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Am J Physiol Heart Circ Physiol 282: H999-H1006, 2002; doi:10.1152/ajpheart.00163.2001
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Vol. 282, Issue 3, H999-H1006, March 2002

Baicalein attenuates oxidant stress in cardiomyocytes

Zuo-Hui Shao1,3, Terry L. Vanden Hoek1,3, Yimin Qin2,3, Lance B. Becker1,3, Paul T. Schumacker2,3, Chang-Qing Li1,3, Lucy Dey4,5, Eugene Barth3,6,7, Howard Halpern3,6,7, Gerald M. Rosen7,8, and Chun-Su Yuan3,4,5

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


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP>·), H2O2, and hydroxyl radicals (14). Baicalein has also been shown to strongly inhibit iron-dependent lipid peroxidation in microsomes (9) and mitochondria (26). In glomerular mesangial cells, damage induced by exogenous H2O2 treatment was inhibited by baicalein (32). Although the specific mechanism of protection was not known, baicalein may be protective against oxidant injury by virtue of its ability to function as an iron chelator (13). By inhibiting the Fe2+-catalyzed Fenton reaction, baicalein may decrease the generation of hydroxyl radicals in the presence of H2O2 (1). Using in vitro methods, Gao et al. (10) reported that baicalein prevented human dermal fibroblast cell damage by ROS and was more effective than the iron chelator deferoxamine, hydroxyl radical scavengers including dimethyl sulfoxide and ethanol, the lipid peroxidase quenching agent alpha -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<UP><SUB>2</SUB><SUP>−</SUP></UP>·, H2O2, and hydroxyl radicals, have been shown to contribute to myocardial ischemia-reperfusion injury (7, 27). A similar role for ROS has been demonstrated in the contractile dysfunction and cell death observed during simulated ischemia-reperfusion and mitochondrial inhibition in a cardiomyocyte model (35-37). The observation that antioxidants confer significant protection in cultured cardiomyocytes suggests that such a model is appropriate for testing whether baicalein protects against oxidant injury. In addition, since many antioxidants previously found to be effective in these settings require preincubation (36) or must be given as a cocktail (39), this model allows testing of whether a single antioxidant administered at the time of oxidant generation can confer protection without preincubation. Such antioxidants could prove useful in modifying injury where other antioxidants have failed because of difficulties of intracellular access (27). Our previous study demonstrated that a water extract of Scutellaria baicalensis could attenuate oxidant stress even when given just at the start of conditions that increase endogenous oxidant generation (32). The present study was designed to extend those findings by examining the antioxidant and cardioprotective properties of one of the principal flavonoids in that extract.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>2</SUB><SUP>−</SUP></UP>· are shown in Fig. 1.


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  		Fig. 1.   A: structure of baicalein (5,6,7-trihydroxy-2-phenyl-4H-1-benzopyran-4-one). B: reaction of 5-methoxycarbonyl-5-methyl-1-pyrroline-N-oxide (MMPO) with O<UP><SUB>2</SUB><SUP>−</SUP></UP>· yielding the spin trap adduct MMPO-OOH.

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 O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and/or hydroxyl radical, to yield fluorescent ethidium (Eth). Eth binds to DNA (Eth-DNA), further amplifying its fluorescence. Thus increases in DHE oxidation to Eth-DNA (i.e., increases in Eth-DNA fluorescence) are suggestive of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation (36). The probe 2',7'-dichlorofluorescin diacetate (DCFH-DA, 5 µM; Molecular Probes) enters the cell, and the acetate group on DCFH-DA is cleaved by cellular esterases, trapping the nonfluorescent 2',7'-dichlorofluorescin (DCFH) inside. Subsequent oxidation by ROS, particularly H2O2 and hydroxyl radical, yields the fluorescent product dichlorofluorescein (DCF) (28). Thus increases in DCFH oxidation to DCF (i.e., increases in DCF fluorescence) suggest H2O2 or hydroxyl radical generation (36).

Measurement of In Vitro ROS Generation

Spin trapping of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· by 110 mM MMPO was achieved as follows. All experiments were carried out in phosphate-buffered (50 mM) saline (PBS) containing 1 mM DTPA to chelate any trace redox active metal ions that contaminate PBS. Xanthine (0.40 mM) was dissolved in PBS. Catalase (300 U/ml) was added to the reaction mixture to eliminate H2O2 produced by the self-dismutation of O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. The reaction was initiated by addition of xanthine oxidase (0.04 U/ml) to the above mixture. This resulted in an initial rate of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· production of ~10 µM/min. The reaction mixture was then loaded into a quartz aqueous flat cell (Wilmad) and installed into a horizontally disposed cavity (TM011) of the electron paramagnetic resonance (EPR) spectrometer (model E-12, Varian Associates, Palo Alto, CA). The ability of baicalein to scavenge O<UP><SUB>2</SUB><SUP>−</SUP></UP>· was also assessed with 10 µM DCFH-DA using a xanthine (0.4 mM)/xanthine oxidase (0.02 U/ml) system to generate O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. In the presence of SOD (200 U/ml), all O<UP><SUB>2</SUB><SUP>−</SUP></UP>· would be expected to quickly become H2O2 and oxidize DCFH to DCF. Baicalein (10 µM) was added to the system to measure the changes of DCF fluorescence using a fluorescence spectrophotometer (excitation 488 nm/emission 529 nm).

Conditions 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
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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  		Fig. 2.   Effect of baicalein on 2',7'-dichlorofluorescin (DCFH) oxidation during brief hypoxia. Cardiomyocytes were allowed to preincubate for 45 min and perfuse for 15 min with normoxia and then exposed to 10 min of hypoxia (3 Torr PO2), followed by 10 min of normoxia. Hypoxia increased in dichlorofluorescein (DCF) fluorescence. By contrast, baicalein given during 10 min of hypoxia produced a concentration-dependent attenuation in DCF fluorescence. , Hypoxia; black-triangle, hypoxia with 25 µM baicalein; , hypoxia with 50 µM baicalein; black-down-triangle , hypoxia with 100 µM baicalein; black-lozenge , hypoxia with 200 µM baicalein. au, Arbitrary units. *P < 0.01; **P < 0.001 compared with hypoxia.

Effect of Baicalein on Oxidant Stress During Simulated Ischemia

Oxidation of DHE to Eth-DNA was used to assess O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and hydroxyl radical generation during simulated ischemia. A significant increase in Eth-DNA fluorescence was seen during 1 h of ischemia (Fig. 3). However, 5 or 10 µM baicalein attenuated Eth-DNA fluorescence from 100% in untreated ischemic cells to 66.7 ± 6.2% (n = 5, P < 0.01) and 48.2 ± 4.6% (n = 5, P < 0.001), respectively. This suggests that baicalein can attenuate oxidant stress due to O<UP><SUB>2</SUB><SUP>−</SUP></UP>· during simulated ischemia.


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  		Fig. 3.   Effect of baicalein on dihydroethidium (DHE) oxidation during simulated ischemia. Ethidium (Eth)-DNA fluorescence significantly increased during 1 h of ischemia. When baicalein was administered during 1 h of ischemia, Eth-DNA fluorescence was decreased. , Ischemia; black-triangle, ischemia with 5 µM baicalein; black-down-triangle , ischemia with 10 µM baicalein. *P < 0.01; **P < 0.001 compared with untreated ischemic cells.

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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  		Fig. 4.   Effect of baicalein on DCFH oxidation during ischemia-reperfusion (I/R). At 30 min of reperfusion after 1 h of ischemia, there was a rapid burst of DCF fluorescence. In cells treated with 25 or 100 µM baicalein, DCF fluorescence was attenuated. , Ischemia-reperfusion; , ischemia-reperfusion with 25 µM baicalein; black-lozenge , ischemia-reperfusion with 100 µM baicalein. *P < 0.01; **P < 0.001 compared with untreated ischemic cells.

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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  		Fig. 5.   Effect of baicalein on cell death during ischemia-reperfusion. When 50 µM baicalein was given only at reperfusion, propidium iodide (PI) uptake was significantly reduced at the end of 3 h of reperfusion. , Ischemia-reperfusion; , ischemia-reperfusion with 50 µM baicalein. **P < 0.001 compared with untreated ischemic cells.

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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  		Fig. 6.   Effect of baicalein on oxidant stress at reperfusion after 1 h of ischemia. There was a rapid burst of DCF fluorescence at 30 min of reperfusion. In cells treated with 25 µM baicalein at reperfusion, DCF fluorescence was attenuated. When the ATP-sensitive K+ (KATP) channel inhibitor 5-hydroxydecanoate (5-HD, 500 µM) was given during equilibration and ischemia-reperfusion and 25 µM baicalein was given only at reperfusion, attenuation of DCF fluorescence was not reversed by 5-HD. , Ischemia-reperfusion; , ischemia-reperfusion with 25 µM baicalein; , ischemia-reperfusion with 25 µM baicalein and 500 µM 5-HD.

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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  		Fig. 7.   Effect of baicalein on oxidant generation at reperfusion after 1 h of ischemia. There was a rapid burst of DCF fluorescence at 30 min of reperfusion. In cells treated with 25 µM baicalein at reperfusion, DCF fluorescence was attenuated. When 1 µM diazepam-binding inhibitor (DBI) was given during equilibration and ischemia-reperfusion and 25 µM baicalein was given only at reperfusion, attenuation of DCF fluorescence was not reversed by DBI. , Ischemia-reperfusion; , ischemia-reperfusion with 25 µM baicalein; , ischemia-reperfusion with 25 µM baicalein and 1 µM DBI.

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<UP><SUB>2</SUB><SUP>−</SUP></UP>· (6). Figure 8 shows that antimycin A increased DCF fluorescence from 232 ± 27 in controls (n = 10) to 1,883 ± 105 at 2 h of exposure as expected. By contrast, DCF fluorescence in cells exposed to 100 µM antimycin A and treated with 10 or 50 µM baicalein was attenuated to 997 ± 75 (n = 10, P < 0.01) or 822 ± 65 (n = 10, P < 0.001), respectively. These findings indicate that baicalein caused a concentration-dependent attenuation of oxidant stress induced by mitochondrial electron transport inhibition with antimycin A. 


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  		Fig. 8.   Effect of baicalein on DCF fluorescence during antimycin A exposure. DCF fluorescence increased after 2 h of 100 µM antimycin A exposure but decreased in cells treated with 10 and 50 µM baicalein. *P < 0.01; **P < 0.001 compared with antimycin A-exposed cells.

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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  		Fig. 9.   Effect of baicalein on cell death during antimycin A exposure. PI uptake in cells exposed to 100 µM antimycin A was increased compared with controls. In cells treated with 10 and 50 µM baicalein, PI uptake was significantly less after 2 h of antimycin A exposure. *P < 0.01; **P < 0.001 compared with antimycin A-exposed cells.

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<UP><SUB>2</SUB><SUP>−</SUP></UP>· to create an adduct (MMPO-OOH) with a unique EPR spectrum. Superoxide was generated in vitro using a xanthine/xanthine oxidase system. In the absence of baicalein, the MMPO-OOH levels increased for the first 15 min, then decreased toward zero as the rate of O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation declined and the MMPO-OOH adduct decayed spontaneously (Fig. 10). Addition of SOD abolished the increase in MMPO-OOH, confirming that the EPR spectrum reflected the spin trapping of O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. Addition of baicalein produced a concentration-dependent attenuation of the EPR spectrum of MMPO-OOH, suggesting that baicalein scavenged O<UP><SUB>2</SUB><SUP>−</SUP></UP>·.


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  		Fig. 10.   Effect of baicalein on intensity of the electron paramagnetic resonance (EPR) spectrum of the MMPO spin trap adduct of superoxide. Superoxide anions (~10 µM/min) generated by 0.40 mM xanthine (X) + xanthine oxidase (XO, 0.04 U/ml) were trapped by MMPO to yield a product with a characteristic EPR spectrum. Intensity of this spectrum was attenuated by baicalein in a dose-dependent manner. , 1.0 µM baicalein; black-triangle, 2.5 µM baicalein; black-down-triangle , 5.0 µM baicalein; black-lozenge , 10 µM baicalein; , 25 µM baicalein; down-triangle, MMPO + X/XO; open circle , MMPO + X/XO + DMSO; triangle , MMPO + PBS with no X/XO; , MMPO + X/XO + superoxide dismutase (SOD).

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<UP><SUB>2</SUB><SUP>−</SUP></UP>· is minimal. Addition of SOD caused a significant increase in DCFH oxidation, suggesting that this probe is susceptible to H2O2. Addition of baicalein to the SOD-containing system caused a marked decrease in the oxidation of DCFH, suggesting that baicalein can scavenge O<UP><SUB>2</SUB><SUP>−</SUP></UP>· and that it does not act as an SOD mimetic (Fig. 11).


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  		Fig. 11.   Effect of baicalein on DCF fluorescence in an X/XO + SOD system. When SOD was added to the X/XO system, DCF fluorescence increased. DCF fluorescence decreased after addition of 10 µM baicalein. , X/XO + SOD; , 10 µM baicalein + X/XO + SOD; open circle , X/XO.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generated by xanthine/xanthine oxidase. The corresponding EPR spectrum of MMPO-OOH is unique for O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. The EPR spectroscopy data indicate that baicalein can scavenge O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. In other in vitro studies, DCFH oxidation by H2O2 was assessed in a xanthine/xanthine oxidase system. DCFH oxidation was dramatically increased in the presence of SOD, indicating that DCFH oxidation by H2O2 was greater than that by O<UP><SUB>2</SUB><SUP>−</SUP></UP>·. In that system, baicalein attenuated the DCFH oxidation, suggesting that baicalein can scavenge O<UP><SUB>2</SUB><SUP>−</SUP></UP>· or H2O2 and that it does not act as an SOD mimetic.

Effect 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 O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generated from residual O2 present during ischemia, since the DHE oxidation can be attenuated by exogenous SOD and is increased by SOD inhibition (3, 38). The attenuation of DHE oxidation seen during ischemia by baicalein further confirms that baicalein can act as a potent O<UP><SUB>2</SUB><SUP>−</SUP></UP>· scavenger.

Effect 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 O<UP><SUB>2</SUB><SUP>−</SUP></UP>·, H2O2, and hydroxyl radicals, are responsible for reperfusion injury after myocardial ischemia (16, 42). Measures of free radical production have detected surges of ROS, particularly hydroxyl radical, during the first few seconds of reperfusion (42). In the present study, we observed a rapid (within 5 min) burst of DCF fluorescence at reperfusion that was reduced after treatment with baicalein. Also, cell death decreased and contractile activity returned in baicalein-treated cells. These observations indicate that baicalein confers significant protection in a model where oxidants are generated within cells during ischemia-reperfusion. Of the individual ROS scavengers evaluated in our system, baicalein has demonstrated the most effective protection against ischemia-reperfusion injury.

Effect 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 O<UP><SUB>2</SUB><SUP>−</SUP></UP>· generation induced by ETC blockade with antimycin A (34) can create a lethal oxidant stress that overwhelms antioxidant defenses. When isolated cardiomyocytes were exposed to antimycin A, we observed an immediate increase in DCF fluorescence and an increase in cell death after 2 h. However, in cells exposed to antimycin A and treated with baicalein, the oxidant stress was attenuated as indicated by a decline in DCF fluorescence, and cell death also decreased in a dose-dependent manner. These findings support the conclusion that baicalein is protective under conditions where intracellular ROS generation is increased.

Mechanism 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.


    ACKNOWLEDGEMENTS

This work was supported by National Institutes of Health Grants HL-03779, HL-35440, HL-32646, RR-12257, and AT-00381.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Afanas'ev, IB, Dorozhko AI, Brodskii V, Kostyuk A, and Potapovitch AI. Chelating and free radical scavenging mechanisms of inhibitory actions of rutin and quercetin in lipid peroxidation. Biochem Pharmacol 38: 1763-1769, 1989[Web of Science][Medline].

2.   Anholt, RHH, De Souza EB, Oster-Granite ML, and Snyder SH. Peripheral-type benzodiazepine receptors: autographic localization in whole-body sections of neonatal rats. J Pharmacol Exp Ther 233: 517-526, 1985[Abstract/Free Full Text].

3.   Becker, LB, Vanden Hoek TL, Shao ZH, Li CQ, and Schumacker PT. Generation of superoxide in cardiomyocytes during ischemia before reperfusion. Am J Physiol Heart Circ Physiol 277: H2240-H2246, 1999[Abstract/Free Full Text].

4.   Bond, JM, Herman B, and Lemasters JJ. Recovery of cultured rat neonatal myocytes from hypercontracture after chemical hypoxia. Res Commun Chem Pathol Pharmacol 71: 195-200, 1991[Web of Science][Medline].

5.  Bonnett EE, Brown RFC, Clark VM, Sutherland IO, and Todd A. Experiments toward the synthesis of corrins. II. The preparation and reactions of Delta 1-pyrroline 1-oxidases. J Chem Soc 2094-2102, 1959.

6.   Boveris, A, Fraga CG, Varsavsky AI, and Koch OR. Increased chemiluminescence and superoxide production in the liver of chronically ethanol-treated rats. Arch Biochem Biophys 227: 534-541, 1983[Web of Science][Medline].

7.   Das, DK. Cellular biochemical and molecular aspects of reperfusion injury. Ann NY Acad Sci 723: 116-127, 1994[Web of Science][Medline].

8.   Ferrari, R, Ceconi C, Curello S, Alfieri O, and Visioli O. Myocardial damage during ischemia and reperfusion. Eur Heart J 14: 25-30, 1993.

9.   Gao, D, Sakurai K, Chen J, and Ogiso T. Protection by baicalein against ascorbic acid-induced lipid peroxidation of rat liver microsomes. Res Commun Mol Pathol Pharmacol 90: 103-114, 1995[Web of Science][Medline].

10.   Gao, D, Tawa R, Masaki H, Okano Y, and Sakurai H. Protective effects of baicalein against reactive oxygen species. Chem Pharm Bull (Tokyo) 46: 1383-1387, 1998[Medline].

11.   Gao, Z, Huang K, Yang X, and Xu H. Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi. Biochim Biophys Acta 1472: 643-650, 1999[Medline].

12.   Goldhaber, JI, and Weiss JN. Oxygen free radicals and cardiac reperfusion abnormalities. Hypertension 20: 118-127, 1992[Abstract/Free Full Text].

13.   Halliwell, B. Antioxidant characterization, methodology and mechanism. Biochem Pharmacol 49: 1341-1348, 1995[Web of Science][Medline].

14.   Hamada, H, Hiramatsu M, Edamatsu R, and Mori A. Free radical scavenging action of baicalein. Arch Biochem Biophys 306: 261-266, 1993[Web of Science][Medline].

15.   Hanasaki, Y, Ogawa S, and Fufui S. The correlation between active oxygen scavenging and antioxidative effects of flavonoids. Free Radic Biol Med 16: 845-850, 1994[Web of Science][Medline].

16.   Hearse, DJ. Reperfusion-induced injury: a possible role for oxidant stress and its manipulation. Cardiovasc Drug Ther 5, Suppl2: 225-235, 1991.

17.   Huang, KC. The Pharmacology of Chinese Herbs (10th ed.). Boca Raton, FL: CRC, 1999.

18.   Kaneko, T, Kaii K, and Matsuo M. Protection of linoleic acid hydroperoxide-induced cytotoxicity by phenolic antioxidants. Free Radic Biol Med 16: 405-409, 1994[Web of Science][Medline].

19.   Kehrer, JP, and Smith CV. Free radicals in biology: sources, reactivities, and roles in the etiology of human diseases. In: Natural Antioxidants in Human Health and Disease, edited by Balz F.. San Diego, CA: Academic, 1994, p. 32.

20.   Kimuya, Y, Kubo M, Tani T, Arichi S, and Okuda H. Studies on Scutellariae radix. IV. Effects on lipid peroxidation in rat liver. Chem Pharm Bull (Tokyo) 29: 2610-2617, 1981[Medline].

21.   Kinnally, KW, Zorov DB, Antonenko YN, Snyde SS, McEner MW, and Tedeschi H. Mitochondrial benzodiazepine receptor linked to inner membrane ion channels by nanomolar actions of ligands. Proc Natl Acad Sci USA 90: 1374-1382, 1993[Abstract/Free Full Text].

22.   Knight, JA. Reactive oxygen species and the neurodegenerative disorders. Ann Clin Lab Sci 27: 11-25, 1997[Abstract].

23.   Liao, FF, Wang HH, Chen MC, Chen CC, and Chen CF. Benzodiazepine binding site-interactive flavones from Scutellaria baicalensis root. Planta Med 64: 571-572, 1998[Web of Science][Medline].

24.   Manach, C, Morand C, Demigne C, Texier O, Regerat F, and Remesy C. Bioavailability of rutin and quercetin in rats. FEBS Lett 409: 12-16, 1997[Web of Science][Medline].

25.   McCord, JM. Oxygen-derived free radicals in post-ischemic tissue injury. N Engl J Med 312: 159-163, 1985[Abstract].

26.   Miyahara, M, Ohtaka H, Katayama H, Tatsumi Y, Miyaichi Y, and Tomimori T. Structure-activity relationship of flavonoids in suppressing rat liver lipid peroxidation. Yakugaku Zasshi 113: 133-154, 1993[Web of Science][Medline].

27.   Opie, LH. Reperfusion injury and its pharmacologic modification. Circulation 80: 1049-1062, 1989[Abstract/Free Full Text].

28.   Rothe, G, and Valet G. Flow cytometric assays of oxidative burst activity in phagocytes. Methods Enzymol 233: 539-548, 1994[Web of Science][Medline].

29.   Saija, A, Scales M, Lanza M, Marzullo D, Bonina F, and Castelli F. Flavonoids as antioxidant agents: importance of their interactions with biomembranes. Free Radic Biol Med 19: 481-486, 1995[Web of Science][Medline].

30.   Sato, T, O'Rourke B, and Marban E. Modulation of mitochondrial ATP-dependent K+ channels by protein kinase C. Circ Res 83: 110-114, 1998[Abstract/Free Full Text].

31.   Shao, ZH, Li CQ, Vanden Hoek TL, Becker LB, Schumacker PT, Wu JA, Attele AS, and Yuan CS. Extract from Scutellaria baicalensis Georgi attenuates oxidant stress in cardiomyocytes. J Mol Cell Cardiol 31: 1885-1895, 1999[Web of Science][Medline].

32.   Shaw, S, Naegeli P, Etter JD, and Weidmann P. Role of intracellular signaling pathways in hydrogen peroxide-induced injury to rat glomerular mesangial cells. Clin Exp Pharmacol Physiol 22: 924-933, 1995[Web of Science][Medline].

33.   Tsai, P, Elas M, Parasca AD, Barth ED, Mailer C, Halpern HJ, and Rosen GM. Carboxy-5-methyl-1-pyrroline N-oxide: a spin trap for the hydroxyl radical. J Chem Soc Perkin Trans 2: 875-880, 2001.

34.   Turrens, JF, and Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J 191: 421-427, 1980[Web of Science][Medline].

35.   Vanden Hoek, TL, Shao ZH, Li CQ, Zak R, Schumacker PT, and Becker LB. Reperfusion injury in cardiac myocytes after simulated ischemia. Am J Physiol Heart Circ Physiol 270: H1334-H1341, 1996[Abstract/Free Full Text].

36.   Vanden Hoek, TL, Shao ZH, Li CQ, Schumacker PT, and Becker LB. Mitochondrial electron transport can become a significant source of oxidative injury in cardiomyocytes. J Mol Cell Cardiol 29: 2441-2450, 1997[Web of Science][Medline].

37.   Vanden Hoek, TL, Li CQ, Shao ZH, Schumacker PT, and Becker LB. Significant levels of oxidants are generated by isolated cardiomyocytes during ischemia prior to reperfusion. J Mol Cell Cardiol 29: 2571-2583, 1997[Web of Science][Medline].

38.   Vanden Hoek, TL, Becker LB, Shao ZH, Li CQ, and Schumacker PT. Reactive oxygen species released from mitochondria during brief hypoxia induce preconditioning in cardiomyocytes. J Biol Chem 273: 18092-18098, 1998[Abstract/Free Full Text].

39.   Vanden Hoek, TL, Becker LB, Shao ZH, Li CQ, and Schumacker PT. Preconditioning in cardiomyocytes protects by attenuating oxidant stress at reperfusion. Circ Res 86: 541-548, 2000[Abstract/Free Full Text].

40.   Wright, G, Reichenbecher V, Green T, Wright GL, and Wang S. Paraquat inhibits the processing of human manganese-dependent superoxide dismutase by SF-9 insect cell mitochondria. Exp Cell Res 234: 78-84, 1997[Web of Science][Medline].

41.   Yoshino, M, and Murakami K. Interaction of iron with polyphenolic compounds: application to antioxidant characterization. Anal Biochem 257: 40-44, 1998[Web of Science][Medline].

42.   Zweier, JL, Flaherty JL, and Weisfeldt ML. Direct measurement of free radical generation following reperfusion of ischemic myocardium. Proc Natl Acad Sci USA 84: 1404-1407, 1987[Abstract/Free Full Text].

Am J Physiol Heart Circ Physiol 282(3):H999-H1006
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