Baicalein protects cardiomyocytes against mitochondrial oxidant injury associated with JNK inhibition and mitochondrial Akt activation.

Am. J. Chin. Med. 2014.42:79-94. Downloaded from www.worldscientific.com by FLINDERS UNIVERSITY LIBRARY on 02/04/15. For personal use only.

The American Journal of Chinese Medicine, Vol. 42, No. 1, 79–94 © 2014 World Scientific Publishing Company Institute for Advanced Research in Asian Science and Medicine DOI: 10.1142/S0192415X14500050

Baicalein Protects Cardiomyocytes Against Mitochondrial Oxidant Injury Associated with JNK Inhibition and Mitochondrial Akt Activation Hsien-Hao Huang,*,† Zuo-Hui Shao,* Chang-Qing Li,* Terry L. Vanden Hoek* and Jing Li* *Department

of Emergency Medicine, Center for Cardiovascular Research University of Illinois Hospital and Health Sciences System Chicago, IL 60612, USA

† Department of Emergency Medicine Taipei Veterans General Hospital and Emergency Medicine College of Medicine, National Yang-Ming University Taipei, Taiwan

Abstract: Baicalein, a flavonoid derived from Scutellaria baicalensis Georgi, possesses cardioprotection against oxidant injury by scavenging reactive oxygen species (ROS). Few studies investigate whether baicalein protection is mediated by attenuating mitochondrial ROS and modulating the prosurvival and proapoptotic signaling. Primary cultured chick cardiomyocytes were used to study the role of baicalein in mitochondrial superoxide (O 2 ) generation and signaling of Akt and JNK. Cells were exposed to H2O2 for 2 h and baicalein was given 2 h prior to and during 2 h of H2O2 exposure. Cell viability was assessed by propidium iodide and DNA fragmentation. H2O2 (500 M) significantly induced 45:3 6:2% of cell death compared to the control (p < 0:001) and resulted in DNA laddering. Baicalein (10, 25 or 50 M) dose-dependently reduced the cell death to 38:7 5:6% (p ¼ 0:226); 31:2 3:9% (p < 0:01); 30:3 5:3% (p < 0:01), respectively. It also attenuated DNA laddering. Further, baicalein decreased intracellular ROS and mitochondrial O 2 generation that was confirmed by superoxide dismutase PEG-SOD and mitochondria electron transport chain complex III inhibitor stigmatellin. In addition, baicalein increased Akt phosphorylation and decreased JNK phosphorylation in H2O2-exposed cells. Moreover, baicalein augmented mitochondrial phosphorylation of Akt Thr308 and GSK3β Ser9, and prevented mitochondrial cytochrome c release assessed by cellular fractionation. Our results Correspondence to: Dr. Jing Li, MD, Department of Emergency Medicine, Centers for Cardiovascular Research, University of Illinois Hospital and Health Sciences System, 808 S. Wood Street, MC 724, Chicago, IL 60612, USA. Tel: (þ1) 312-413-1704, Fax: (þ1) 312-996-8599, E-mail: [email protected] or to Dr. Terry L. Vanden Hoek, MD, Department of Emergency Medicine, Centers for Cardiovascular Research, University of Illinois Hospital and Health Sciences System, 808 S. Wood Street, MC 724, Chicago, IL 60612, USA. Tel: (þ1) 312-9666560, Fax: (þ1) 312-413-0289, E-mail: [email protected]

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H.-H. HUANG et al. suggest that baicalein cardioprotection may involve an attenuation of mitochondrial O 2 and an increase in mitochondrial phosphorylation of Akt and GSK3β while decreasing JNK activation. Keywords: Baicalein; Cardiomyocyte; Mitochondrial Superoxide; JNK; Akt.


Introduction Baicalein (5, 6, 7-trihydroxyflavone) is a flavone, a type of flavonoid, originally isolated from the roots of Scutellaria baicalensis Georgi (Huangqin). It has been reported that baicalein possesses a potent antioxidant property to scavenge reactive oxygen species (ROS), including O 2 , OH and hydrogen peroxide (H2O2) and also act as a chelator of redox-active metal ions (Collis et al., 1996; Gao et al., 1999; Valko et al., 2006). We and others demonstrated that baicalein ameliorated the oxidant injury induced by ischemia/ reperfusion (I/R), a scenario similar to clinical conditions, such as myocardial infarction, stroke and cardiac arrest (Shao et al., 1999, 2002; Chang et al., 2007, 2013). Accumulating evidence indicates that baicalein is a potent ROS scavenger and protects cell and tissue from oxidant injury in multiple organs (Shao et al., 1999; Liu et al., 2010). Fewer reports have shown its role in regulating mitochondrial ROS generation, in particular, the species and sites. Mitochondria are a major source of ROS production. The superoxide anion, mainly produced during oxidant stress such as ischemia and hypoxia, is a result of increased electron leak from the impaired mitochondrial electron transport chain (ETC) and is massively increased from ETC complex III upon reperfusion. The produced superoxide is converted to H2O2, which in turn is reduced to hydroxyl radical through the Fenton reaction (Zweier et al., 1994; Vanden Hoek et al., 1997a). Excessive ROS production leads to a release of cytochrome c to the cytosol that triggers apoptosis (Boveris et al., 1976; Lesnefsky et al., 2001). Therefore, reducing mitochondrial ROS production can potentially ameliorate cell death. Akt/PKB is a 57kD threonine/serine kinase that plays a key role in regulating survival (Shiraishi et al., 2004; Mullonkal and Toledo-Pereyra, 2007). The PI3K/Akt pathway promotes cellular survival by phosphorylating and inhibiting death-inducing proteins (Mullonkal and Toledo-Pereyra, 2007). Akt activation, via its effects on mitochondrial associated proteins including glycogen synthase kinase-3beta (GSK3β), can attenuate mitochondrial ROS generation (Juhaszova et al., 2000; Ohori et al., 2008; Song et al., 2009). It has been reported that Akt exerts its protection by translocating the active Akt into nuclei and mitochondria (Andjelkovic et al., 1997; Bijur and Jope, 2003). On the contrary, JNK is a c-Jun N-terminal kinase/stress activated protein kinase and its activation is associated with elevated reactive oxygen intermediates and oxidant-induced apoptosis (Dougherty et al., 2004). JNK can oppose Akt in affecting mitochondrial function and enhance cell death (Wang et al., 2006). Therefore, one mechanism of cell survival is to activate Akt and deactivate JNK. The effects of baicalein on these two pathways in cardiomyocytes and the mitochondrial activation of Akt and JNK remain unexplored.

BAICALEIN ON OXIDANT INJURY IN CARDIOMYOCYTES

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In this study, we aimed to determine whether (1) baicalein attenuates H2O2-induced mitochondrial oxidant injury in cardiomyocytes, (2) this protection involves regulating both prosurvival and proapoptotic signaling, such as Akt and JNK, and (3) baicalein modulates mitochondrial phosphorylation of Akt and its downstream target GSK3β.

Materials and Methods


Chemicals The reagents including H2O2, baicalein, propidium iodide (PI), digitonin, PEG-SOD, stigmatellin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Trypsin, 6-carboxy-2 0 , 7 0 -dichlorodihydrofluorescein diacetate (6-carboxy-H2DCFDA) and MitoSOX red were obtained from Invitrogen (Carlsbad, CA, USA). The fractionation kit was purchased from EMD Biosciences (San Diego, CA, USA). DNA extraction kit was obtained from Qiagen (Valencia, CA, USA). All antibodies except for RhoGD1 (BD Transduction Laboratories, San Diego, CA), VDAC1 (EMD Biosciences, San Diego, CA, USA), alphasarcomeric actin (Sigma, St. Louis, MO, USA) and -tubulin (NeoMarkers, Fremont, CA, USA) were purchased from Cell Signaling Technology (Danvers, MA, USA). All materials for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were obtained from Bio-Rad (Bio-Rad Laboratories, Richmond, CA, USA), except SuperSignal (Thermol Scientific, Rockford, IL, USA). Cardiomyocyte Isolation Embryonic chick ventricular cardiomyocytes isolated from 10-day-old chick embryos were prepared, as previously described (Vanden Hoek et al., 1996). In brief, the hearts were removed; the ventricles were minced and enzymatically digested with 0.025% trypsin. Following cell isolation, cardiomyocytes (0:7 10 6 ) were placed on 25 mm glass coverslips and incubated at 37 C in the culture medium (54% balanced salt solution, 40% medium 199, 6% heat-inactivated fetal bovine serum, 100 U/ml penicillin and 100 g/ml streptomycin). Cardiomyocyte purity was assessed by immunofluorescent staining for alpha-sarcomeric actin. All experiments were performed with the 4–5 day cultured cells, at which point viability exceeded 95%. Video/Fluorescent Microscopy A Nikon TE 2000-U inverted phase/epifluorescent microscope was used for cell imaging. Fluorescent images were acquired from a cooled Cool-SNAP-ES camera (Photometrics, Tuscon, AZ, USA) and changes in fluorescent intensity were quantified with MetaMorph software (Universal Imaging, Downington, PA, USA).

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Viability Assay Cell viability was assessed with the exclusion fluorescent dye, propidium iodide (5 M, Sigma) measured at excitation (Ex) 540 nm/emission (Em) 590 nm using a Nikon TE 2000-U inverted phase/epifluorescent microscope (Photometrics). This dye exhibited no toxicity in control cells even after a ten-hour exposure. At the end of the experiment, all cells on the coverslip were permeabilized with digitonin. Measurement of propidium iodide (PI) fluorescence was done with an average of three random fields on each coverslip at the end of H2O2 exposure and baicalein (98%, dissolved in DMSO) treatment, and after 1 h digitonin exposure. Percentage cell death (PI uptake) was expressed as the PI fluorescence relative to the maximal value seen after digitonin exposure (100%). Each experiment was repeated in different batches of cells. DNA Fragmentation Analysis Genomic DNA was extracted using a DNA extraction kit. Briefly, cardiomyocytes (4–5 10 6 ) were harvested and washed twice with cold PBS. Following the isolation of genomic DNA, the DNA was precipitated by isopropanol and washed with ethanol. The extracted DNA (3–5 g) was loaded onto a 2% agarose gel and run at 80 V for 45 min in Tris-acetate-EDTA (pH 8.3) buffer. The gel was visualized and photographed by a LAS3000 Imaging system (Fujifilm, Japan). Measurement of Intracellular ROS The intracellular probe 6-carboxy-2 0 , 7 0 -dichlorodihydrofluorescein diacetate (6-carboxyH2DCFDA) was used to monitor intracellular ROS. It is a non-fluorescent and cell permeable analog that is oxidized to highly fluorescent carboxy-dichlorofluorescein (carboxy-DCF) as measured at Ex 488 nm/Em 520 nm and expressed in arbitrary units (a.u.). This carboxylated form is more cell permeant than the classic H2DCFDA (DeAtley et al., 1999). Before the experiments, the medium was changed to phenol red-free M199 with Earle’s salts and the carboxy-H2DCFDA was added with the final concentration of 1 M. Cells were incubated for 30 min and washed with balance salt solution and then maintained with 2 ml of culture medium. Measurement of Mitochondrial Superoxide MitoSOX red was used for measuring mitochondrial superoxide (O 2 ). It was live-cell permeant and rapidly oxidized by superoxide in mitochondria with exhibition of red fluorescence. Cardiomyocytes were incubated with MitoSOX red (10 M) for 15 min during the last 15 min of H2O2 treatment. After washing twice with balanced salt solution, fluorescence images were acquired in an Olympus DSU “fixed cell” spinning disk confocal microscope with a 100x/1.35 oil immersion objective at Green (FITC, GFP) filter Ex 485/ 20 nm/Em 525/30 nm. The image and statistical analysis was performed with Image J (ver 1.38, NIH). Polyethylene glycol-superoxide dismutase and stigmatellin were used.


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Mitochondrial and Cytosolic Fractionation


Mitochondrial and cytosolic fractions were extracted using a cytosol/mitochondria fraction kit. Cardiomyocytes were washed twice with PBS and the pellets were suspended in the cytosol extraction buffer. Cells were homogenized with a tissue grinder. After centrifugation for 10 min at 700 g, the supernatants were transferred and centrifuged at 10,000 g for 30 min at 4 C. The resulting supernatant was used as the soluble cytosolic fraction. The pellet was suspended in the mitochondrial extraction buffer to generate the mitochondrial fraction. The fractions were analyzed by a Western blot. Western Blot Analysis The cells were treated and harvested in buffer containing 1% Triton-100, 20 mM Tris, 137 mM NaCl, 2 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 50 mM NaF, 1 mM Na3VO4, 200 mM PMSF, and 1x protease inhibitor cocktail. Protein concentrations were determined using the Bradford assay. Thirty micrograms protein lysates were resolved on SDS-Page gel and transferred to nitrocellulose membranes. After blocking in 5% milk/TBST, proteins were probed with antibodies against phosphorylated Akt (Thr308 and Ser473), phosphorylated JNK/SAPK (p46, p54) and -tubulin, then exposed to HRPlinked secondary antibodies, finally exposed to SuperSignal and visualized by X-ray film. For fractionation experiments, proteins from cytosolic and mitochondrial fractions were probed with antibodies against p-Akt Thr308, p-JNK, phosphorylated GSK3βser9, and cytochrome c, RhoGD1 and VDAC1. Densitometry was performed using Image J software (ver 1.38, NIH). Statistical Analysis Results are expressed as mean S.E. An individual experiment (n) was the result of observations of a single field of 500 cells on a coverslip. For comparison among the different treatment groups, the one-way ANOVA was used with post-hoc examination by Turkey test. A p < 0:05 was considered statistically significant. Results Baicalein Decreased Cell Death Induced by H2O2 Exposure H2O2 is a dominant form of ROS in cells. It has previously been used as a model of I/R injury in various cell types (Dossumbekova et al., 2008; Chou et al., 2010; Takagi et al., 2010). In this study, we used H2O2 to induce oxidant injury in cardiomyocytes to determine whether baicalein attenuates H2O2-induced cell death. H2O2 (500 M) exposure for 2 h caused significant cell death (45:3 6:2% vs. 8:6 2:2% in control, n ¼ 8, p < 0:001). Baicalein treatment at 10, 25 or 50 M for 2 h prior to and during 2 h H2O2 exposure resulted in a reduction of cell death to 38:7 5:6% (n ¼ 8, p ¼ 0:226), 31:2 3:9%


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(A)

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Figure 1. Baicalein protected cardiomyocytes against H2O2-induced injury. (A) Cardiomyocytes were treated with baicalein followed by H2O2 exposure and cell viability was determined by propidium iodide (PI) uptake. H2O2 induced significant cell death. **p < 0:001 vs. control. Baicalein (10, 25 or 50 M) was given 2 h prior to and during 2 h of H2O2 exposure markedly decreased cell death. *p < 0:01 vs. H2O2 alone. Data presented are mean standard error of eight independent experiments. (B) Baicalein suppressed the nucleosomal DNA fragmentation followed by H2O2 exposure analyzed by agarose gel electrophoresis. A ladder of DNA fragmentation induced by H2O2 (500 M) was reduced by baicalein (25 M). Data represented three independent experiments.

(n ¼ 8, p < 0:01) and 30:3 5:3% (n ¼ 8, p < 0:01), respectively (Fig. 1A). Baicalein alone had no effect on cell death (9:4 3:6% vs. 8:6 2:2% in control). Further, baicalein reduced a ladder-like DNA fragmentation (Fig. 1B). These results suggest that baicalein protects cardiomyocytes against H2O2-induced cell injury in a dose-dependent fashion. The treatment of baicalein 2 h prior to and during 2 h H2O2 was used for the proceeding experiments. Baicalein Attenuated Intracellular Oxidant Generation Induced by H2O2 Exposure The 6-carboxy-H2DCFDA was used to measure intracellular oxidant generation induced by H2O2. Figure 2 showed that H2O2 induced a rapid and significant increase of DCF fluorescence from 221 32 a.u. at baseline to 984 95 a.u. (n ¼ 6, p < 0:001). Baicalein treatment (10, 25 or 50 M) caused a dose-dependent attenuation in DCF fluorescence to 903 93 (n ¼ 6, p ¼ 0:354), 679 83 (n ¼ 6, p < 0:05) and 603 119 (n ¼ 6, p < 0:05), respectively. Baicalein alone had no effect on DCF fluorescence. Both 25 and 50 M of baicalein decreased DCF fluorescence and cell death to a comparable level, thus, the lower dose of 25 M was chosen for the proceeding experiments. Baicalein Attenuated Mitochondrial Superoxide Generation Induced by H2O2 Exposure We used MitoSOX red, a specific O 2 indicator (Mukhopadhyay et al., 2007), to determine if baicalein attenuates mitochondrial superoxide generation. MitoSOX red was live-cell permeant and rapidly oxidized by O 2 in mitochondria with exhibition of red fluorescence. This oxidation is prevented by superoxide dismutases, enzymes catalyzing



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Figure 2. Baicalein attenuated H2O2-induced oxidant generation. The intracellular oxidant production was determined by 6-carboxy-H2DCFDA (1 M). H2O2 (500 M) induced an increase in DCF fluorescence that was dose-dependently decreased by baicalein treatment (10, 25 or 50 M). **p < 0:001 vs. control; # p < 0:05 vs. H2O2 alone. The fluorescence intensity values were averaged from three different fields and analyzed with MetaMorph® software. Data presented are mean standard error of six independent experiments.

the dismutation of superoxide into oxygen and hydrogen peroxide. As illustrated in Fig. 3A, MitoSOX red fluorescence was significantly increased in H2O2-exposed cells compared to the control. Baicalein markedly reduced the MitoSOX red fluorescence (p < 0:05, n ¼ 5) (Fig. 3B) and it alone had no effect on the MitoSOX red fluorescence

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Figure 3. Baicalein attenuated mitochondrial superoxide measured by MitoSOX red. (A) A representative image of MitoSOX red fluorescence. H2O2 (500 M) caused an increased MitoSOX red fluorescence that was attenuated by baicalein. Co-treatment of H2O2 and PEG-SOD (200 unit/ml) or stigmatellin (STG, 50 nM) also attenuated the red fluorescence. Baicalein alone did not increase red fluorescence compared to the control. (B) The intensity analysis of MitoSOX red fluorescence. H2O2 increased the MitoSOX red fluorescence that was significantly decreased by baicalein (25 M). *p < 0:01 vs. control; # p < 0:05 vs. H2O2. Baicalein alone was not significantly different from the control. Data presented are mean S.E. of five independent experiments.

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(p ¼ 0:935, n ¼ 5), suggesting that baicalein attenuated H2O2-induced mitochondrial O 2 generation. This observation was further confirmed by polyethylene glycol superoxide dismutase (PEG-SOD, 200 unit/ml), which converts O 2 to H2O2, thereby decreasing the red fluorescence (Fig. 3A). To identify the site of O 2 generation, stigmatellin (50 nM), a mitochondrial ETC complex III inhibitor that inhibits electron flow through the quinol oxidation site of complex III, was employed. The MitoSOX red fluorescence was partially attenuated compared to H2O2 alone, suggesting that baicalein cardioprotection against H2O2-induced oxidative injury may be partially associated with attenuating O 2 that is originally generated from the mitochondrial ETC complex III. Baicalein Enhanced Akt Phosphorylation Akt is a key survival kinase in mediating cardioprotection against H2O2-induced and I/R injury (Wang et al., 2009). We observed that H2O2 induced an increased Akt phosphorylation at both Thr308 (Fig. 4A) and Ser473 sites (Fig. 4B) compared to the control. Baicalein further enhanced p-Akt Thr308 and p-Akt Ser473. Densitometric analysis showed that baicalein significantly increased p-Akt Thr308 (Fig. 4A, n ¼ 4, p < 0:01) and resulted in a trend toward an increase in p-Akt Ser473 during H2O2 exposure (Fig. 4B, n ¼ 4, p ¼ 0:716). Baicalein alone had no effect on Akt phosphorylation. Baicalein Decreased JNK Phosphorylation Induced by H2O2 Exposure H2O2 has been demonstrated to induce oxidative stresses, cellular injury and apoptosis through JNK pathways (Mizukami et al., 2001). Activation of JNK by oxidant stress

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Figure 4. Baicalein enhanced Akt phosphorylation (p-Akt) after H2O2 exposure. (A) H2O2 (500 M) increased pAkt Thr308 that was further enhanced by baicalein (25 M) treatment. *p < 0:01 vs. H2O2 alone. (B) Baicalein induced a trend toward increased p-Akt Ser473. p ¼ 0:216 vs. H2O2 alone. Tubulin was used as a loading control. Data presented are mean S.E. of four independent experiments.



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Figure 5. Baicalein decreased H2O2-induced JNK phosphorylation analyzed by Western blot. Baicalein (25 M) decreased p-JNK that was induced by H2O2. *p < 0:01. Tubulin was used as a loading control. Data presented are mean S.E. of four independent experiments.

is initiated in the mitochondria and involves ROS generation (Dougherty et al., 2004). Figure 5 showed that JNK phosphorylation was significantly increased after H2O2 exposure (p < 0:001, n ¼ 4), which was attenuated by baicalein (n ¼ 4, p < 0:01). Baicalein Increased the Mitochondrial Phosphorylation of Akt and GSK-3β Akt has been reported to preserve the mitochondrial function against oxidative injury (Shao et al., 2010) and the phosphorylation of GSK3β, a key downstream target of the PI3kinase/Akt survival signaling pathway, reflects Akt activity (Lin et al., 2007; Song et al., 2009). Signal emanating from plasma membrane receptors or generated by stress rapidly modulates Akt and Gsk-3β in mitochondria (Bijur and Jope, 2003). To test whether baicalein protection involves the increased mitochondrial Akt activation and GSK-3β phosphorylation, the treated-cells were fractionated into cytosolic and mitochondrial fractions. Immunoblotting for VDAC1 and RhoGDI, as mitochondrial and cytosolic markers respectively, indicated the cross contamination in these two fractions to be minimal. Figure 6A showed that baicalein treatment increased the p-Akt in both cytosolic and mitochondrial fractions in H2O2-exposed cells compared to H2O2 alone (n ¼ 4, p < 0:05, p < 0:01). Similarly, mitochondrial p-GSK3β Ser9 was significantly increased (Fig. 6B, n ¼ 4, p < 0:001) while cytosolic p-GSK3β Ser9 had a trend of increase (Fig. 6B, n ¼ 4, p ¼ 0:226) with baicalein treatment. In contrast, JNK phosphorylation was not observed in mitochondria (data not shown). Again, baicalein alone did not alter the basal phosphorylation of Akt and GSK3β compared to the control.


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Figure 6. Baicalein enhanced the phosphorylation of mitochondrial Akt Thr308 (p-Akt Thr308) and GSK3β Ser9 (p-GSK3β) measured by cellular fractionation of cytosol and mitochondria. Baicalein (25 M) increased both cytosolic and mitochondrial p-Akt Thr308. # p < 0:05, *p < 0:01 vs. H2O2 alone. Data are presented as mean standard error of four individual experiments. (B) Baicalein (25 M) increased p-GSK3β Ser9 in mitochondria with a trend increase in cytosol. **p < 0:001 vs. H2O2 alone cells; p ¼ 0:217 vs. H2O2 alone. Data are presented as mean standard error of four individual experiments.

Baicalein Reduced Cytochrome c Release Induced by H2O2 Exposure Cytochrome c is used as an indicator of mitochondrial damage, and H2O2 has been shown to increase cytochrome c release (Madesh and Hajnoczky, 2001). Under normal conditions,

Figure 7. Baicalein reduced mitochondrial cytochrome c release assessed by cellular fractionation of cytosol and mitochondria. H2O2 triggered an increased level of cytochrome c in cytosol. **p < 0:001 vs. control. Baicalein (25 M) decreased cytosol cytochrome c level and preserved mitochondrial cytochrome c level. **p < 0:001 vs. H2O2 alone; # p < 0:05 vs. H2O2 alone. Anti-RhoGD1 and VDAC1 were used to show cross contamination in these two fractions to be minimal. Data are showed as mean S.E. of four individual experiments.



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cytochrome c resides in the space between the outer membrane and inner membrane of mitochondria. When mitochondria are damaged, cytochrome c is leaked out of the outer membrane and released into the cytosol. To examine the role of baicalein in protecting mitochondrial integrity, cytochrome c was measured in both cytosolic and mitochondrial fractions. As depicted in Fig. 7, in control cells, cytochrome c was detected in the mitochondrial fraction, but not in the cytosolic fraction. In H2O2 exposed cells, the level of cytochrome c in the mitochondria was reduced, while an increase of cytochrome c in cytosolic fraction was observed. Baicalein pretreatment retained cytochrome c in mitochondria and decreased cytochrome c release to cytosol, indicating an amelioration of mitochondria damage. Quantitative analysis (Fig. 7) showed that in the cytosolic fraction, H2O2 resulted in an increase of cytochrome c compared to the control (n ¼ 4, p < 0:001) that was markedly attenuated by the baicalein (n ¼ 4, p < 0:001). In contrast, mitochondrial cytochrome c was preserved in baicalein-treated H2O2-exposed cells (n ¼ 4, p < 0:05). Baicalein alone had little effect on the release of cytochrome c (n ¼ 4, p ¼ 0:75). Discussion The present study investigated the effect of baicalein on H2O2-induced I/R injury in cardiomyocytes. We demonstrated that baicalein reduced DNA fragmentation; attenuated mitochondrial superoxide generation from mitochondria ETC complex III; upregulated the phosphorylation of mitochondrial Akt and its downstream target GSK3β while downregulating JNK activation. Role of Baicalein in ROS Generation Mitochondrial ETC is a major source of generating oxidants that contribute to I/R injury (Turrens and Boveris, 1980). Our prior work showed that the mitochondrial ETC caused a significant oxidative injury when cardiomyocytes were exposed to I/R and antioxidants attributed to protection against oxidant stress (Vanden Hoek et al., 1996, 1997b). In this study, H2O2 in cardiomyocytes induced a significant ROS production measured by DCF fluorescence, which was attenuated in a dose-dependent manner after baicalein treatment. This observation is consistent with previous work that baicalein exerted its protection via scavenging O 2 , OH and H2O2 in cells and in cell-free systems as measured by electric spin resonance (Shao et al., 2002) and baicalein attenuated intracellular ROS in various cells (Shao et al., 1999; Dossumbekova et al., 2008). However, few reports described the role of baicalein in regulating mitochondrial ROS. O 2 is the primary ROS produced by mitochondria (Zhang and Gutterman, 2007), therefore we examined the effect of baicalein on mitochondrial O 2 . Indeed, H2O2 induced an increase of the red fluorescence measured by MitoSOX, which was reduced by baicalein to almost the baseline level, suggesting baicalein predominantly acts to reduce mitochondrial O 2 . Since mitochondria continu ously generate O 2 at about 3–5% of total O2 consumption due to an electron leak caused by mitochondrial complexes I and III (Turrens and Boveris, 1980), we used a superoxide

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dismutase PEG-SOD and showed that red fluorescence was significantly reduced. Further, stigmatellin, a mitochondrial ETC complex III inhibitor, markedly reduced H2O2-induced MitoSOX red fluorescence, indicating that baicalein acts at mitochondrial ETC complex III. The involvement of Complex I was not investigated here because its inhibitor rotenone abolished the ROS burst, but did not confer any cardioprotection in our previous study (Anderson et al., 2006).


Effect of Baicalein on Prosurvival and Proapoptotic Pathways As depicted in Fig. 4, baicalein enhanced both p-Akt Thr308 and p-Akt Ser473 suggesting that baicalein protection may involve the increase of Akt signaling by activating upstream activators of PDK1 and mTORC2 (Sale and Sale, 2008), or inhibiting the phosphatases that regulate Akt. The lipid phosphatase PTEN regulates Akt phosphorylation at both Thr308 and Ser473 sites through the dephosphorylation of PIP3 (Hamada et al., 1993). The PHdomain phosphatases (PHLPPs) dephosphorylates Akt at Ser473 (Gao et al., 2005) while PP2A acts on Thr308 (Kuo et al., 2008). We observed that Akt phosphorylation at both Thr308 and Ser473 sites was enhanced by baicalein, suggesting that its protection perhaps is mediated by the activation of PI3K or inhibition of PTEN (Liu et al., 2010). The possibility that baicalein protection is mediated by suppressing PTEN activity is supported by a recent study in which a rapid increase in the intracellular reactive oxygen species level and nitrotyrosine formation induced by oxygen and glucose deprivation was counteracted by baicalein. The reduction of phosphorylation Akt and GSK3β was also restored by baicalein. As a consequence, Bcl-2/Bcl-xL-associated death protein was phosphorylated and its activity was inhibited (Liu et al., 2010). In addition to the role of baicalein on prosurvival signaling, the effect of baicalein on proapoptotic signaling JNK activation was also examined. We observed that baicalein suppressed H2O2-induced JNK activation. Although JNK phosphorylation was not detected in mitochondria, increased JNK activation has been implicated to prone the cell to undergo apoptosis in cardiomyocytes by our recent finding that baicalein ameliorated doxorubicin-induced cardiotoxicity by inhibiting JNK activation (Chang et al., 2011). These results indicate a unique role of baicalein in balancing the survival and death pathways. Similar to Akt, baicalein may regulate JNK at multiple levels at MAP3Ks such as apoptosis signal-regulated kinase (ASK1), MAP2K including MKK4 and MKK7, and MAPK such as JNK family members (Davis, 2000; Kyriakis, 2001). The effect of baicalein on the regulation of Akt and JNK could also occur in a crosstalk manner, as described that Akt can decrease the activation of ASK1/JNK cascade via phosphorylating ASK1 at Ser83 during oxidative stress (Wang et al., 2007). The mechanism of how baicalein activates the putative Akt survival pathway and JNK death pathway warrants further studies. Baicalein Regulation in Mitochondrial Phosphorylation of Akt and GSK3β In this study, we provided the first evidence that baicalein cardioprotection may involve an increase of Akt phosphorylation in both the cytosol and mitochondria, which was



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associated with the reduction of mitochondrial O 2 generation. The increased mitochondrial Akt may have a direct effect on mitochondria protection via stimulating the phosphorylation of hexokinase-II and concomitantly inhibiting the ability of Ca 2þ to induce cytochrome c release; as reported by Miyamoto et al., that increased mitochondrial Akt promotes its antioxidant protection in response to leukemia inhibitory factor LIF (Miyamoto et al., 2008). Akt activation, via its effects on mitochondrial associated proteins including GSK3β, can also attenuate mitochondrial ROS generation and confer cellular protection (Juhaszova et al., 2000; Ohori et al., 2008; Song et al., 2009). Phosphorylation of GSK3β, one of the first Akt downstream identified, was also increased in both cytosol and mitochondrial. This increased Phosphorylation of GSK3β may confer its protection via increasing the threshold for mPTP opening via preserving hexokinase II in mPTP complex (Juhaszova et al., 2008; Miyamoto et al., 2008), and suppressing p53-mediated apoptosis (Nishihara et al., 2007). Furthermore, the increased phosphorylation of GSK3β in mitochondria may provide more substrates accessible for phosphorylation (Bijur and Jope, 2003). Our results showed that baicalein increased phosphorylation of Akt and GSK3β, which was associated with the reduction of cytochrome c release from the mitochondria to the cytosol. As a result, DNA fragmentation, an indicator of cell apoptosis, was inhibited. We also showed that increased phosphorylation of Akt and GSK3β was related to the reduction of mitochondrial ROS generation that is consistent with a previous finding (Halestrap et al., 2007). In conclusion, baicalein protected cardiomyocytes against H2O2-induced oxidant injury by attenuating ROS, DNA fragmentation, and cell death. This cardioprotection may be associated with decreasing mitochondrial ROS, increasing Akt phosphorylation while decreasing JNK phosphorylation, and enhancing the phosphorylation of mitochondrial Akt and GSk3β. Baicalein may serve as a therapeutic strategy in treating oxidant injury. Acknowledgments This work is supported by the National Health Institute grant AT003441. References Anderson, T.C., C.Q. Li, Z.H. Shao, T. Hoang, K.C. Chan, K.J. Hamann, L.B. Becker and T.L. Vanden Hoek. Transient and partial mitochondrial inhibition for the treatment of postresuscitation injury: getting it just right. Crit. Care Med. 34: S474–S482, 2006. Andjelkovic, M., D.R. Alessi, R. Meier, A. Fernandez, N.J. Lamb, M. Frech, P. Cron, P. Cohen, J.M. Lucocq and B.A. Hemmings. Role of translocation in the activation and function of protein kinase B. J. Biol. Chem. 272: 31515–31524, 1997. Bijur, G.N. and R.S. Jope. Rapid accumulation of Akt in mitochondria following phosphatidylinositol 3-kinase activation. J. Neurochem. 87: 1427–1435, 2003. Boveris, A., E. Cadenas and A.O. Stoppani. Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem. J. 156: 435–444, 1976. Chang, W.T., J. Li, H.H. Haung, H. Liu, M. Han, S. Ramachandran, C.Q. Li, W.W. Sharp, K.J. Hamann, C.S. Yuan, T.L. Hoek and Z.H. Shao. Baicalein protects against doxorubicin-induced


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Cryptotanshinone protects against adriamycin-induced mitochondrial dysfunction in cardiomyocytes.

Echinochrome A protects mitochondrial function in cardiomyocytes against cardiotoxic drugs.

Ibutilide protects against cardiomyocytes injury via inhibiting endoplasmic reticulum and mitochondrial stress pathways.

reoxygenation injury via mitochondrial pathway mediated by SIRT1.

Akt protects the heart against ischaemia-reperfusion injury by modulating mitochondrial morphology.

Creatine protects against mitochondrial dysfunction associated with HIV-1 Tat-induced neuronal injury.

Mitochondrial CaMKII inhibition in airway epithelium protects against allergic asthma.

reperfusion injury via inhibition of NF-κB pathway in mice.

Reoxygenation-Induced Mitochondrial Dysfunction in H9c2 Cardiomyocytes.

MiR-25 protects cardiomyocytes against oxidative damage by targeting the mitochondrial calcium uniporter.

reperfusion.

Taurine protects against oxidant injury to rat alveolar pneumocytes.

Inhaled Carbon Monoxide Protects against the Development of Shock and Mitochondrial Injury following Hemorrhage and Resuscitation.

DNaseI protects against Paraquat-induced acute lung injury and pulmonary fibrosis mediated by mitochondrial DNA.

AMPK activation by isorhamnetin protects hepatocytes against oxidative stress and mitochondrial dysfunction.

PPARα activation protects against cholestatic liver injury.

CXCR4 attenuates cardiomyocytes mitochondrial dysfunction to resist ischaemia-reperfusion injury.

Adiponectin protects against acetaminophen-induced mitochondrial dysfunction and acute liver injury by promoting autophagy in mice.

GSK-3β Pathway.

Nrf2 signaling.

eNOS.

Keap1 pathway.

Sirt3 protects cortical neurons against oxidative stress via regulating mitochondrial Ca2+ and mitochondrial biogenesis.

Ginsenoside Rb3 protects cardiomyocytes against ischemia-reperfusion injury via the inhibition of JNK-mediated NF-κB pathway: a mouse cardiomyocyte model.