraldehyde dehydrogenase545306.5744%109/5616/661.681.9AFEPATGR,
AGAPNGLFNVVQGGAATGQFLCQHR
Protein synthesis
473P6303960 kDa heat shock protein (mitochondrial)610885.9120%61/568/301.371.54AAVEEGIVLGGGCALLR,
ISSVQSIVPALEIANAHR
Chaperon
1010P60711Actin (cytoplasmic)420525.2923%54/516/37−1.51−1.27GYSFTTTAER,
SYELPDGQVITIGNER
Cytoskeleton
704P11884Aldehyde dehydrogenase, mitochondria569666.6341%143/5616/541.121.56RVTLELGGK,
SGQQEGAK
Redox regulation
990P54921Alpha-soluble NSF attachment protein336275.350%120/5613/399.857.69QAEAMALLAEAER,
IEEACEIYAR
Transport
1002P55260Annexin A4361685.3139%145/5113/28−1.92−2.35GDTSGDYR,
WGTDEVK
Transport/Ca2+
396P48037Annexin A6761065.3952%253/5636/74−1.35−1.73YELTGKFER,
AINEAYKEDYHK
Transport/Ca2+
1116P35426Cell division protein kinase 4340066.0954%198/5615/323.53.22VTLVFEHIDQDLR,
VPNGGAAGGGLPVSTVR
Cell cycle
1319P47875Cysteine and glycine-rich protein 1214558.951%58/566/44−7.38−11.9NLDSTTVAVHGEEIYCK,
GLESTTLADKDGEIYCK
Cytoskeleton regulation
1323P47875Cysteine and glycine-rich protein 1214558.954%91/567/4013.7712.77TVYFAEEVQCEGNSFHK,
HEEAPGHRPTTNPNASK
Cytoskeleton regulation
416Q5XI50E3 ubiquitin-protein ligase MARCH7769327.6416%53/519/44−2.86−3.98MVSGNRGTSLNDSYHSR,
CTGSLQYVHQECMK
Protein degradation
709P62630Elongation factor 1-alpha 1504249.129%87/5611/35−2.01−1.1EHALLAYTLGVK,
STTTGHLIYK
Protein synthesis
1244O08719Ena/VASP-like protein421838.7420%59/516/432.271.63WVPIKPGQQGFSR,
VKPAGSVNDVGLDALDLDRMK
Cytoskeleton regulation
305Q99PF5Far upstream element-binding protein 2744666.3841%169/5620/43−2.09−2.25ERDQGGFGDR.
IGQQPQQPGAPPQQDYTK
Gene expression
886P97590Galectin-7153336.4329%52/516/43−1.41−1.58MPSSNVRSVEVGGDVQLHSVK,
MSATHHK
Apoptosis
1251Q9Z1B2Glutathione S-transferase Mu 5270676.3350%76/5615/54−4.85−9.44ITQSNAILR,
VDIMENQIMDFR
Redox regulation
1255P42930Heat shock protein beta-1229366.1256%147/5612/65−3.9−4.49KYTLPPGVDPTLVSSSLSPEGTLTVEA,
VPFSLLR
Chaperon
954Q6RUG5Islet cell autoantigen 1-like protein492985.2331%54/519/53−1.64−1.6MDSFEHLRPEDSQSVVSRMQK,
DASQELDPDTFK
Unknown
1240O35760Isopentenyl-diphosphate Delta-isomerase 1267215.5738%61/566/58−1.8−1.31MPEINASNLDEK,
AELGIPLEEVDLNEMNYLTR
Lipid synthesis
1237Q63279Keratin, type I cytoskeletal 19446095.2125%51/5110/734.023.7QGPGPFRDYSQYFK,
MSVEADINGLRR
Cytoskeleton
1219Q6QLM7Kinesin heavy chain isoform 5A1176425.5615%54/5112/43−2−2.3SLTEYMQTVELKK,
MAETNNECSIKVLCR
Transport
418P56536Kinesin heavy chain isoform 5C (Fragment)273765.8725%63/516/40−2.7−3.79FVSSPEEVMDVIDEGK,
NRHVAVTNMNEHSSR
Transport
349P48679Lamin-A745646.5448%221/5632/68−2.04−2.28LQDEMLRR,
LESSESR
Cytoskeleton
1263Q6AYP2Microfibrillar-associated protein 3-like458044.913%55/517/3012.22DEVYTIPNSLKR,
VTQFKTMEFAR
Sperm development
1355P13832Myosin regulatory light chain RLC-A199404.6762%102/5613/522.181.82DGFIDKEDLHDMLASMGK,
GNFNYIEFTR
Muscle contraction
1357Q64122Myosin regulatory light polypeptide 9197654.842%68/5110/482.121.5EAFNMIDQNR,
KGNFNYVEFTR
Muscle contraction
1276Q63716Peroxiredoxin-1 223238.2741%77/567/44−1.84−2.56ADEGISFR,
MSSGNAKIGHPAPSFK
Redox regulation
1213P97562Peroxisomal acyl-coenzyme A oxidase 2775487.6417%53/5110/412.191.96HGMHAFIVPIR,
LAWSLGWSEDGPER
lipid metabolism
1198P25113Phosphoglycerate mutase 1289286.6740%72/518/28−1.66−5.8YADLTEDQLPSCESLKDTIAR,
VLIAAHGNSLR
Glycolysis
1202P25113Phosphoglycerate mutase 1289286.6760%169/5122/591.411.56HGESAWNLENR,
FSGWYDADLSPAGHEEAK
Glycolysis
1405P62963Profilin-1151198.4675%94/5611/44−3.011.12EGVHGGLINK,
EGVHGGLINKK
Cytoskeleton regulation
1120P18420Proteasome subunit alpha type-1297846.1544%115/5612/28−1.37−1.53NQYDNDVTVWSPQGR
QECLDSR
Protein degradation
1242P40112Proteasome subunit beta type-3232356.1543%60/519/50−3.09−1.49LNLYELKEGR,
NCVAIAADRR
Protein degradation
1300P34067Proteasome subunit beta type-4293496.4534%70/5612/626.236.45FDCGVVIAADMLGSYGSLAR,
VNDSTMLGASGDYADFQYLK
Protein degradation
1321P28075Proteasome subunit beta type-5287386.5235%75/568/4815.0413.13GMGLSMGTMICGWDKR,
RGPGLYYVDSEGNR
Protein degradation
522P11598Protein disulfide-isomerase A3570445.8826%77/5611/381.311.69GFPTIYFSPANK,
IFRDGEEAGAYDGPR
Redox regulation
1301Q6IML7Rab and DnaJ domain-containing protein313298.7226%54/516/30−3.01−2.61EPLKSLR,
CIDESEGRLWAESR
Signal transduction
781P29315Ribonuclease inhibitor516534.6757%174/5618/64−1.56−1.42LSLQNCSLTEAGCGVLPDVLR,
LQLEYCNLTATSCEPLASVLR
Gene expression
952P62138Serine/threonine-protein phosphatase PP1-alpha catalytic subunit382295.9453%163/5616/49−1.44−1.52TFTDCFNCLPIAAIVDEK,
IYGFYDECK
Signal transduction
397P48721Stress-70 protein (mitochondrial)740975.9733%103/5621/79−2.12−4.1VCQGER,
DNMALQR
Chaperon
437O35814Stress-induced-phosphoprotein 1631586.420%88/5112/621.261.56AAALEFLNR,
TLLSDPTYR
Transport
1300P83941Transcription elongation factor B polypeptide 1126364.7445%56/565/626.236.45AMLSGPGQFAENETNEVNFR,
EIPSHVLSKVCMYFTYK
Gene expression
1208Q91Y78Ubiquitin carboxyl-terminal hydrolase isozyme L3262785.0163%124/5613/43−1.64−1.62HLENYDAIR,
VDLHFIALVHVDGHLYELDGR
Protein degradation

Figure 8 shows the functional distribution of identified proteins from 2D-DIGE results. Most of proteins identified using MALDI-TOF MS are related to the cytoskeleton (9%), redox regulation (9%), and protein degradation (14%), implying that quercetin can reverse ROS damage to the cytoskeleton and redox homeostasis in cardiomyocytes.

3.7. Verification by Immunoblotting and Immunostaining

The levels of the alpha-soluble NSF attachment protein (α-SNAP) and cell division protein kinase 4 (CDK4) were examined by immunoblotting or immunostaining to validate the results of 2D-DIGE analysis. These results indicate that α-SNAP and CDK4 were overexpressed in response to H2O2. However, quercetin suppressed ROS-induced α-SNAP and CDK4 protein expression in H9C2 cells (Figures 9(a) and 9(b)). These data are consistent with 2D-DIGE results.

4. Discussions

Cardiovascular diseases have become a primary health concern worldwide in recent years. Ischemia/reperfusion injury in cardiomyocytes, which leads to excess ROS generation, is a particularly serious result of cardiovascular diseases. Many studies have focused on how to alleviate ischemia reperfusion-induced ROS in cardiomyocytes. For example, many plant molecules, including resveratrol, quercetin, sasanquasaponin, proanthocyanidin, safflower, and orientin, function as protectors in ischemia/reperfusion-damaged cardiomyocytes [1923]. However, the role of quercetin in the ischemia/reperfusion injury of cardiomyocytes remains unclear.

According to previous reports, Src kinase regulates many cell signals, including cell adhesion, migration, proliferation, and apoptosis [24, 25]. During oxidative stress, Src kinase induces cell death by inactivating PI-3 K, cell migration, and spreading [26]. PP1, a Src kinase inhibitor, can rescue ROS-damaged H9C2 cells by inhibiting cell apoptosis and enhancing cell adhesion/viability [2]. However, the inhibition of Src kinase activity with PP1 is generally unsuitable for mammalian cells. Alternatively, in our findings, H9C2 cells pretreated with quercetin for 1 h are protected against H2O2-induced apoptosis in this study. The role of quercetin in H2O2-treated cardiomyocytes is to inhibit inflammatory response and maintain cell physiology, including morphology, redox status, and metabolism, by regulating Src kinase, FAK, and STAT3.

The results of this study indicate that H2O2 stimulates the tyrosine phosphorylation of Src kinase and FAK, which affect cell morphology and tight junction proteins, leading to cell detachment [27]. Quercetin, however, inhibits the tyrosine phosphorylation of Src kinase and FAK which maintain cell-cell interaction and morphology. Many studies have shown that quercetin protects retina, testis, neuron, cerebral, and cardiovascular cells from ischemia/reperfusion injury [2831]. This study further demonstrates that quercetin increases migration and survival in H2O2-treated cardiomyocytes (Figure 2).

α-SNAP is a component of the soluble N-ethylmaleimide-sensitive fusion factor attachment protein receptors (SNAREs) complex required for vesicular transport between the endoplasmic reticulum and the Golgi apparatus. The major function of α-SNAP is to recycle the SNARE complex. Several reports have shown that SNARE-dependent trafficking is required for integrin signaling through a FAK/Src/PI3 K-dependent pathway [32], and the inhibition of SNARE-mediated exocytosis attenuates ischemia/reperfusion injury [33]. α-SNAP may play a critical role in regulating Src kinase signaling and inducing ischemia/reperfusion injury in cardiomyocytes. This study shows that α-SNAP was robust to overexpression (9.85-fold) in 5 mM H2O2-treated H9C2 cells. However, pretreatment with quercetin reduced H2O2-induced α-SNAP expression. Quercetin inhibits ROS-induced α-SNAP overexpression in cardiomyocytes, which could be effectively applied for protecting cardiomyocytes from oxidative stress (Figure 10).

The major functions of the Ena/VASP-like (Evl) protein include the regulation of cytoskeletal dynamics and organization axon guidance, platelet aggregation, cell motility, and cell adhesion [34, 35]. However, several studies have shown that the Evl protein has another function in homologous pairing and strand exchange through interaction with RAD51 and RAD51B [36]. Because H2O2 targets DNA, oxidative stress causes base damage such as strand breaking in DNA. At this moment, the repaired mechanisms, including base excision repair (BER), transcription-coupled repair (TCR), mismatch repair (MMR), nonhomologous end-joining (NHEJ), translesion synthesis (TLS), global genome repair (GGR), and homologous recombination (HR), will been turned on [37]. ROS-treated cells exhibited DNA damage, stimulating homologous recombination. In this case, Evl expression increased in cardiomyocytes, but quercetin pretreatment reduced the expression of ROS-induced Evl (Figure 10). This suggests that quercetin may stabilize the DNA structure of ROS-damaged cardiomyocytes.

Isopentenyl-diphosphate delta-isomerase 1, which is located in peroxisomes, catalyzes the isomerization of 1,3-allylic rearrangement of the homoallylic substrate isopentenyl (IPP) to dimethylallyl diphosphate (DMAPP), which is a strong electrophile allylic isomer. DMAPP is also an important product in the synthesis of many lipophilic molecules such as sterols, ubiquinones, and terpenoids. Yochem et al. demonstrated that losing idi-1 gene is lethal in Caenorhabditis elegans, leading to accumulated and enlarged lysosomes and autophagosomes [38]. This study shows that ROS-treated block isopentenyl-diphosphate delta-isomerase 1 expression may induce cell death; however, quercetin pretreatment reversed isopentenyl-diphosphate delta-isomerase 1 expression in H9C2 cell (Figure 10).

Elongation factor 1-alpha (EF-1 alpha) is a multifunctions protein that promotes peptide synthesis through GTP-dependent binding of aminoacyl-tRNA to the A-site of ribosomes and binds to filamentous actin [39] and severs microtubules, leading to abnormal tetraploid cells and cell death [40]. In 1996, GaŁasiński demonstrated that quercetin prevents the peptide elongation by interacting with EF-1 alpha in plant [41]. The present data show that H2O2 downregulates the expression of EF-1 alpha in H9C2 cells, whereas quercetin pretreatment reverses the expression of EF-1 alpha. Quercetin can prevent ROS-induced cytoskeleton damage and promote protein synthesis in cardiomyocytes (Figure 10).

Cellular antioxidant enzymes including superoxide dismutases (Mn-SOD and CuZn-SOD), catalase (CAT), peroxidases, and glutathione S-transferases regulate redox homeostasis in mammalian cells. Catalase and peroxidases scavenge H2O2 or convert it to hydroxyl radicals. Superoxide dismutases convert superoxide anions (O2-) to H2O2. The observation of the oxidative state in this study demonstrates that ROS inhibits the MnSOD expression that leads to O2- accumulation in cell. However, quercetin pretreatment not only reduces ROS production, but also prevents MnSOD expression in H2O2-treated H9C2 cells (Figure 3).

Inflammation contributes to the pathophysiology of cardiac ischemia/reperfusion injury. Myocardial ischemia and reperfusion, sepsis, viral myocarditis, and immune rejection induce the inflammatory response [11]. Cardiac ischemia/reperfusion is an acute inflammatory response that may activate phospholipase A2, metabolizing arachidonic acid into inflammatory factors by cyclooxygenases (COX-1 and COX-2), cytochrome P450, and lipoxygenase. These enzymes increase ROS production in the mitochondria. Xanthine oxidase and NADPH oxidase, which produce ROS in cells, result in inflammatory gene expression. These results suggest that quercetin blocks ROS-induced inflammatory responses such as COX-2, which converts arachidonic acid to prostaglandin (Figure 10).

STAT3, which belongs to the STAT protein family, is a protein transcription factor regulating many downstream signals for cell survival, apoptosis, proliferation, angiogenesis, and metabolic and anti-oxidative pathways [42]. Previous reports mentioned that oxidative stress activates the JAK2/STAT3/IL6 signal pathway in obese Zucker rats’ fatty livers [15]. Furthermore, ROS production in hepatoma cells infected with Hepatitis C virus (HCV) activated STAT3 through JAK, Src kinase, and p38 MAP kinase pathways [43], and the decreased phosphorylation of p38 MAPK blocks the oxidative stress-induced senescence of myeloid leukemic cells [44]. PI-3K/AKT pathways, playing an important role in cell survival, proliferation, and growth, were activated by IL-1, leading to the proinflammatory gene activation of NF-κB regulation. This study shows that H2O2 induced the phosphorylation of Src kinase, AKT, p38, and STAT3 (pY-705 and pS-727) in cardiomyocytes inhibited by pretreatment with quercetin. Quercetin protects H9C2 cells from ROS-induced hyperinflammatory responses that inhibit the activation of Src, p38, and STAT3 (Figure 10).

In summary, this study shows that quercetin inhibits Src kinase, a potential therapeutic target in vitro, and kinases such as FAK, p38, and STAT3. Thus, quercetin has comprehensive effects on cardiomyocyte. The inhibition of an inflammatory response through STAT3 inactivation in cardiomyocyte may be beneficial for an ischemia/reperfusion injury model. Hence, quercetin should be tested in an animal model to verify its therapeutic role.

Abbreviations

2D-DIGE:Two-dimensional differential gel electrophoresis
FBS:Fetal bovine serum
:Hydrogen peroxide
MS:Mass spectrometry
MALDI-TOF MS:Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry
ROS:Reactive oxygen species
STAT3: Signal transducer and activator of transcription 3
DCFH-DA: 2,7-Dichlorofluorescein diacetate
FACS: Fluorescence activated cell sorting
FAK: Focal adhesion kinase.

Conflict of Interests

The authors confirm that there is no conflict of interests.

Acknowledgments

This work was supported by NSC Grants (99-2311-B-007-002, 100-2311-B-007-005, and 101–2311-B-007-011) from National Science Council, Taiwan, NTHU and CGH Grant (100N2723E1) from National Tsing Hua University, NTHU Booster Grant (99N2908E1) from National Tsing Hua University, Toward Word-ClassUniversity project from National Tsing Hua University (100N2051E1), and VGHUST Grant (99-P5-22) from Veteran General Hospitals University System of Taiwan.

References

  1. H. Takano, Y. Zou, H. Hasegawa, H. Akazawa, T. Nagai, and I. Komuro, “Oxidative stress-induced signal transduction pathways in cardiac myocytes: involvement of ROS in heart diseases,” Antioxidants and Redox Signaling, vol. 5, no. 6, pp. 789–794, 2003. View at: Google Scholar
  2. H. C. Chou, Y. W. Chen, T. R. Lee et al., “Proteomics study of oxidative stress and Src kinase inhibition in H9C2 cardiomyocytes: a cell model of heart ischemia-reperfusion injury and treatment,” Free Radical Biology and Medicine, vol. 49, no. 1, pp. 96–108, 2010. View at: Publisher Site | Google Scholar
  3. M. J. Morgan and Z. G. Liu, “Reactive oxygen species in TNFα-induced signaling and cell death,” Molecules and Cells, vol. 30, no. 1, pp. 1–12, 2010. View at: Publisher Site | Google Scholar
  4. F. Rusnak and T. Reiter, “Sensing electrons: protein phosphatase redox regulation,” Trends in Biochemical Sciences, vol. 25, no. 11, pp. 527–529, 2000. View at: Publisher Site | Google Scholar
  5. A. W. Boots, G. R. M. M. Haenen, and A. Bast, “Health effects of quercetin: from antioxidant to nutraceutical,” European Journal of Pharmacology, vol. 585, no. 2-3, pp. 325–337, 2008. View at: Publisher Site | Google Scholar
  6. J. Sanhueza, J. Valdes, R. Campos, A. Garrido, and A. Valenzuela, “Changes in the xanthine dehydrogenase/xanthine oxidase ratio in the rat kidney subjected to ischemia-reperfusion stress: preventive effect of some flavonoids,” Research Communications in Chemical Pathology and Pharmacology, vol. 78, no. 2, pp. 211–218, 1992. View at: Google Scholar
  7. B. Sun, G. B. Sun, J. Xiao et al., “Isorhamnetin inhibits H2O2-induced activation of the intrinsic apoptotic pathway in H9c2 cardiomyocytes through scavenging reactive oxygen species and ERK inactivation,” Journal of Cellular Biochemistry, vol. 113, pp. 473–485, 2012. View at: Publisher Site | Google Scholar
  8. M. Kyaw, M. Yoshizumi, K. Tsuchiya, K. Kirima, and T. Tamaki, “Antioxidants inhibit JNK and p38 MAPK activation but not ERK 1/2 activation by angiotensin II in rat aortic smooth muscle cells,” Hypertension Research, vol. 24, no. 3, pp. 251–261, 2001. View at: Publisher Site | Google Scholar
  9. D. Staedler, E. Idrizi, B. H. Kenzaoui, and L. Juillerat-Jeanneret, “Drug combinations with quercetin: doxorubicin plus quercetin in human breast cancer cells,” Cancer Chemother Pharmacol, vol. 68, pp. 1161–1172, 2011. View at: Publisher Site | Google Scholar
  10. H. Kaiserová, T. Šimůnek, W. J. F. van der Vijgh, A. Bast, and E. Kvasničková, “Flavonoids as protectors against doxorubicin cardiotoxicity: role of iron chelation, antioxidant activity and inhibition of carbonyl reductase,” Biochimica et Biophysica Acta, vol. 1772, no. 9, pp. 1065–1074, 2007. View at: Publisher Site | Google Scholar
  11. D. J. Marchant, J. H. Boyd, D. C. Lin, D. J. Granville, F. S. Garmaroudi, and B. M. McManus, “Inflammation in myocardial diseases,” Circulation Research, vol. 110, pp. 126–144, 2012. View at: Publisher Site | Google Scholar
  12. G. Muthian and J. J. Bright, “Quercetin, a flavonoid phytoestrogen, ameliorates experimental allergic encephalomyelitis by blocking IL-12 signaling through JAK-STAT pathway in T lymphocyte,” Journal of Clinical Immunology, vol. 24, no. 5, pp. 542–552, 2004. View at: Publisher Site | Google Scholar
  13. A. Tyagi, C. Agarwal, L. D. Dwyer-Nield, R. P. Singh, A. M. Malkinson, and R. Agarwal, “Silibinin modulates TNF-alpha and IFN-gamma mediated signaling to regulate COX2 and iNOS expression in tumorigenic mouse lung epithelial LM2 cells,” Molecular Carcinogenesis, vol. 51, no. 10, pp. 832–842, 2012. View at: Publisher Site | Google Scholar
  14. T. Yagi, H. Yoshioka, T. Wakai, T. Kato, T. Horikoshi, and H. Kinouchi, “Activation of signal transducers and activators of transcription 3 in the hippocampal CA1 region in a rat model of global cerebral ischemic preconditioning,” Brain Research, vol. 1422, pp. 39–45, 2011. View at: Publisher Site | Google Scholar
  15. G. S. Dikdan, S. C. Saba, A. N. Dela Torre, J. Roth, S. Wang, and B. Koneru, “Role of oxidative stress in the increased activation of signal transducers and activators of transcription-3 in the fatty livers of obese Zucker rats,” Surgery, vol. 136, no. 3, pp. 677–685, 2004. View at: Publisher Site | Google Scholar
  16. C. C. Chen, Y. C. Lu, Y. W. Chen et al., “Hemopexin is up-regulated in plasma from type 1 diabetes mellitus patients: role of glucose-induced ROS,” Journal of Proteomics, vol. 75, no. 12, pp. 3760–3777, 2012. View at: Publisher Site | Google Scholar
  17. C. L. Wu, H. C. Chou, C. S. Cheng et al., “Proteomic analysis of UVB-induced protein expression- and redox-dependent changes in skin fibroblasts using lysine- and cysteine-labeling two-dimensional difference gel electrophoresis,” Journal of Proteomics, vol. 75, pp. 1991–2014, 2012. View at: Publisher Site | Google Scholar
  18. K. Raedschelders, D. M. Ansley, and D. D. Chen, “The cellular and molecular origin of reactive oxygen species generation during myocardial ischemia and reperfusion,” Pharmacology & Therapeutics, vol. 133, pp. 230–255, 2012. View at: Google Scholar
  19. J. T. Hwang, D. Y. Kwon, O. J. Park, and M. S. Kim, “Resveratrol protects ROS-induced cell death by activating AMPK in H9c2 cardiac muscle cells,” Genes and Nutrition, vol. 2, no. 4, pp. 323–326, 2008. View at: Publisher Site | Google Scholar
  20. Z. Liao, D. Yin, W. Wang et al., “Cardioprotective effect of sasanquasaponin preconditioning via bradykinin-NO pathway in isolated rat heart,” Phytotherapy Research, vol. 23, no. 8, pp. 1146–1153, 2009. View at: Publisher Site | Google Scholar
  21. Z. H. Shao, K. R. Wojcik, A. Dossumbekova et al., “Grape seed proanthocyanidins protect cardiomyocytes from ischemia and reperfusion injury via Akt-NOS signaling,” Journal of Cellular Biochemistry, vol. 107, no. 4, pp. 697–705, 2009. View at: Publisher Site | Google Scholar
  22. S. Y. Han, H. X. Li, X. Ma, K. Zhang, Z. Z. Ma, and P. F. Tu, “Protective effects of purified safflower extract on myocardial ischemia in vivo and in vitro,” Phytomedicine, vol. 16, no. 8, pp. 694–702, 2009. View at: Publisher Site | Google Scholar
  23. N. Lu, Y. Sun, and X. Zheng, “Orientin-induced cardioprotection against reperfusion is associated with attenuation of mitochondrial permeability transition,” Planta Medica, vol. 77, no. 10, pp. 984–991, 2011. View at: Publisher Site | Google Scholar
  24. S. Huveneers and E. H. J. Danen, “Adhesion signaling—crosstalk between integrins, Src and Rho,” Journal of Cell Science, vol. 122, no. 8, pp. 1059–1069, 2009. View at: Publisher Site | Google Scholar
  25. A. B. Stein, X. L. Tang, Y. Guo, Y. T. Xuan, B. Dawn, and R. Bolli, “Delayed adaptation of the heart to stress: late preconditioning,” Stroke, vol. 35, no. 11, pp. 2676–2679, 2004. View at: Publisher Site | Google Scholar
  26. M. A. Krasilnikov, “Phosphatidylinositol-3 kinase sependent pathways: the role in control of cell growth, survival, and malignant transformation,” Biochemistry, vol. 65, no. 1, pp. 59–67, 2000. View at: Google Scholar
  27. S. Miravet, J. Piedra, J. Castaño et al., “Tyrosine phosphorylation of plakoglobin causes contrary effects on its association with desmosomes and adherens junction components and modulates β-catenin-mediated transcription,” Molecular and Cellular Biology, vol. 23, no. 20, pp. 7391–7402, 2003. View at: Publisher Site | Google Scholar
  28. M. Aldemir, G. Ozgun, E. Onen, E. Okulu, and O. Kayigil, “Quercetin has a protective role on histopathological findings on testicular ischaemia-reperfusion injury in rats,” Andrologia, vol. 44, supplement 1, pp. 479–483, 2012. View at: Publisher Site | Google Scholar
  29. D. Dekanski, V. Selakovic, V. Piperski, Z. Radulovic, A. Korenic, and L. Radenovic, “Protective effect of olive leaf extract on hippocampal injury induced by transient global cerebral ischemia and reperfusion in Mongolian gerbils,” Phytomedicine, vol. 18, pp. 1137–1143, 2011. View at: Google Scholar
  30. A. K. Pandey, P. P. Hazari, R. Patnaik, and A. K. Mishra, “The role of ASIC1a in neuroprotection elicited by quercetin in focal cerebral ischemia,” Brain Research, vol. 1383, pp. 289–299, 2011. View at: Publisher Site | Google Scholar
  31. X. Cao, M. Liu, J. Tuo, D. Shen, and C. C. Chan, “The effects of quercetin in cultured human RPE cells under oxidative stress and in Ccl2/Cx3cr1 double deficient mice,” Experimental Eye Research, vol. 91, no. 1, pp. 15–25, 2010. View at: Publisher Site | Google Scholar
  32. M. Skalski, N. Sharma, K. Williams, A. Kruspe, and M. G. Coppolino, “SNARE-mediated membrane traffic is required for focal adhesion kinase signaling and Src-regulated focal adhesion turnover,” Biochimica et Biophysica Acta, vol. 1813, no. 1, pp. 148–158, 2011. View at: Publisher Site | Google Scholar
  33. J. W. Calvert, S. Gundewar, M. Yamakuchi et al., “Inhibition of N-ethylmaleimide-sensitive factor protects against myocardial ischemia/reperfusion injury,” Circulation Research, vol. 101, no. 12, pp. 1247–1254, 2007. View at: Publisher Site | Google Scholar
  34. L. D. Hu, H. F. Zou, S. X. Zhan, and K. M. Cao, “EVL (Ena/VASP-like) expression is up-regulated in human breast cancer and its relative expression level is correlated with clinical stages,” Oncology Reports, vol. 19, no. 4, pp. 1015–1020, 2008. View at: Google Scholar
  35. S. J. Wanner, M. C. Danos, J. L. Lohr, and J. R. Miller, “Molecular cloning and expression of Ena/Vasp-like (Evl) during Xenopus development,” Gene Expression Patterns, vol. 5, no. 3, pp. 423–428, 2005. View at: Publisher Site | Google Scholar
  36. M. Takaku, S. Machida, N. Hosoya et al., “Recombination activator function of the novel RAD51- and RAD51B-binding protein, human EVL,” Journal of Biological Chemistry, vol. 284, no. 21, pp. 14326–14336, 2009. View at: Publisher Site | Google Scholar
  37. G. Slupphaug, B. Kavli, and H. E. Krokan, “The interacting pathways for prevention and repair of oxidative DNA damage,” Mutation Research, vol. 531, no. 1-2, pp. 231–251, 2003. View at: Publisher Site | Google Scholar
  38. J. Yochem, D. H. Hall, L. R. Bell, E. M. Hedgecock, and R. K. Herman, “Isopentenyl-diphosphate isomerase is essential for viability of Caenorhabditis elegans,” Molecular Genetics and Genomics, vol. 273, no. 2, pp. 158–166, 2005. View at: Publisher Site | Google Scholar
  39. T. Izawa, Y. Fukata, T. Kimura, A. Iwamatsu, K. Dohi, and K. Kaibuchi, “Elongation factor-1α is a novel substrate of Rho-associated kinase,” Biochemical and Biophysical Research Communications, vol. 278, no. 1, pp. 72–78, 2000. View at: Publisher Site | Google Scholar
  40. Y. Kobayashi and S. Yonehara, “Novel cell death by downregulation of eEF1A1 expression in tetraploids,” Cell Death and Differentiation, vol. 16, no. 1, pp. 139–150, 2009. View at: Publisher Site | Google Scholar
  41. W. Gałasiński, “Eukaryotic polypeptide elongation system and its sensitivity to the inhibitory substances of plant origin,” Proceedings of the Society for Experimental Biology and Medicine, vol. 212, no. 1, pp. 24–37, 1996. View at: Google Scholar
  42. H. Wang, F. Lafdil, X. Kong, and B. Gao, “Signal transducer and activator of transcription 3 in liver diseases: a novel therapeutic target,” International Journal of Biological Sciences, vol. 7, no. 5, pp. 536–550, 2011. View at: Google Scholar
  43. G. Waris, J. Turkson, T. Hassanein, and A. Siddiqui, “Hepatitis C virus (HCV) constitutively activates STAT-3 via oxidative stress: role of STAT-3 in HCV replication,” Journal of Virology, vol. 79, no. 3, pp. 1569–1580, 2005. View at: Publisher Site | Google Scholar
  44. Y. Xiao, P. Zou, J. Wang, H. Song, J. Zou, and L. Liu, “Lower phosphorylation of p38 MAPK blocks the oxidative stress-induced senescence in myeloid leukemic CD34+CD38- cells,” Journal of Huazhong University of Science and Technology, vol. 32, no. 3, pp. 328–333, 2012. View at: Publisher Site | Google Scholar

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