ORIGINAL ARTICLE
Cytoprotection of Baicalein Against Oxidative Stress-induced Cardiomyocytes Injury Through the Nrf2/Keap1 Pathway Guozhen Cui, PhD,*† Sharon Chui Wah Luk, PhD,* Ronald Adolphus Li, PhD,‡ Ken Kwok Keung Chan, PhD,‡ Si Wan Lei, MSc,* Liang Wang, MSc,* Huifang Shen, PhD,† George Pak Heng Leung, PhD,§ and Simon Ming Yuen Lee, PhD*
Abstract: Baicalein is one of the major flavonoids found in the root of Scutellaria baicalensis Georgi. Previous studies suggest that baicalein displays protective effect on experimental cardiac models in vitro and in vivo. However, the mode of action remains unclear. Here, we showed that baicalein conferred cardioprotective effect against oxidative stress-induced cell injury in H9c2 cells and human embryonic stem cells-derived cardiomyocytes. Immunoprecipitation with anti-NF-E2–related factor 2 (Nrf2) antibody in baicalein-treated cells demonstrated that baicalein effectively disrupted the association between Nrf2 and Kelch-like epichlorohydrin-associated protein 1 (Keap1). In addition, the unbounded Nrf2 translocated from cytoplasm to nucleus and increased Nrf2/heme oxygenase-1 (HO-1) content in a time-dependent manner. Moreover, antioxidant response element transcriptional activity was enhanced by baicalein treatment, and the Nrf2 siRNA transfection could block the cytoprotective effect of baicalein. Taken together, these results demonstrate that baicalein protected cardiomyocytes against oxidative stress-induced cell injury through the Nrf2/Keap1 pathway. Key Words: baicalein, cardioprotection, oxidative stress, Nrf2 (J Cardiovasc Pharmacol 2015;65:39–46)
Received for publication May 1, 2014; accepted August 5, 2014. From the *State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China; †Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai, China; ‡Stem Cell and Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; and §Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China. Supported by the Science and Technology Development Fund of Macao SAR (Ref. No. 014/2011/A1), Overseas and Hong Kong, Macau Young Scholars Collaborative Research Fund by the Natural National Science Foundation of China (Ref. No. 81328025), Research Committee, University of Macau, the Research Grant Council of HKSAR (TBRS, T13-706/11), SCRMC, and Faculty Cores of the University of Hong Kong. The authors report no conflicts of interest. Reprints: George Pak Heng Leung, PhD, Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, 999077 China (e-mail: [email protected]), or and Simon Ming Yuen Lee, PhD, State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, 999078 China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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INTRODUCTION Cardiovascular diseases are the leading cause of death worldwide, and majority of the cases are ischemic heart disease (IHD).1,2 IHD develops when cholesterol in the blood begins to accumulate on the walls of the arteries, which narrows the arteries and eventually blocks the blood flow. A decrease in the amount of oxygen supplied to the heart muscle may result in myocardial infarction.3 Because of the size of the effected population, an effective strategy to reduce the global burden of IHD is urgently needed.4 Fukuda et al5 had demonstrated that Human Embryonic Kidney cells treated with tert-butyl hydroperoxide (t-BHP), a hydrogen peroxide donor, can mimic oxidative stress in myocardial ischemia. H9c2 cell line derived from embryonic rat heart maintains some features of the cardiomyocytes.6,7 Hence, it has been extensively used as an in vitro heart model. In this study, we used t-BHP–treated H9c2 as a myocardial ischemia model to study the protective mechanism against oxidative stress in patients with IHD. Transcriptional factor NF-E2–related factor 2 (Nrf2) is thought to act as a cellular defense system against oxidative stress.8 Under normal or unstressed condition, Nrf2 is kept in the cytoplasm by a cluster of proteins, Keap1 and Cullin 3 (Cul3). Keap1 is a substrate adapter, which helps Cul3 ubiquitinate Nrf2.9 When Nrf2 is ubiquitinated, it is transported to the proteasome where it is degraded, and the components are recycled. Under oxidative or electrophilic stress, the cysteine residues in Keap1 are disrupted, thus upsetting the Keap1-Cul3 ubiquitination system. When Nrf2 is not ubiquitinated, it will build up in the cytoplasm and translocates into the nucleus. In the nucleus, it will form heterodimer with small Maf protein and binds to the antioxidant response element (ARE), an upstream promoter region of many antioxidative genes, and initiates their transcription, thereby conferring broader protective roles.10,11 Baicalein is one of the main flavonoids derived from the root of S. baicalensis Georgi. It exhibits multiple pharmacological activities, such as antioxidation,12,13 anti-inflammatory,14 anticancer,15 and antiallergic effect.16 Because of its antioxidative and/or anti-inflammatory activities, most studies have been focused on its protective action in neurodegenerative disease.17,18 In the last decade, the cardioprotective effect of baicalein on various in vitro and in vivo experimental models has been reported. It was demonstrated that baicalein exerts protective effect against hypoxia-reoxygenation and doxorubicin-induced cell damage in chick cardiomyocytes.19–21 In addition, the recent www.jcvp.org |
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studies by Zong et al22 revealed that baicalein also attenuates cardiac hypertrophy. However, its molecular mechanism has not been clearly established, some had suggested that it is mediated by antioxidant and others had suggested by prooxidant.21,23,24 The aim of this study was to examine the molecular mechanism of the protective effect of baicalein in oxidative stress-induced cell injury in cardiomyocytes.
MATERIALS AND METHODS Materials Baicalein (98% purity) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Gibco/life Technologies (Grand Island, NY). A 5-(6-)chloromethyl-20 ,70 -dichlo-rodihydrofluorescein diacetate (CM-H2DCFDA), LIVE/DEAD Viability/Cytotoxicity Assay Kit, and Opti-MEM I Reduced-Serum Medium were supplied by Invitrogen (Eugene, OR). Nrf2-siRNA and scrambled RNA were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Cignal Antioxidant Response Reporter (luciferase) Kit and SureFECT Transfection Reagent were purchased from SABiosciences (Frederick, MD). The DualLuciferase Reporter Assay System was purchased from Promega (Madison, WI). All other chemicals and reagents used in this study are analytical grade.
H9c2 Cell Culture Rat embryonic heart-derived H9c2 cells are widely used to investigate the molecular mechanism is involved in cardiomyocyte survival. The H9c2 cell line was purchased from America Tissue Type Collection (Manassas, VA). Cells were grown in DMEM medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in a 75-cm2 tissue culture flask at 378C, 5% CO2. The cells were fed every 2–3 days, and they were subcultured when reached 70%–80% confluence.
Live/Dead Cell Viability Staining and MTT Assay To measure cell viability, H9c2 cells were seeded in 96well plate at a density of 5 · 103 cells per well. They were grown at 378C for 24 hours in complete medium until confluence is above 80%. Cells were pretreated with various concentrations of baicalein (0.3–30 mM) for 4 hours before they were treated with 150 mM t-BHP for 4 hours. Cell viability staining was performed using the Live/Dead viability/ cytotoxicity kit according to manufacturer’s instructions. Fluorescence imaging of cells was performed using an IN Cell Analyzer 2000 (GE Healthcare), and cell survival was quantified using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay.
with 5 mM CM-H2DCFDA at 378C for 20 minutes in the dark; and then, cells were treated with baicalein (30 mM) for 4 hours and exposed to t-BHP for 1 hour. The fluorescence intensity was measured at 530 nm using a fluorescence microplate reader.
Human ESC-CMs Culture The human embryonic stem cell line (HES-2) was obtained from the WiCell Research Institute (Madison, WI, http://www.wicell.org), and they were induced as previously described.25,26 This differentiation protocol yields more than 90% spontaneous beating cardiospheres. Cardiospheres were cultured in suspension with cardiomyocytes maintenance medium in low attachment 6-well plate (CLS3471; Corning Costar Corp, Cambridge, MA). The maintenance medium contains 500 mL basic liquid media StemPro 34 (Invitrogen Corp, Grand Island, CA), 2.5 mL ascorbic acid (10 mg/mL; final concentration of 50 mg/mL), 1% L-glutamine, 1% penicillin, and 1% streptomycin. For the cardioprotective study, the spontaneous contracting cardiospheres were dissociated into single cells and plated in a gelatin-coated 96-well plate and cultured for 2 days in H1-DIF medium containing 10% fetal bovine serum, 77% DMEM, 1% Penicillin/Streptomycin, 1% nonessential amino acids, and 1% L-glutamine.
Hypoxia-induced Cell Injury in In Vitro Study The protective effect of baicalein was further confirmed by pretreating H9c2 cells and human embryonic stem cells-derived cardiomyocytes (hESC-CMs) with 3, 10, or 30 mM of baicalein or vehicle for 2 hours before hypoxia treatment. To mimic pathological ischemic injury in in vitro, both cell lines were subjected to hypoxia for 3 hours (94.5% N2, 0.5% O2, and 5% CO2) using a hypoxia workstation INVIVO 2400 with Ruskin hypoxic gas mixer (Biotrace, Bridgend, United Kingdom). Cellular injury was determined by measuring the activity of lactate dehydrogenase (LDH) released into the incubation medium. Released LDH activity was determined by the cytotoxicity detection kit according to the manufacturer’s instructions. Absorbance was measured using a microplate reader at 490 nm.
Preparation of Whole Cell, Cytosolic, and Nucleus Extracts For the preparation of the whole cell lysate, H9c2 cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated on ice for 30 minutes with radio-immunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, MO) and 1% Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN). Cell lysates were centrifuged at 12,500g for 20 minutes at 48C. Supernatant was collected. The subcellular fraction was obtained using a nucleus extraction kit (Cayman Chemical, Ann Arbor, MI) according to previously published method.27 Protein concentration was quantified using the bicinchoninic acid (BCA) protein quantification kit (Pierce Chemical Co, Rockford, IL).
Detection of Intracellular Reactive Oxygen Species
Immunoblot Analysis
Intracellular reactive oxygen species (ROS) in H9c2 cells was evaluated using CM-H2DCFDA with a fluorescence microplate reader. Cells were seeded in a 96-well plate and incubated
Lysates from H9c2 cells were immunoblotted according to the previously described method.28 After blocking with 5% nonfat dry milk, membrane was incubated overnight at
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48C with primary antibodies. Anti-Keap1, anti-b-actin, and horseradish peroxidase-conjugated antirabbit antibodies were purchased from Cell Signaling Technology (Beverly, MA). HO-1 monoclonal antibody was purchased from Abcam (Cambridge, MA), and anti-Nrf2 monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). After incubation with primary antibodies, membranes were incubated with secondary antibodies for 1 hour. To confirm equal protein loading, membranes were incubated with anti-b–actin antibody. Proteins were detected using an advanced enhanced chemiluminescence (ECL) system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Semiquantification of the proteins was performed with densitometric analysis using Quantity One (Biorad).
(Cell Signaling Technology, Beverly, MA) for 4 hours at 48C. Immune complexes were boiled for 5 minutes with ·2 loading buffer, followed by immunoblot analysis with Keap1 and Nrf2 antibodies.
Immunofluorescence Staining
Baicalein Increased Cell Viability and Decreased ROS Release in t-BHP–treated H9c2 Cells
Cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Then cells were washed with PBS containing 1% bovine serum albumin and incubated with blocking buffer (0.3% Triton X-100% and 1% bovine serum albumin in PBS) for 45 minutes at room temperature. After blocking, cells were incubated with anti-Nrf2 antibody (1:200) overnight at 48C and with Alexa Fluor 488-conjugated antibody (1:200) (Invitrogen, Eugene, OR) for 45 minutes at 378C. To visualize the nucleus, cells were counterstained with 300 nM 40 ,6-diamidino2-phenylindole (DAPI) for 10 minutes. The fluorescence images were captured using a fluorescence microscope.
Transient Transfection With Nrf2 siRNA
H9c2 cells were seeded at 5 · 103 cells per well in a 96-well plate to achieve 40%–60% confluence and transfected with 10 pm Nrf2 siRNA or scrambled siRNA using SureFECT transfection reagent according to manufacturer’s instructions. The transfected cells were treated with baicalein for 4 hours and then with 150 mM t-BHP for 4 hours. Cell viability was determined using MTT assay.
Nrf2 Transcriptional Activity The Nrf2 transcriptional activity was monitored using the ARE reporter assay. This system consists of Nrf2responsive luciferase construct and constitutively expresses Renilla construct in a 40:1 ratio (0.1 mg/mL). The Renilla luciferase reporter gene is used to normalize transfection efficiencies and to monitor cell viability. The H9c2 cells were seeded in a 96-well plate at a density of 5 · 103 cells per well. After 24 hours, H9c2 cells were transfected with 150 ng of ARE reporter construct for 12 hours using SureFECT transfection reagent according to manufacturer’s protocol. After another 12 hours, the cells were treated with either vehicle (0.1% dimethyl sulfoxide [DMSO]) or baicalein for 4 hours. Firefly and Renilla luciferase activities were measured using the Dual-Glo luciferase assay system.
Immunoprecipitation To determine the amount of Keap1-bound Nrf2, whole cell lysates were incubated with anti-Nrf2 antibody overnight at 48C under agitation. The antigen–antibody complex was pulled down after incubation with protein G-agarose 2014 Lippincott Williams & Wilkins
Statistical Analysis All assays were performed at least 3 times. Values were expressed as mean 6 SD. Statistical analysis was performed by one-way analysis of variance followed by Tukey’s multiple comparison tests using Graph Pad Prism 5.0. A value of P , 0.05 was considered to be statistically significant.
RESULTS
To determine whether baicalein alone has a toxic effect in H9c2 cells, cell viability was evaluated after 4 hours incubation with baicalein (0.3–30 mM) using fluorescence imaging. The results of the assay indicated that baicalein did not exhibit any toxic effect in H9c2 cells at the concentration tested (Fig. 1A). The morphological characteristics of the baicalein-treated cells are similar to the control. The testing method was also used to study the protective effect of baicalein in t-BHP–treated H9c2 cells. Fluorescence images and the results of the MTT assay both showed that pretreatment with baicalein could increase the cell viability of the t-BHP–treated H9c2 cells (Figs. 1A, B). The MTT assay showed that pretreatment with baicalein significantly increased cell viability in a concentration-dependent manner, P , 0.01 at 3 mM and P , 0.001 at 10 and 30 mM (Fig. 1B). The cytoprotective effect of baicalein in oxidative stress-induced by t-BHP treatment was further evaluated using CM-H2DCFDA. The results showed that treatment with 150 mM t-BHP triggered rapid ROS accumulation in cells. In contrast, pretreatment with 3, 10, or 30 mM baicalein significantly reduced the levels of ROS, and it was in a concentration-dependent manner, P , 0.001 (Fig. 1C).
Baicalein Prevented Against Hypoxia-induced Cell Injury in H9c2 Cells and hESC-CMs To mimic pathological ischemic injury in in vitro, H9c2 cells, and hESC-CMs were subjected to hypoxia for 3 hours (94.5% N2, 0.5% O2, and 5% CO2). The protective effect of baicalein was studied by pretreating the cells with 3, 10, or 30 mM of baicalein for 2 hours before hypoxia treatment. It demonstrated that hypoxia increased LDH release by more than 2-fold versus the normoxia group (P , 0.05) in both cell lines (Figs. 1D, E). As shown in Figure 1D, incubation with 30 mM of baicalein displayed significant protective effects against hypoxia-induced cell injury in H9c2 cells (P , 0.05). In contrast, pretreatment with baicalein before hypoxia significantly reduced the release of LDH in all 3 tested concentrations in hESC-CMs (P , 0.05). However, there was no significant difference in the reduction of LDH release amongst the 3 baicalein dosage groups (3, 10, 30 mM). All 3 groups showed a similar level of LDH release (Fig. 1E). www.jcvp.org |
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FIGURE 1. Protective effects of baicalein against oxidative stress-induced cell injury. A, Representative images of H9c2 cells after 4 hours incubation of control, baicalein (30 mM), t-BHP (150 mM), or t-BHP (150 mM) in the presence of baicalein (30 mM). Live cells were stained green, and the nuclei of the dead cells were stained red. B, Cell viability was significantly reduced with 4-hour incubation of 150 mM t-BHP. Pretreatment with baicalein increased cell viability in a concentration-dependent manner in H9c2 cells. C, ROS was significantly reduced with the pretreatment of 3, 10, or 30 mM baicalein in H9c2 cells (P , 0.001). Baicalein attenuated hypoxiainduced cell injury in H9c2 cells (D) and in hESC-CMs (E). #P , 0.05 versus normoxia group, *P , 0.05 versus hypoxia group. ***P , 0.001 versus hypoxia group.
Baicalein Increased Nrf2 and HO-1 Protein Expression in a Concentration- and Time-dependent Manner in H9c2 Cells Under stress conditions, adaptive survival response will trigger the upregulation of the cytoprotective genes through transcription factors, such as Nrf2 to protect cells from detrimental oxidant-induced cellular and tissue death.29 To investigate whether the cytoprotective effect of baicalein was mediated through the activation of Nrf2, proteins content of Nrf2, and antioxidant enzyme, HO-1 were measured. H9c2 cells were incubated with either 3, 10, or 30 mM baicalein for 4 hours or with 30 mM baicalein for 1, 2, and 4 hours. After 4 hours of 10 and 30 mM baicalein treatment, the expression level of both Nrf2 and HO-1 proteins was significantly increased as compared with the control, P , 0.05 (Fig. 2A). Similarly, treatment with 30 mM baicalein for 4 hours significantly increased both the Nrf2 and HO-1 proteins expression with P , 0.05 (Figs. 2A, B). The data suggested that it requires about 4 hours of either 10 or 30 mM baicalein pretreatment to significantly increase the proteins expression of Nrf2 and HO-1 in H9c2 cells.
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Baicalein Activated Nrf2 Pathway in H9c2 Cells Under stressed condition, Nrf2 is translocated to the nucleus and binds to the ARE located in the promoter region of various antioxidant and detoxifying enzymes.29 The localization of Nrf2 was studied using immunofluorescence and immunoblot assays. The results of the cell immunofluorescence assay demonstrated that baicalein treatment increased the accumulation of Nrf2 in the nucleus, which is the active form of Nrf2 (Fig. 3B). Similar result was also observed in the immunoblot assay; it showed that baicalein treatment promoted the accumulation of Nrf2 in the nucleus fraction and a reduction in the cytoplasmic fraction in a time-dependent manner (Fig. 3A). The translocation of Nrf2 to the nucleus was observed at 2 hours of 30 mM baicalein treatment, and it is more prominent at 4 hours. Nrf2-driven transcriptional activity induced by baicalein was also investigated using the ARE-luciferase reporter assay. The result showed that baicalein significantly increased the ARE–luciferase transcription activity in a concentrationdependent manner with P , 0.05 in all 3 baicalein concentrations (3, 10, and 30 mM; Fig. 3C). This was in agreement 2014 Lippincott Williams & Wilkins
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FIGURE 2. Baicalein significantly increased proteins expression of Nrf2 and HO-1 in a concentration- and time-dependent manner. A, H9c2 cells were incubated with baicalein at 3, 10, or 30 mM for 4 hours or (B) at 30 mM baicalein for 1, 2, or 4 hours. Immunoblot analysis was performed using Nrf2 and HO-1 antibodies as described in Methods. Results expressed as mean 6 SD of 3 independent experiments, #P , 0.05 versus control group.
with the results of the immunofluorescence and immunoblot assays, which showed an increase of Nrf2 in the nucleus fraction.
Nrf2 siRNA Transfection Abolished the Cytoprotective Effect of Baicalein in H9c2 Cells Nrf2 siRNA was used to investigate whether baicalein exerted cytoprotection against oxidative DNA damageinduced by t-BHP treatment is Nrf2-dependent. As shown in Figure 3D, Nrf2 siRNA significantly decreased cell viability with P , 0.05 in baicalein and t-BHP–treated cells and inhibited the cytoprotective effect of baicalein. However, the scrambled control siRNA did not affect the cell viability in these cells. Thus, the result suggested that the cytoprotective effect of baicalein against oxidative stress-induced cell injury was dependent on Nrf2 pathway (Fig. 4).
Baicalein Disrupted the Nrf2/Keap1 Complex The effect of baicalein on Nrf2/Keap1 complex was examined using immunoprecipitation with anti-Nrf2 antibody, and the Keap1 protein was quantified using immunoblot analysis. The results showed that baicalein effectively disrupted the interaction between Nrf2 and Keap1 in a timedependent manner (Fig. 3E). The association between Keap1 and Nrf2 began to decrease after 1-hour treatment of 30 mM baicalein, and it continues to decrease at 2 and 4 hours. This was in agreement with the results of the immunofluorescence and immunoblot assays, which showed an increase of Nrf2 in the nucleus fraction. 2014 Lippincott Williams & Wilkins
DISCUSSION In the past decade, several reports had demonstrated that baicalein has a cytoprotective effect in neonatal rat and chick cardiomyocytes; however, there is no agreement on its precise mechanism of action. In this study, we showed that baicalein effectively protected H9c2 cells against t-BHP– treated cell injury and protected both H9c2 and hESC-CMs against hypoxia-induced cell injury. The data demonstrate that baicalein exerts its cytoprotection through the activation of the Nrf2/Keap1 signaling pathway, which is in agreement with our previous finding that the same pathway is involved in the protective effect against neurotoxin-induced cell injury in vitro by baicalein.30 Myocardial death results from ischemia/reperfusion are associated with the overproduction of ROS.31 Thus, in theory, by decreasing the production of ROS, it could attenuate ROS-induced myocardial dysfunction. In this study, cells exposed to t-BHP or hypoxia were used as the oxidative stress models for heart ischemia and/or reperfusion-induced myocardial damage. The data showed that pretreatment of H9c2 cells with baicalein significantly inhibited t-BHP–induced ROS in a concentration-dependent manner and resulted in an increase of cell viability. Similar result was also observed in both cell lines using the LDH assay. LDH is an indicator for cells viability because when tissue breakdown, LDH is released. Our result showed that baicalein obviously reduced LDH release in hypoxia-exposed H9c2 cells and hESC-CMs (Figs. 1D, E). To the best of our knowledge, this is the first report showing that baicalein reduced cell injury in t-BHP–treated H9c2 cells and in hypoxia-exposed H9c2 cells and hESC-CMs. www.jcvp.org |
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FIGURE 3. The effects of baicalein on Nrf2/Keap1 signaling pathway. A, Immunoblot assay of the nucleus and cytoplasmic extractions showed an increase of Nrf2 in the nucleus fraction and a decrease in the cytoplasmic fraction. B, Immunofluorescence staining of H9c2 cells treated with 30 mM baicalein for 4 hours. Left panel: red fluorescence showed the localization of Nrf2; Middle panel: Stained nucleus with DAPI; Right panel: Merged images of red and blue filters. C, H9c2 cells were transfected with ARE-reporter luciferase plasmid as described in Methods and treated with baicalein (3, 10, or 30 mM). After 4 hours, cells were collected, and the luciferase activity was analyzed. Baicalein treatment (3, 10, or 30 mM) significantly increased the ARE–luciferase activity (P , 0.01). D, Transfection of Nrf2-siRNA significantly reduced the protective effect of baicalein (P , 0.05). E, The Nrf2/ Keap1 complex was disrupted by baicalein. H9c2 cells were treated with 30 mM baicalein for 1, 2, or 4 hours; cell lysates were prepared for immunoprecipitaion (IP) using Nrf2 antibody. The Nrf2-associated Keap1 was assessed through immunoblot analysis (IB). The precipitates were blotted with anti-Nrf2 antibody to ensure that an equal amount of Nrf2 was pulled down under each condition. #P , 0.001 versus control group, ***P , 0.001 versus t-BHP group, *P , 0.001 versus BAI + t-BHP group.
Transcription factor Nrf2 was suggested to serve as a promising therapeutic target for cardiovascular diseases. Research by Calvert et al32 has shown that endogenously produced hydrogen sulfide (H2S) mediates cardioprotective effect against cardiac ischemia/reperfusion through Nrf2 signaling in vivo. Nrf2 activators tert-butylhydroquinone (30 mM) and sulforaphane (20 mM) induce Nrf2 nucleus translocation to confer the protective effects in H9c2 cardiomyoblast cells and HK2 renal tubular epithelial cells, respectively.33,34 The effective concentrations of these 2 compounds are similar to that of baicalein (10–30 mM) found in this study. It was also shown that the cardioprotective effect of MG132 and 4-hydroxy-2-nonenal was blocked in Nrf2 knockout mice.35,36 In addition, HO-1, a downstream target of the Nrf2 pathway, has shown to have an important cardioprotective adaptation that can counterbalance the pathological changes in mice’s chronic heart failure.37,38 However, the protective characteristics of Nrf2 signaling pathway could also promote cancer growth and drug resistance in tumor
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cells.39 This discrepancy might be explained by the different cell types used. Nonetheless, there is a possibility that baicalein pretreatment could also promote tumor growth despite its reported potential anticancer activity through inducing cell cycle arrest and apoptosis.40 The role of Nrf2 pathway activated by baicalein in mediating the anticancer effects would be another interesting area that is worth investigating in the future. This study provides evidence that baicalein protects t-BHP–treated H9c2 cells by facilitating the dissociation of the Nrf2/Keap1 complex. The unbounded Nrf2 protein translocates into the nucleus and binds to the ARE where it initiates the transcription of HO-1. This is in agreement with the observation that Nrf2 siRNA can abolish the cytoprotective effect of baicalein in t-BHP–treated H9c2 cells. Hence, the data suggested that the protective effect of baicalein is mediated through the Nrf2/Keap1 signaling pathway. There are several kinases including phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases that act 2014 Lippincott Williams & Wilkins
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FIGURE 4. Proposed mechanism of cytoprotective effect against oxidative stress-induced cardiomyocytes injury by baicalein. Baicalein activates Nrf2 by facilitating the dissociation of Nrf2/ Keap1 complex, which leads to the nucleus translocation of Nrf2. Nucleus Nrf2 then binds to AREs and initiates the upregulation of HO-1 to confer the cytoprotective effect against oxidative stress-induced cardiomyocytes injury.
as the primary sensors for the activation of the Nrf2 pathway.41,42 Whether baicalein activates the Nrf2 signaling pathway by binding directly to Nrf2 and/or Keap1 or an upstream element such as phosphatidylinositol 3-kinase remains to be determined. In summary, our results demonstrated that baicalein possesses protective effect against oxidative stress-induced cell injury in H9c2 cells and hESC-CMs. This protective effect, in part, is regulated by Nrf2-dependent signaling pathway. These results, combined with previous publications showing the important role of Nrf2 in protection against cardiovascular disease, suggested that baicalein might be a candidate for the prevention of myocardial damage in IHD. REFERENCES 1. Mathers CD, Boerma T, Ma Fat D. Global and regional causes of death. Br Med Bull. 2009;92:7–32. 2. Lopez AD, Murray CCJL. The global burden of disease, 1990-2020. Nat Med. 1998;4:1241–1243. 3. Reimer KA, Jennings RB. The changing anatomic reference base of evolving myocardial infarction. Underestimation of myocardial collateral blood flow and overestimation of experimental anatomic infarct size due to tissue edema, hemorrhage and acute inflammation. Circulation. 1979; 60:866–876. 4. Smith SC Jr. Reducing the global burden of ischemic heart disease and stroke: a challenge for the cardiovascular community and the United Nations. Circulation. 2011;124:278–279. 5. Fukuda K, Davies SS, Nakajima T, et al. Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels. Circ Res. 2005;97:1262–1269. 6. Yu B, Poirier LA, Nagy LE. Mobilization of GLUT-4 from intracellular vesicles by insulin and K(+) depolarization in cultured H9c2 myotubes. Am J Physiol. 1999;277:E259–E267. 7. Yu B, Schroeder A, Nagy LE. Ethanol stimulates glucose uptake and translocation of GLUT-4 in H9c2 myotubes via a Ca(2+)-dependent mechanism. Am J Physiol Endocrinol Metab. 2000;279:E1358–E1365. 8. Li N, Alam J, Venkatesan MI, et al. Nrf2 is a key transcription factor that regulates antioxidant defense in macrophages and epithelial cells: protecting against the proinflammatory and oxidizing effects of diesel exhaust chemicals. J Immunol. 2004;173:3467–3481. 9. Kobayashi A, Kang MI, Okawa H, et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate for proteasomal degradation of Nrf2. Mol Cell Biol. 2004;24:7130–7139. 10. Itoh K, Chiba T, Takahashi S, et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997;236:313–322.
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11. Baird L, Dinkova-Kostova AT. The cytoprotective role of the Keap1Nrf2 pathway. Arch Toxicol. 2011;85:241–272. 12. Shieh DE, Liu LT, Lin CC. Antioxidant and free radical scavenging effects of baicalein, baicalin and wogonin. Anticancer Res. 2000;20: 2861–2865. 13. Li XX, He GR, Mu X, et al. Protective effects of baicalein against rotenone-induced neurotoxicity in PC12 cells and isolated rat brain mitochondria. Eur J Pharmacol. 2012;674:227–233. 14. Lin CC, Shieh DE. The anti-inflammatory activity of Scutellaria rivularis extracts and its active components, baicalin, baicalein and wogonin. Am J Chin Med. 1996;24:31–36. 15. Handzlik J, Bajda M, Zygmunt M, et al. Antiarrhythmic properties of phenylpiperazine derivatives of phenytoin with alpha(1)-adrenoceptor affinities. Bioorg Med Chem. 2012;20:2290–2303. 16. Kimata M, Shichijo M, Miura T, et al. Effects of luteolin, quercetin and baicalein on immunoglobulin E-mediated mediator release from human cultured mast cells. Clin Exp Allergy. 2000;30:501–508. 17. Mu X, He GR, Cheng YX, et al. Baicalein exerts neuroprotective effects in 6-hydroxydopamine-induced experimental parkinsonism in vivo and in vitro. Pharmacol Biochem Behav. 2009;92:642–648. 18. Lee HJ, Noh YH, Lee DY, et al. Baicalein attenuates 6-hydroxydopamineinduced neurotoxicity in SH-SY5Y cells. Eur J Cell Biol. 2005;84:897–905. 19. Tu IH, Yen HTD, Cheng HW, et al. Baicalein protects chicken embryonic cardiomyocyte against hypoxia-reoxygenation injury via mu- and delta- but not kappa-opioid receptor signaling. Eur J Pharmacol. 2008; 588:251–258. 20. Chang WT, Li J, Haung HH, et al. Baicalein protects against doxorubicin-induced cardiotoxicity by attenuation of mitochondrial oxidant injury and JNK activation. J Cell Biochem. 2011;112:2873–2881. 21. Shao ZH, Vanden Hoek TL, Qin YM, et al. Baicalein attenuates oxidant stress in cardiomyocytes. Am J Physiol Heart Circ Physiol. 2002;282: H999–H1006. 22. Zong J, Zhang DP, Zhou H, et al. Baicalein protects against cardiac hypertrophy through blocking MEK-ERK1/2 signaling. J Cell Biochem. 2013;114:1058–1065. 23. Woo AYH, Cheng CHK, Waye MMY. Baicalein protects rat cardiomyocytes from hypoxia/reoxygenation damage via a prooxidant mechanism. Cardiovasc Res. 2005;65:244–253. 24. Chang WT, Li J, Vanden Hoek MS, et al. Baicalein preconditioning protects cardiomyocytes from ischemia-reperfusion injury via mitochondrial oxidant signaling. Am J Chin Med. 2013;41:315–331. 25. Yang L, Soonpaa MH, Adler ED, et al. Human cardiovascular progenitor cells develop from a KDR plus embryonic-stem-cell-derived population. Nature. 2008;453:524–526. 26. Lieu DK, Fu JD, Chiamvimonvat N, et al. Mechanism-based facilitated maturation of human pluripotent stem cell-derived cardiomyocytes. Circ Arrhythm Electrophysiol. 2013;6:191–201. 27. Cui G, Shan L, Hung M, et al. A novel Danshensu derivative confers cardioprotection via PI3K/Akt and Nrf2 pathways. Int J Cardiol. 2013; 168:1349–1359.
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28. Cui W, Cui GZ, Li W, et al. Bis(12)-hupyridone, a novel multifunctional dimer, promotes neuronal differentiation more potently than its monomeric natural analog huperzine A possibly through alpha7 nAChR. Brain Res. 2011;1401:10–17. 29. Li JQ, Ichikawa T, Janicki JS, et al. Targeting the Nrf2 pathway against cardiovascular disease. Expert Opin Ther Targets. 2009;13: 785–794. 30. Zhang Z, Cui W, Li G, et al. Baicalein protects against 6-OHDA-induced neurotoxicity through activation of Keap1/Nrf2/HO-1 and involving PKCalpha and PI3K/AKT signaling pathways. J Agric Food Chem. 2012;60:8171–8182. 31. Zheng XH, Zhao XF, Zhao X, et al. Determination of the main biloactive metabolites of Radix salvia miltiorrhizae in compound Danshen dripping pills and the tissue distribution of Danshensu in rabbit by SPE-HPLC-MS. J Sep Sci. 2007;30:851–857. 32. Calvert JW, Jha S, Gundewar S, et al. Hydrogen sulfide mediates cardioprotection through Nrf2 signaling. Circ Res. 2009;105:365–374. 33. He X, Kan H, Cai L, et al. Nrf2 is critical in defense against high glucoseinduced oxidative damage in cardiomyocytes. J Mol Cell Cardiol. 2009; 46:47–58. 34. Yoon HY, Kang NI, Lee HK, et al. Sulforaphane protects kidneys against ischemia-reperfusion injury through induction of the Nrf2-dependent phase 2 enzyme. Biochem Pharmacol. 2008;75:2214–2223.
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J Cardiovasc Pharmacol Volume 65, Number 1, January 2015 35. Dreger H, Westphal K, Weller A, et al. Nrf2-dependent upregulation of antioxidative enzymes: a novel pathway for proteasome inhibitormediated cardioprotection. Cardiovasc Res. 2009;83:354–361. 36. Zhang Y, Sano M, Shinmura K, et al. 4-Hydroxy-2-nonenal protects against cardiac ischemia-reperfusion injury via the Nrf2-dependent pathway. J Mol Cell Cardiol. 2010;49:576–586. 37. Wang GW, Hamid T, Keith RJ, et al. Cardioprotective and antiapoptotic effects of heme oxygenase-1 in the failing heart. Circulation. 2010;121: 1912–1925. 38. He XH, Wang Y, Yan XT, et al. Transduction of PEP-1-heme oxygenase-1 fusion protein reduces myocardial ischemia/reperfusion injury in rats. J Cardiovasc Pharmacol. 2013;62:436–442. 39. Kensler TW, Wakabayashi N. Nrf2: friend or foe for chemoprevention? Carcinogenesis. 2010;31:90–99. 40. Li-Weber M. New therapeutic aspects of flavones: the anticancer properties of Scutellaria and its main active constituents Wogonin, Baicalein and Baicalin. Cancer Treat Rev. 2009;35:57–68. 41. Lee JS, Surh YJ. Nrf2 as a novel molecular target for chemoprevention. Cancer Lett. 2005;224:171–184. 42. Na HK, Kim EH, Jung JH, et al. (-)-Epigallocatechin gallate induces Nrf2-mediated antioxidant enzyme expression via activation of PI3K and ERK in human mammary epithelial cells. Arch Biochem Biophys. 2008;476:171–177.
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Cytoprotection of Baicalein Against Oxidative Stress-induced Cardiomyocytes Injury Through the Nrf2/Keap1 Pathway Guozhen Cui, PhD,*† Sharon Chui Wah Luk, PhD,* Ronald Adolphus Li, PhD,‡ Ken Kwok Keung Chan, PhD,‡ Si Wan Lei, MSc,* Liang Wang, MSc,* Huifang Shen, PhD,† George Pak Heng Leung, PhD,§ and Simon Ming Yuen Lee, PhD*
Abstract: Baicalein is one of the major flavonoids found in the root of Scutellaria baicalensis Georgi. Previous studies suggest that baicalein displays protective effect on experimental cardiac models in vitro and in vivo. However, the mode of action remains unclear. Here, we showed that baicalein conferred cardioprotective effect against oxidative stress-induced cell injury in H9c2 cells and human embryonic stem cells-derived cardiomyocytes. Immunoprecipitation with anti-NF-E2–related factor 2 (Nrf2) antibody in baicalein-treated cells demonstrated that baicalein effectively disrupted the association between Nrf2 and Kelch-like epichlorohydrin-associated protein 1 (Keap1). In addition, the unbounded Nrf2 translocated from cytoplasm to nucleus and increased Nrf2/heme oxygenase-1 (HO-1) content in a time-dependent manner. Moreover, antioxidant response element transcriptional activity was enhanced by baicalein treatment, and the Nrf2 siRNA transfection could block the cytoprotective effect of baicalein. Taken together, these results demonstrate that baicalein protected cardiomyocytes against oxidative stress-induced cell injury through the Nrf2/Keap1 pathway. Key Words: baicalein, cardioprotection, oxidative stress, Nrf2 (J Cardiovasc Pharmacol 2015;65:39–46)
Received for publication May 1, 2014; accepted August 5, 2014. From the *State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao, China; †Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai, China; ‡Stem Cell and Regenerative Medicine Consortium, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China; and §Department of Pharmacology and Pharmacy, Li Ka Shing Faculty of Medicine, University of Hong Kong, Hong Kong, China. Supported by the Science and Technology Development Fund of Macao SAR (Ref. No. 014/2011/A1), Overseas and Hong Kong, Macau Young Scholars Collaborative Research Fund by the Natural National Science Foundation of China (Ref. No. 81328025), Research Committee, University of Macau, the Research Grant Council of HKSAR (TBRS, T13-706/11), SCRMC, and Faculty Cores of the University of Hong Kong. The authors report no conflicts of interest. Reprints: George Pak Heng Leung, PhD, Department of Pharmacology and Pharmacy, The University of Hong Kong, Hong Kong, 999077 China (e-mail: [email protected]), or and Simon Ming Yuen Lee, PhD, State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, 999078 China (e-mail: [email protected]). Copyright © 2014 by Lippincott Williams & Wilkins
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INTRODUCTION Cardiovascular diseases are the leading cause of death worldwide, and majority of the cases are ischemic heart disease (IHD).1,2 IHD develops when cholesterol in the blood begins to accumulate on the walls of the arteries, which narrows the arteries and eventually blocks the blood flow. A decrease in the amount of oxygen supplied to the heart muscle may result in myocardial infarction.3 Because of the size of the effected population, an effective strategy to reduce the global burden of IHD is urgently needed.4 Fukuda et al5 had demonstrated that Human Embryonic Kidney cells treated with tert-butyl hydroperoxide (t-BHP), a hydrogen peroxide donor, can mimic oxidative stress in myocardial ischemia. H9c2 cell line derived from embryonic rat heart maintains some features of the cardiomyocytes.6,7 Hence, it has been extensively used as an in vitro heart model. In this study, we used t-BHP–treated H9c2 as a myocardial ischemia model to study the protective mechanism against oxidative stress in patients with IHD. Transcriptional factor NF-E2–related factor 2 (Nrf2) is thought to act as a cellular defense system against oxidative stress.8 Under normal or unstressed condition, Nrf2 is kept in the cytoplasm by a cluster of proteins, Keap1 and Cullin 3 (Cul3). Keap1 is a substrate adapter, which helps Cul3 ubiquitinate Nrf2.9 When Nrf2 is ubiquitinated, it is transported to the proteasome where it is degraded, and the components are recycled. Under oxidative or electrophilic stress, the cysteine residues in Keap1 are disrupted, thus upsetting the Keap1-Cul3 ubiquitination system. When Nrf2 is not ubiquitinated, it will build up in the cytoplasm and translocates into the nucleus. In the nucleus, it will form heterodimer with small Maf protein and binds to the antioxidant response element (ARE), an upstream promoter region of many antioxidative genes, and initiates their transcription, thereby conferring broader protective roles.10,11 Baicalein is one of the main flavonoids derived from the root of S. baicalensis Georgi. It exhibits multiple pharmacological activities, such as antioxidation,12,13 anti-inflammatory,14 anticancer,15 and antiallergic effect.16 Because of its antioxidative and/or anti-inflammatory activities, most studies have been focused on its protective action in neurodegenerative disease.17,18 In the last decade, the cardioprotective effect of baicalein on various in vitro and in vivo experimental models has been reported. It was demonstrated that baicalein exerts protective effect against hypoxia-reoxygenation and doxorubicin-induced cell damage in chick cardiomyocytes.19–21 In addition, the recent www.jcvp.org |
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studies by Zong et al22 revealed that baicalein also attenuates cardiac hypertrophy. However, its molecular mechanism has not been clearly established, some had suggested that it is mediated by antioxidant and others had suggested by prooxidant.21,23,24 The aim of this study was to examine the molecular mechanism of the protective effect of baicalein in oxidative stress-induced cell injury in cardiomyocytes.
MATERIALS AND METHODS Materials Baicalein (98% purity) was obtained from the National Institute for the Control of Pharmaceutical and Biological Products (Beijing, China). Dulbecco’s modified Eagle’s medium (DMEM), penicillin, and streptomycin were purchased from Gibco/life Technologies (Grand Island, NY). A 5-(6-)chloromethyl-20 ,70 -dichlo-rodihydrofluorescein diacetate (CM-H2DCFDA), LIVE/DEAD Viability/Cytotoxicity Assay Kit, and Opti-MEM I Reduced-Serum Medium were supplied by Invitrogen (Eugene, OR). Nrf2-siRNA and scrambled RNA were supplied by Santa Cruz Biotechnology (Santa Cruz, CA). Cignal Antioxidant Response Reporter (luciferase) Kit and SureFECT Transfection Reagent were purchased from SABiosciences (Frederick, MD). The DualLuciferase Reporter Assay System was purchased from Promega (Madison, WI). All other chemicals and reagents used in this study are analytical grade.
H9c2 Cell Culture Rat embryonic heart-derived H9c2 cells are widely used to investigate the molecular mechanism is involved in cardiomyocyte survival. The H9c2 cell line was purchased from America Tissue Type Collection (Manassas, VA). Cells were grown in DMEM medium with 10% fetal bovine serum, 100 U/mL penicillin, and 100 U/mL streptomycin in a 75-cm2 tissue culture flask at 378C, 5% CO2. The cells were fed every 2–3 days, and they were subcultured when reached 70%–80% confluence.
Live/Dead Cell Viability Staining and MTT Assay To measure cell viability, H9c2 cells were seeded in 96well plate at a density of 5 · 103 cells per well. They were grown at 378C for 24 hours in complete medium until confluence is above 80%. Cells were pretreated with various concentrations of baicalein (0.3–30 mM) for 4 hours before they were treated with 150 mM t-BHP for 4 hours. Cell viability staining was performed using the Live/Dead viability/ cytotoxicity kit according to manufacturer’s instructions. Fluorescence imaging of cells was performed using an IN Cell Analyzer 2000 (GE Healthcare), and cell survival was quantified using MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) assay.
with 5 mM CM-H2DCFDA at 378C for 20 minutes in the dark; and then, cells were treated with baicalein (30 mM) for 4 hours and exposed to t-BHP for 1 hour. The fluorescence intensity was measured at 530 nm using a fluorescence microplate reader.
Human ESC-CMs Culture The human embryonic stem cell line (HES-2) was obtained from the WiCell Research Institute (Madison, WI, http://www.wicell.org), and they were induced as previously described.25,26 This differentiation protocol yields more than 90% spontaneous beating cardiospheres. Cardiospheres were cultured in suspension with cardiomyocytes maintenance medium in low attachment 6-well plate (CLS3471; Corning Costar Corp, Cambridge, MA). The maintenance medium contains 500 mL basic liquid media StemPro 34 (Invitrogen Corp, Grand Island, CA), 2.5 mL ascorbic acid (10 mg/mL; final concentration of 50 mg/mL), 1% L-glutamine, 1% penicillin, and 1% streptomycin. For the cardioprotective study, the spontaneous contracting cardiospheres were dissociated into single cells and plated in a gelatin-coated 96-well plate and cultured for 2 days in H1-DIF medium containing 10% fetal bovine serum, 77% DMEM, 1% Penicillin/Streptomycin, 1% nonessential amino acids, and 1% L-glutamine.
Hypoxia-induced Cell Injury in In Vitro Study The protective effect of baicalein was further confirmed by pretreating H9c2 cells and human embryonic stem cells-derived cardiomyocytes (hESC-CMs) with 3, 10, or 30 mM of baicalein or vehicle for 2 hours before hypoxia treatment. To mimic pathological ischemic injury in in vitro, both cell lines were subjected to hypoxia for 3 hours (94.5% N2, 0.5% O2, and 5% CO2) using a hypoxia workstation INVIVO 2400 with Ruskin hypoxic gas mixer (Biotrace, Bridgend, United Kingdom). Cellular injury was determined by measuring the activity of lactate dehydrogenase (LDH) released into the incubation medium. Released LDH activity was determined by the cytotoxicity detection kit according to the manufacturer’s instructions. Absorbance was measured using a microplate reader at 490 nm.
Preparation of Whole Cell, Cytosolic, and Nucleus Extracts For the preparation of the whole cell lysate, H9c2 cells were washed with ice-cold phosphate-buffered saline (PBS) and incubated on ice for 30 minutes with radio-immunoprecipitation assay (RIPA) lysis buffer containing 1% phenylmethylsulfonyl fluoride (Sigma Chemical Co., St. Louis, MO) and 1% Protease Inhibitor Cocktail (Roche Applied Science, Indianapolis, IN). Cell lysates were centrifuged at 12,500g for 20 minutes at 48C. Supernatant was collected. The subcellular fraction was obtained using a nucleus extraction kit (Cayman Chemical, Ann Arbor, MI) according to previously published method.27 Protein concentration was quantified using the bicinchoninic acid (BCA) protein quantification kit (Pierce Chemical Co, Rockford, IL).
Detection of Intracellular Reactive Oxygen Species
Immunoblot Analysis
Intracellular reactive oxygen species (ROS) in H9c2 cells was evaluated using CM-H2DCFDA with a fluorescence microplate reader. Cells were seeded in a 96-well plate and incubated
Lysates from H9c2 cells were immunoblotted according to the previously described method.28 After blocking with 5% nonfat dry milk, membrane was incubated overnight at
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48C with primary antibodies. Anti-Keap1, anti-b-actin, and horseradish peroxidase-conjugated antirabbit antibodies were purchased from Cell Signaling Technology (Beverly, MA). HO-1 monoclonal antibody was purchased from Abcam (Cambridge, MA), and anti-Nrf2 monoclonal antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). After incubation with primary antibodies, membranes were incubated with secondary antibodies for 1 hour. To confirm equal protein loading, membranes were incubated with anti-b–actin antibody. Proteins were detected using an advanced enhanced chemiluminescence (ECL) system (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom). Semiquantification of the proteins was performed with densitometric analysis using Quantity One (Biorad).
(Cell Signaling Technology, Beverly, MA) for 4 hours at 48C. Immune complexes were boiled for 5 minutes with ·2 loading buffer, followed by immunoblot analysis with Keap1 and Nrf2 antibodies.
Immunofluorescence Staining
Baicalein Increased Cell Viability and Decreased ROS Release in t-BHP–treated H9c2 Cells
Cells were washed with ice-cold PBS and fixed with 4% paraformaldehyde for 10 minutes at room temperature. Then cells were washed with PBS containing 1% bovine serum albumin and incubated with blocking buffer (0.3% Triton X-100% and 1% bovine serum albumin in PBS) for 45 minutes at room temperature. After blocking, cells were incubated with anti-Nrf2 antibody (1:200) overnight at 48C and with Alexa Fluor 488-conjugated antibody (1:200) (Invitrogen, Eugene, OR) for 45 minutes at 378C. To visualize the nucleus, cells were counterstained with 300 nM 40 ,6-diamidino2-phenylindole (DAPI) for 10 minutes. The fluorescence images were captured using a fluorescence microscope.
Transient Transfection With Nrf2 siRNA
H9c2 cells were seeded at 5 · 103 cells per well in a 96-well plate to achieve 40%–60% confluence and transfected with 10 pm Nrf2 siRNA or scrambled siRNA using SureFECT transfection reagent according to manufacturer’s instructions. The transfected cells were treated with baicalein for 4 hours and then with 150 mM t-BHP for 4 hours. Cell viability was determined using MTT assay.
Nrf2 Transcriptional Activity The Nrf2 transcriptional activity was monitored using the ARE reporter assay. This system consists of Nrf2responsive luciferase construct and constitutively expresses Renilla construct in a 40:1 ratio (0.1 mg/mL). The Renilla luciferase reporter gene is used to normalize transfection efficiencies and to monitor cell viability. The H9c2 cells were seeded in a 96-well plate at a density of 5 · 103 cells per well. After 24 hours, H9c2 cells were transfected with 150 ng of ARE reporter construct for 12 hours using SureFECT transfection reagent according to manufacturer’s protocol. After another 12 hours, the cells were treated with either vehicle (0.1% dimethyl sulfoxide [DMSO]) or baicalein for 4 hours. Firefly and Renilla luciferase activities were measured using the Dual-Glo luciferase assay system.
Immunoprecipitation To determine the amount of Keap1-bound Nrf2, whole cell lysates were incubated with anti-Nrf2 antibody overnight at 48C under agitation. The antigen–antibody complex was pulled down after incubation with protein G-agarose 2014 Lippincott Williams & Wilkins
Statistical Analysis All assays were performed at least 3 times. Values were expressed as mean 6 SD. Statistical analysis was performed by one-way analysis of variance followed by Tukey’s multiple comparison tests using Graph Pad Prism 5.0. A value of P , 0.05 was considered to be statistically significant.
RESULTS
To determine whether baicalein alone has a toxic effect in H9c2 cells, cell viability was evaluated after 4 hours incubation with baicalein (0.3–30 mM) using fluorescence imaging. The results of the assay indicated that baicalein did not exhibit any toxic effect in H9c2 cells at the concentration tested (Fig. 1A). The morphological characteristics of the baicalein-treated cells are similar to the control. The testing method was also used to study the protective effect of baicalein in t-BHP–treated H9c2 cells. Fluorescence images and the results of the MTT assay both showed that pretreatment with baicalein could increase the cell viability of the t-BHP–treated H9c2 cells (Figs. 1A, B). The MTT assay showed that pretreatment with baicalein significantly increased cell viability in a concentration-dependent manner, P , 0.01 at 3 mM and P , 0.001 at 10 and 30 mM (Fig. 1B). The cytoprotective effect of baicalein in oxidative stress-induced by t-BHP treatment was further evaluated using CM-H2DCFDA. The results showed that treatment with 150 mM t-BHP triggered rapid ROS accumulation in cells. In contrast, pretreatment with 3, 10, or 30 mM baicalein significantly reduced the levels of ROS, and it was in a concentration-dependent manner, P , 0.001 (Fig. 1C).
Baicalein Prevented Against Hypoxia-induced Cell Injury in H9c2 Cells and hESC-CMs To mimic pathological ischemic injury in in vitro, H9c2 cells, and hESC-CMs were subjected to hypoxia for 3 hours (94.5% N2, 0.5% O2, and 5% CO2). The protective effect of baicalein was studied by pretreating the cells with 3, 10, or 30 mM of baicalein for 2 hours before hypoxia treatment. It demonstrated that hypoxia increased LDH release by more than 2-fold versus the normoxia group (P , 0.05) in both cell lines (Figs. 1D, E). As shown in Figure 1D, incubation with 30 mM of baicalein displayed significant protective effects against hypoxia-induced cell injury in H9c2 cells (P , 0.05). In contrast, pretreatment with baicalein before hypoxia significantly reduced the release of LDH in all 3 tested concentrations in hESC-CMs (P , 0.05). However, there was no significant difference in the reduction of LDH release amongst the 3 baicalein dosage groups (3, 10, 30 mM). All 3 groups showed a similar level of LDH release (Fig. 1E). www.jcvp.org |
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FIGURE 1. Protective effects of baicalein against oxidative stress-induced cell injury. A, Representative images of H9c2 cells after 4 hours incubation of control, baicalein (30 mM), t-BHP (150 mM), or t-BHP (150 mM) in the presence of baicalein (30 mM). Live cells were stained green, and the nuclei of the dead cells were stained red. B, Cell viability was significantly reduced with 4-hour incubation of 150 mM t-BHP. Pretreatment with baicalein increased cell viability in a concentration-dependent manner in H9c2 cells. C, ROS was significantly reduced with the pretreatment of 3, 10, or 30 mM baicalein in H9c2 cells (P , 0.001). Baicalein attenuated hypoxiainduced cell injury in H9c2 cells (D) and in hESC-CMs (E). #P , 0.05 versus normoxia group, *P , 0.05 versus hypoxia group. ***P , 0.001 versus hypoxia group.
Baicalein Increased Nrf2 and HO-1 Protein Expression in a Concentration- and Time-dependent Manner in H9c2 Cells Under stress conditions, adaptive survival response will trigger the upregulation of the cytoprotective genes through transcription factors, such as Nrf2 to protect cells from detrimental oxidant-induced cellular and tissue death.29 To investigate whether the cytoprotective effect of baicalein was mediated through the activation of Nrf2, proteins content of Nrf2, and antioxidant enzyme, HO-1 were measured. H9c2 cells were incubated with either 3, 10, or 30 mM baicalein for 4 hours or with 30 mM baicalein for 1, 2, and 4 hours. After 4 hours of 10 and 30 mM baicalein treatment, the expression level of both Nrf2 and HO-1 proteins was significantly increased as compared with the control, P , 0.05 (Fig. 2A). Similarly, treatment with 30 mM baicalein for 4 hours significantly increased both the Nrf2 and HO-1 proteins expression with P , 0.05 (Figs. 2A, B). The data suggested that it requires about 4 hours of either 10 or 30 mM baicalein pretreatment to significantly increase the proteins expression of Nrf2 and HO-1 in H9c2 cells.
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Baicalein Activated Nrf2 Pathway in H9c2 Cells Under stressed condition, Nrf2 is translocated to the nucleus and binds to the ARE located in the promoter region of various antioxidant and detoxifying enzymes.29 The localization of Nrf2 was studied using immunofluorescence and immunoblot assays. The results of the cell immunofluorescence assay demonstrated that baicalein treatment increased the accumulation of Nrf2 in the nucleus, which is the active form of Nrf2 (Fig. 3B). Similar result was also observed in the immunoblot assay; it showed that baicalein treatment promoted the accumulation of Nrf2 in the nucleus fraction and a reduction in the cytoplasmic fraction in a time-dependent manner (Fig. 3A). The translocation of Nrf2 to the nucleus was observed at 2 hours of 30 mM baicalein treatment, and it is more prominent at 4 hours. Nrf2-driven transcriptional activity induced by baicalein was also investigated using the ARE-luciferase reporter assay. The result showed that baicalein significantly increased the ARE–luciferase transcription activity in a concentrationdependent manner with P , 0.05 in all 3 baicalein concentrations (3, 10, and 30 mM; Fig. 3C). This was in agreement 2014 Lippincott Williams & Wilkins
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FIGURE 2. Baicalein significantly increased proteins expression of Nrf2 and HO-1 in a concentration- and time-dependent manner. A, H9c2 cells were incubated with baicalein at 3, 10, or 30 mM for 4 hours or (B) at 30 mM baicalein for 1, 2, or 4 hours. Immunoblot analysis was performed using Nrf2 and HO-1 antibodies as described in Methods. Results expressed as mean 6 SD of 3 independent experiments, #P , 0.05 versus control group.
with the results of the immunofluorescence and immunoblot assays, which showed an increase of Nrf2 in the nucleus fraction.
Nrf2 siRNA Transfection Abolished the Cytoprotective Effect of Baicalein in H9c2 Cells Nrf2 siRNA was used to investigate whether baicalein exerted cytoprotection against oxidative DNA damageinduced by t-BHP treatment is Nrf2-dependent. As shown in Figure 3D, Nrf2 siRNA significantly decreased cell viability with P , 0.05 in baicalein and t-BHP–treated cells and inhibited the cytoprotective effect of baicalein. However, the scrambled control siRNA did not affect the cell viability in these cells. Thus, the result suggested that the cytoprotective effect of baicalein against oxidative stress-induced cell injury was dependent on Nrf2 pathway (Fig. 4).
Baicalein Disrupted the Nrf2/Keap1 Complex The effect of baicalein on Nrf2/Keap1 complex was examined using immunoprecipitation with anti-Nrf2 antibody, and the Keap1 protein was quantified using immunoblot analysis. The results showed that baicalein effectively disrupted the interaction between Nrf2 and Keap1 in a timedependent manner (Fig. 3E). The association between Keap1 and Nrf2 began to decrease after 1-hour treatment of 30 mM baicalein, and it continues to decrease at 2 and 4 hours. This was in agreement with the results of the immunofluorescence and immunoblot assays, which showed an increase of Nrf2 in the nucleus fraction. 2014 Lippincott Williams & Wilkins
DISCUSSION In the past decade, several reports had demonstrated that baicalein has a cytoprotective effect in neonatal rat and chick cardiomyocytes; however, there is no agreement on its precise mechanism of action. In this study, we showed that baicalein effectively protected H9c2 cells against t-BHP– treated cell injury and protected both H9c2 and hESC-CMs against hypoxia-induced cell injury. The data demonstrate that baicalein exerts its cytoprotection through the activation of the Nrf2/Keap1 signaling pathway, which is in agreement with our previous finding that the same pathway is involved in the protective effect against neurotoxin-induced cell injury in vitro by baicalein.30 Myocardial death results from ischemia/reperfusion are associated with the overproduction of ROS.31 Thus, in theory, by decreasing the production of ROS, it could attenuate ROS-induced myocardial dysfunction. In this study, cells exposed to t-BHP or hypoxia were used as the oxidative stress models for heart ischemia and/or reperfusion-induced myocardial damage. The data showed that pretreatment of H9c2 cells with baicalein significantly inhibited t-BHP–induced ROS in a concentration-dependent manner and resulted in an increase of cell viability. Similar result was also observed in both cell lines using the LDH assay. LDH is an indicator for cells viability because when tissue breakdown, LDH is released. Our result showed that baicalein obviously reduced LDH release in hypoxia-exposed H9c2 cells and hESC-CMs (Figs. 1D, E). To the best of our knowledge, this is the first report showing that baicalein reduced cell injury in t-BHP–treated H9c2 cells and in hypoxia-exposed H9c2 cells and hESC-CMs. www.jcvp.org |
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FIGURE 3. The effects of baicalein on Nrf2/Keap1 signaling pathway. A, Immunoblot assay of the nucleus and cytoplasmic extractions showed an increase of Nrf2 in the nucleus fraction and a decrease in the cytoplasmic fraction. B, Immunofluorescence staining of H9c2 cells treated with 30 mM baicalein for 4 hours. Left panel: red fluorescence showed the localization of Nrf2; Middle panel: Stained nucleus with DAPI; Right panel: Merged images of red and blue filters. C, H9c2 cells were transfected with ARE-reporter luciferase plasmid as described in Methods and treated with baicalein (3, 10, or 30 mM). After 4 hours, cells were collected, and the luciferase activity was analyzed. Baicalein treatment (3, 10, or 30 mM) significantly increased the ARE–luciferase activity (P , 0.01). D, Transfection of Nrf2-siRNA significantly reduced the protective effect of baicalein (P , 0.05). E, The Nrf2/ Keap1 complex was disrupted by baicalein. H9c2 cells were treated with 30 mM baicalein for 1, 2, or 4 hours; cell lysates were prepared for immunoprecipitaion (IP) using Nrf2 antibody. The Nrf2-associated Keap1 was assessed through immunoblot analysis (IB). The precipitates were blotted with anti-Nrf2 antibody to ensure that an equal amount of Nrf2 was pulled down under each condition. #P , 0.001 versus control group, ***P , 0.001 versus t-BHP group, *P , 0.001 versus BAI + t-BHP group.
Transcription factor Nrf2 was suggested to serve as a promising therapeutic target for cardiovascular diseases. Research by Calvert et al32 has shown that endogenously produced hydrogen sulfide (H2S) mediates cardioprotective effect against cardiac ischemia/reperfusion through Nrf2 signaling in vivo. Nrf2 activators tert-butylhydroquinone (30 mM) and sulforaphane (20 mM) induce Nrf2 nucleus translocation to confer the protective effects in H9c2 cardiomyoblast cells and HK2 renal tubular epithelial cells, respectively.33,34 The effective concentrations of these 2 compounds are similar to that of baicalein (10–30 mM) found in this study. It was also shown that the cardioprotective effect of MG132 and 4-hydroxy-2-nonenal was blocked in Nrf2 knockout mice.35,36 In addition, HO-1, a downstream target of the Nrf2 pathway, has shown to have an important cardioprotective adaptation that can counterbalance the pathological changes in mice’s chronic heart failure.37,38 However, the protective characteristics of Nrf2 signaling pathway could also promote cancer growth and drug resistance in tumor
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cells.39 This discrepancy might be explained by the different cell types used. Nonetheless, there is a possibility that baicalein pretreatment could also promote tumor growth despite its reported potential anticancer activity through inducing cell cycle arrest and apoptosis.40 The role of Nrf2 pathway activated by baicalein in mediating the anticancer effects would be another interesting area that is worth investigating in the future. This study provides evidence that baicalein protects t-BHP–treated H9c2 cells by facilitating the dissociation of the Nrf2/Keap1 complex. The unbounded Nrf2 protein translocates into the nucleus and binds to the ARE where it initiates the transcription of HO-1. This is in agreement with the observation that Nrf2 siRNA can abolish the cytoprotective effect of baicalein in t-BHP–treated H9c2 cells. Hence, the data suggested that the protective effect of baicalein is mediated through the Nrf2/Keap1 signaling pathway. There are several kinases including phosphatidylinositol 3-kinase, protein kinase C, and mitogen-activated protein kinases that act 2014 Lippincott Williams & Wilkins
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Cytoprotection of Baicalein
FIGURE 4. Proposed mechanism of cytoprotective effect against oxidative stress-induced cardiomyocytes injury by baicalein. Baicalein activates Nrf2 by facilitating the dissociation of Nrf2/ Keap1 complex, which leads to the nucleus translocation of Nrf2. Nucleus Nrf2 then binds to AREs and initiates the upregulation of HO-1 to confer the cytoprotective effect against oxidative stress-induced cardiomyocytes injury.
as the primary sensors for the activation of the Nrf2 pathway.41,42 Whether baicalein activates the Nrf2 signaling pathway by binding directly to Nrf2 and/or Keap1 or an upstream element such as phosphatidylinositol 3-kinase remains to be determined. In summary, our results demonstrated that baicalein possesses protective effect against oxidative stress-induced cell injury in H9c2 cells and hESC-CMs. This protective effect, in part, is regulated by Nrf2-dependent signaling pathway. These results, combined with previous publications showing the important role of Nrf2 in protection against cardiovascular disease, suggested that baicalein might be a candidate for the prevention of myocardial damage in IHD. REFERENCES 1. Mathers CD, Boerma T, Ma Fat D. Global and regional causes of death. Br Med Bull. 2009;92:7–32. 2. Lopez AD, Murray CCJL. The global burden of disease, 1990-2020. Nat Med. 1998;4:1241–1243. 3. Reimer KA, Jennings RB. The changing anatomic reference base of evolving myocardial infarction. Underestimation of myocardial collateral blood flow and overestimation of experimental anatomic infarct size due to tissue edema, hemorrhage and acute inflammation. Circulation. 1979; 60:866–876. 4. Smith SC Jr. Reducing the global burden of ischemic heart disease and stroke: a challenge for the cardiovascular community and the United Nations. Circulation. 2011;124:278–279. 5. Fukuda K, Davies SS, Nakajima T, et al. Oxidative mediated lipid peroxidation recapitulates proarrhythmic effects on cardiac sodium channels. Circ Res. 2005;97:1262–1269. 6. Yu B, Poirier LA, Nagy LE. Mobilization of GLUT-4 from intracellular vesicles by insulin and K(+) depolarization in cultured H9c2 myotubes. Am J Physiol. 1999;277:E259–E267. 7. Yu B, Schroeder A, Nagy LE. Ethanol stimulates glucose uptake and translocation of GLUT-4 in H9c2 myotubes via a Ca(2+)-dependent mechanism. Am J Physiol Endocrinol Metab. 2000;279:E1358–E1365. 8. Li N, Alam J, Venkatesan MI, et al. Nrf2 is a key transcription factor that regulates antioxidant defense in macrophages and epithelial cells: protecting against the proinflammatory and oxidizing effects of diesel exhaust chemicals. J Immunol. 2004;173:3467–3481. 9. Kobayashi A, Kang MI, Okawa H, et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate for proteasomal degradation of Nrf2. Mol Cell Biol. 2004;24:7130–7139. 10. Itoh K, Chiba T, Takahashi S, et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun. 1997;236:313–322.
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