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1 Section of Emergency Medicine and 2 Pulmonary Critical Care, Department of Medicine, 3 Emergency Resuscitation Center, 4 Department of Anesthesia and Critical Care and 5 Tang Center for Herbal Medicine Research, 6 Department of Radiation and Cellular Oncology, and 7 Center for Low Frequency EPR Imaging for In Vivo Physiology, University of Chicago, Chicago, Illinois 60637; and 8 Department of Pharmaceutical Sciences, University of Maryland School of Pharmacy, and Medical Biotechnology Center, University of Maryland, Baltimore, Maryland 21210
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Flavonoids within Scutellaria baicalensis may be potent antioxidants on the basis of our studies of S. baicalensis extract. To further this work, we studied the antioxidative effects of baicalein, a flavonoid component of S. baicalensis, in a chick cardiomyocyte model of reactive oxygen species (ROS) generation during hypoxia, simulated ischemia-reperfusion, or mitochondrial complex III inhibition with antimycin A. Oxidant stress was measured by oxidation of the intracellular probes 2',7'-dichlorofluorescin diacetate and dihydroethidium. Viability was assessed by propidium iodide uptake. Baicalein attenuated oxidant stress during all conditions studied and acted within minutes of treatment. For example, baicalein given only at reperfusion dose dependently attenuated the ROS burst at 5 min after 1 h of simulated ischemia. It also decreased subsequent cell death at 3 h of reperfusion from 52.3 ± 2.5% in untreated cells to 29.4 ± 3.0% (with return of contractions; P < 0.001). In vitro studies using electron paramagnetic resonance spectroscopy with the spin trap 5-methoxycarbonyl-5-methyl-1-pyrroline-N-oxide revealed that baicalein scavenges superoxide but does not mimic the effects of superoxide dismutase. We conclude that baicalein can scavenge ROS generation in cardiomyocytes and that it protects against cell death in an ischemia-reperfusion model when given only at reperfusion.
Scutellaria baicalensis; ischemia; reactive oxygen species; 2',7'-dichlorofluorescin diacetate; antimycin A; dihydroethidium; mitochondria
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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BAICALEIN
(5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one) is one of
the major flavonoids of Scutellaria baicalensis, an herb
used in Chinese and Japanese medical applications. Among its biological
activities, baicalein has been reported to exhibit antioxidant effects
(9, 15, 20, 41). In this regard, it has been reported to
scavenge reactive oxygen species (ROS), including superoxide
(O
-tocopherol (vitamin E), and the xanthine oxidase inhibitor
allopurinol. Collectively, these studies support the conclusion that
baicalein exhibits antioxidant properties, although the specific
mechanism of action is not fully understood. In addition, antioxidant
studies of such in vitro or exogenous oxidant models may not predict
success in treating endogenous intracellular oxidant stress.
There is evidence that intracellular ROS generation may contribute to
the pathogenesis of cellular injury during ischemia and
reperfusion in a number of tissues (12, 22). ROS may
interact degeneratively with cellular components, including nucleic
acids, proteins, and lipids, to compromise structure and function
(19). Moreover, the resulting functional defects may
depend on the specific microdomains where the oxidants are generated.
Although baicalein has been shown to confer protection against
exogenously applied oxidants, its ability to attenuate cell injury has
not been demonstrated in a pathophysiological model where the oxidants
generated within the cell contribute to the cellular dysfunction.
Reactive species, including O
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METHODS |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Diethylenetriaminepentaacetic acid (DTPA), xanthine, xanthine
oxidase, catalase, superoxide dismutase (SOD), baicalein (98% purity),
and antimycin A were purchased from Sigma (St. Louis, MO),
5-hydroxydecanoate-Na (5-HD) from Biomol (Plymouth Meeting, PA), and
diazepam-binding inhibitor from Calbiochem (San Diego, CA). The spin
trap 5-methoxycarbonyl-5-methyl-1-pyrroline-N-oxide (MMPO)
was synthesized on the basis of methods described previously (5,
33). The structure of baicalein and the reaction of MMPO with
O
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Cardiomyocyte System
Embryonic ventricular cardiomyocytes were prepared as described previously (35). Briefly, heart ventricles from 10-day-old chick embryos were removed, minced, enzymatically dispersed with 0.025% trypsin (GIBCO, New York, NY), and centrifuged to yield 4-5 × 105 cells/embryo. Cells (0.7 × 106) were pipetted onto glass coverslips, incubated, and grown into contractile layers. Synchronous contractions were seen by the third day in culture. Contamination by fibroblasts was reduced by preplating, and myocyte phenotype was confirmed using antimyosin heavy chain monoclonal antibodies (CCM-52). Experiments were performed with 3- to 5-day cardiac cell culture, at which point viability was >99%.Perfusion System
Coverslips with synchronously contracting cells were placed inside a Sykes-Moore flow-through chamber (1.2 ml volume; Bellco Glass, Vineland, NJ). The chamber and inflow tubing were maintained at 37°C. Flow rate (0.25 ml/min), pH, and PO2 of the perfusate were controlled. Hypoxic conditions were verified with an optical method of phosphorescence quenching (Oxyspot, Medical Systems, Greenvale, NY). To minimize O2 leaks, the perfusate was supplied to the chamber by stainless steel tubing to prevent diffusive entry of O2 through the tube wall.Perfusate Composition
Standard perfusate consisted of buffered salt solutions (BSS) with 100 Torr PO2, 40 Torr PCO2, pH 7.4, 4.0 meq K+/l, and 5.6 mM glucose. Simulated ischemia consisted of BSS containing no glucose, with 20 mM 2-deoxyglucose added to inhibit glycolysis and 8 meq K+/l. This was bubbled with 80% N2-20% CO2 to produce <3 Torr PO2, 144 Torr PCO2, and final pH 6.8. Hypoxic medium was bubbled with 95% N2-5% CO2 and without glucose. Reperfusion was carried out with standard BSS.Video/Fluorescent Microscopy
Cells were imaged with an Olympus IMT-2 inverted phase/epifluorescent microscope equipped with Hoffman Modulation optics to accentuate the surface topology of the cells. This facilitated detection of contractile movement in the confluent layer of cells. Cell contractions were observed as described previously (32, 36). The criteria for a return of contraction were met if contractions were observed throughout the cell field after 3 h of reperfusion. A single field of cells was monitored for contractions throughout each experiment. Phase-contrast images were recorded for contraction analysis with a charge-coupled device camera. Fluorescence was measured using a cooled slow-scanning personal computer-controlled camera (Hamamatsu, Hamamatsu City, Japan) coupled with Image-One software (Image pro Plus) for quantification of changes in emission fluorescence.Viability Assay
Cell viability was quantified over time using the nuclear stain propidium iodide (PI, 5 µM; Molecular Probes, Eugene, OR), an exclusion fluorescent dye that binds to chromatin on loss of membrane integrity. This method is similar in principle to trypan blue staining and has been reported to predict the transition from reversible to irreversible cell injury in cultured cardiomyocytes (4). PI is not toxic to cells over a course of 8 h, permitting its addition to the perfusate throughout the experiment. At the end of each experiment using PI, all nuclei in a field of ~500 cells were stained by permeabilization with 300 µM digitonin. Percent loss of viability (i.e., cell death) over time was expressed relative to the maximal value seen after digitonin exposure (100%).Measurement of Intracellular ROS Generation
Intracellular oxidant stress was monitored by measuring changes in fluorescence resulting from intracellular probe oxidation. Dihydroethidium (DHE, 2 µM; Molecular Probes) enters the cell and can be oxidized by ROS, including OMeasurement of In Vitro ROS Generation
Spin trapping of OConditions Used to Generate ROS and Induce Oxidant Injury
Brief hypoxia protocol. Cardiomyocytes were preincubated for 45 min with 5 µM DCFH-DA, perfused for 15 min with standard BSS, and then exposed to hypoxia for 10 min and normoxia for 10 min. Baicalein was added to the perfusate only during the hypoxia phase.
Simulated ischemia protocol. Cells were loaded with 2 µM DHE, equilibrated for 30 min with standard BSS, and then exposed to 1 h of simulated ischemia. Baicalein at various doses was given during the ischemia phase.
Simulated ischemia-reperfusion. Cells were loaded with 5 µM DCFH-DA, equilibrated for 30 min with normoxia, and then exposed to 1 h of ischemia, followed by 30 min of reperfusion. Baicalein was added only during reperfusion. Cells were loaded with 5 µM PI, equilibrated for 30 min with normoxia, and exposed to 1 h of ischemia and 3 h of reperfusion and then to 300 µM digitonin for 1 h. Baicalein was added only during reperfusion.
Mitochondrial inhibition. Cells were loaded with 10 µM DCFH-DA or 5 µM PI and exposed to 100 µM antimycin A alone or with baicalein for 2 h in multiple culture dishes.
Data Analysis
Data were collected, and simple descriptive analyses were performed. An individual experiment (n) was the result of observations of a single field of ~500 cells on a coverslip. Replicates were performed on separate coverslips. Values are means ± SE. For test of significance, analysis of variance and two-tailed unpaired t-test were performed, with P < 0.05 considered to be significant.| |
RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Effect of Baicalein on Oxidant Stress During Brief Hypoxia
Oxidant stress was assessed by measuring oxidation of DCFH to DCF. As shown in Fig. 2, brief hypoxia caused a rapid and significant increase in DCF fluorescence (measured in arbitrary units) from 0.71 ± 0.11 (SE) at baseline to 1.70 ± 0.07 (n = 6, P < 0.01) at 10 min of hypoxia. Baicalein, at 25, 50, 100, or 200 µM, given at the start of hypoxia caused a dose-dependent attenuation in DCF fluorescence to 1.57 ± 0.09 (n = 6, P > 0.01), 1.33 ± 0.05 (n = 6, P < 0.01), 0.84 ± 0.10 (n = 6, P < 0.001), and 0.62 ± 0.05 (n = 6, P < 0.001), respectively. These findings suggest that baicalein caused a concentration-dependent attenuation of H2O2 and hydroxyl radicals during transient hypoxia.
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Effect of Baicalein on Oxidant Stress During Simulated Ischemia
Oxidation of DHE to Eth-DNA was used to assess O
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Effect of Baicalein on Oxidant Stress, Cell Viability, and Contractile Function During Ischemia and Reperfusion
A rapid burst of DCF fluorescence was observed at 30 min of reperfusion after 1 h of ischemia (Fig. 4). In cells treated with 25 or 100 µM baicalein only during reperfusion, DCF fluorescence was attenuated from 2.88 ± 0.21 in untreated ischemic cells (n = 5) to 2.11 ± 0.09 (n = 5, P < 0.01). In cells treated with 100 µM baicalein, DCF fluorescence was further attenuated to 1.43 ± 0.07 (n = 5, P < 0.001). These findings suggest that baicalein given only at reperfusion attenuated oxidant stress during reperfusion in a dose-dependent fashion.
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As reported previously, cell death in this model of simulated
ischemia-reperfusion occurred primarily during the reperfusion phase, whereas minimal cell death was seen during the ischemia phase (35). Significant cell death again was evident
during 3 h of reperfusion after 1 h of ischemia. In
cells treated with 50 µM baicalein given only during reperfusion, PI
uptake decreased from 52.3 ± 2.5% in untreated ischemic
cells (n = 6) to 29.4 ± 3.0% (n = 3, P < 0.001; Fig. 5).
Contractile activity returned after reperfusion (3 of 3 experiments in
treated cells), whereas there was no recovery of contraction in
untreated cells (0 of 6 experiments in controls). Thus baicalein
significantly reduced cell death and enhanced the return of
contraction.
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Mechanism of Baicalein Effect on Reperfusion ROS
ATP-sensitive K+ (KATP) channels have been implicated in protection of cardiomyocytes against ischemia-reperfusion injury. Indeed, our own recent work suggests that KATP channel opening just at reperfusion can attenuate oxidant generation and confer significant cardioprotection (39). To determine whether KATP channels were responsible for the protection afforded by baicalein, a KATP channel inhibitor, 5-HD, was given during equilibration and ischemia-reperfusion. DCF fluorescence was attenuated from 2.88 ± 0.21 in untreated ischemic cells (n = 5) to 2.03 ± 0.06 (n = 3, P < 0.01) in baicalein (25 µM)-treated cells also given 500 µM 5-HD. This response was not different from that seen with baicalein alone (from 2.88 ± 0.21 in untreated cells to 2.11 ± 0.09, n = 5, P < 0.01; Fig. 6). Thus inhibition of the KATP channel did not abolish the protective effect of baicalein.
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Baicalein has been reported to have an affinity for the benzodiazepine
binding site of GABAA receptors (23).
Interestingly, the benzodiazepine receptors in peripheral tissues such
as adrenals, kidney, and heart (2) enhance mitochondrial
processing of human manganese-dependent SOD (Mn-SOD) precursor protein.
Wright et al. (40) suggested a possible redox-related
mechanism of mitochondrial protein import that may lead to less
efficient precursor protein uptake by mitochondria under severely
oxidizing conditions. In our study, we tested whether the antioxidant
effect of baicalein is related to this benzodiazepine binding site of
peripheral benzodiazepine receptors. When 25 µM baicalein was given
during the ischemia and reperfusion phases, DCF fluorescence
was attenuated from 2.50 ± 0.13 to 1.76 ± 0.10 (n = 3, P < 0.01). In cells treated
with 1 µM diazepam-binding inhibitor and 25 µM baicalein, DCF
fluorescence was also attenuated to 1.81 ± 0.13 (n = 3, P < 0.01; Fig.
7). Thus inhibition of the benzodiazepine
receptor did not abolish the protective effect of baicalein.
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Effect of Baicalein on Oxidant Stress and Cell Viability During Mitochondrial Electron Transport Inhibition
Antimycin A inhibits mitochondrial electron transport through complex III at a site that enhances generation of O
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Figure 9 shows that cell death was
significantly increased at the end of 2 h of antimycin A exposure
from 4.8 ± 1.5% in controls (n = 10) to
51.1 ± 3.2% (n = 10). In cells exposed to 100 µM antimycin A and treated with 10 or 50 µM baicalein, cell death
was decreased to 25.5 ± 2.3% (n = 10, P < 0.01) and 20.1 ± 1.8% (n = 10, P < 0.001), respectively. Thus baicalein decreased
cell death during mitochondrial electron transport inhibition with
antimycin A.
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ROS Scavenging by Baicalein
Fluorophores such as DCFH and DHE can potentially be oxidized by multiple ROS, so they lack the specificity required to implicate a particular oxidant. Accordingly, studies were carried out using the spin trap MMPO, which reacts with O
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With the use of in vitro studies with DCFH-DA in a xanthine/xanthine
oxidase system, minimal oxidation of DCFH was observed in the absence
of SOD, suggesting that DCFH oxidation by O
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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S. baicalensis is a widely used herb in traditional medical systems of China and Japan (17). The major constituents of S. baicalensis are flavonoids, a group of polyhydroxy phenols (20). Flavonoids of S. baicalensis, which include baicalein, baicalin, wogonin, and skullcapflavones I and II, have been associated with antioxidant and other pharmacological effects. Among them, baicalein has attracted considerable attention, because it has a variety of interesting activities. As a polyphenol, which belongs to the flavone subgroup, it potentially has potent free radical scavenging and antioxidant effects because of its o-trihydroxyl structure in the A ring (11). The antioxidant effectiveness of phenolic compounds may relate to their ability to enter cells and to orient in biomembranes (18). Flavonoids anchor to the polar heads of membrane phospholipids, forming reversible physicochemical complexes (29). The degree of glycosylation is one characteristic that affects various properties of some flavonoids, particularly their hydrophobicity (24). For example, the glycosidic group of rutin, a flavonol, makes it unable to penetrate model membranes (29). Baicalein, being free of sugar moieties, is more lipid soluble and may be able to penetrate membranes with greater ease. This lipophilic characteristic of baicalein may explain why its antioxidant effects could be seen within minutes of treatment against the various conditions under which the endogenous generation of ROS was increased.
Effect of Baicalein on Oxidant Stress During Moderate Hypoxia
We previously reported that 10 min of hypoxia in chick cardiomyocytes elicited an increase in mitochondrial ROS generation, as evidenced by oxidation of the intracellular fluorescent probe DCFH (38). These low levels of ROS appear to participate as signaling messengers. In the present study, increases in DCFH oxidation during hypoxia were attenuated in a dose-dependent manner after addition of baicalein to the perfusate, suggesting that baicalein can attenuate oxidant signaling in cells. To rule out the alternative possibility that baicalein might interfere with DCFH oxidation measurements, EPR spectroscopy experiments were carried out using MMPO as a spin trap for OEffect of Baicalein on Oxidant Stress During Ischemia
As reported previously (36, 37), an increase in the rate of DHE oxidation was seen during the ischemic phase in the present study. Our work has suggested that this oxidant stress is due to OEffect of Baicalein on Reperfusion Oxidants and Reperfusion Injury
Evidence from animal studies suggested that reperfusion of ischemic areas and the readmission of O2 may cause further tissue damage (25). ROS, including OEffect of Baicalein on Oxidant Stress During Mitochondrial Electron Transport Chain Inhibition
Under physiological conditions, oxidant generation from the mitochondrial electron transport chain (ETC) is balanced by cellular antioxidant defenses (8). However, increases in OMechanism of Baicalein Antioxidant Activity
Cellular antioxidants can act by inhibiting free radical formation, by directly scavenging radicals, or by enhancing cellular antioxidant mechanisms. Baicalein may protect by one or more of these mechanisms. In a previous study, we found that the mitochondrial KATP channel modulates oxidant generation at the start of reperfusion (39). When the mitochondrial KATP channel opener pinacidil was added at the start of reperfusion, it abrogated oxidant generation at reperfusion and reduced cell death. The mitochondrial KATP channel inhibitor 5-HD (30) blocked this effect, which suggests that activation of this channel may regulate oxidant generation or antioxidant defenses in the cell. To test whether this channel is involved in the protective effects of baicalein, we tested whether 5-HD could abolish the antioxidant effects conferred by that flavonoid. DCF fluorescence increased during reperfusion after simulated ischemia in cardiomyocytes. However, attenuation of the reperfusion oxidant burst by baicalein was not blocked by 5-HD, indicating that the antioxidant action of baicalein is not mediated by activation of the mitochondrial KATP channel.Studies have also reported that the mitochondrial benzodiazepine receptor (mBzR) regulates multiple conductance channel activity. Moreover, mBzR agonists potentiate multiple conductance channel electrical activity (21) and enhance mitochondrial processing of Mn-SOD precursor protein (40). We therefore tested whether mBzR inhibition could abolish baicalein's antioxidant effect. However, no effect of mBzR inhibition was detected, suggesting that this system is not involved.
In summary, our results show that baicalein attenuated oxidant stress in cardiomyocytes during hypoxia, ischemia, ischemia-reperfusion, and mitochondrial ETC inhibition. Antioxidant action is associated with increased cardiomyocyte survival and contractile function during ischemia-reperfusion. Our results also show that the antioxidant effect of baicalein is not associated with the mitochondrial KATP channel or the mBzR but is associated with significant superoxide scavenging.
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ACKNOWLEDGEMENTS |
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This work was supported by National Institutes of Health Grants HL-03779, HL-35440, HL-32646, RR-12257, and AT-00381.
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FOOTNOTES |
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Address for reprint requests and other correspondence: C.-S. Yuan, Dept. of Anesthesia and Critical Care, The University of Chicago, 5841 S. Maryland Ave., MC 4028, Chicago, IL 60637 (E-mail: cyuan{at}midway.uchicago.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpheart.00163.2001
Received 8 March 2001; accepted in final form 10 October 2001.
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RESULTS
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, Hypoxia; 
,
hypoxia with 25 µM baicalein; 
, hypoxia with 50 µM
baicalein; 
, hypoxia with 100 µM baicalein;
, hypoxia with 200 µM baicalein. au, Arbitrary units.
*P < 0.01; **P < 0.001 compared with
hypoxia.
,
ischemia-reperfusion with 25 µM baicalein and 500 µM
5-HD.
, MMPO + X/XO; 
, MMPO + X/XO + DMSO; 
, MMPO + PBS with no X/XO;
