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Cardiovascular Research 2005 65(1):244-253; doi:10.1016/j.cardiores.2004.09.027
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Copyright © 2004, European Society of Cardiology

Baicalein protects rat cardiomyocytes from hypoxia/reoxygenation damage via a prooxidant mechanism

Anthony Y.H. Woo, Christopher H.K. Cheng1 and Mary M.Y. Waye1,*

Department of Biochemistry, The Croucher Laboratory for Human Genomics, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong SAR, China

* Corresponding author. Tel.: +852 2609 6874; fax: +852 2603 7732. Email address: mary-waye{at}cuhk.edu.hk

Received 12 July 2004; revised 14 September 2004; accepted 28 September 2004


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objectives: Baicalin and its aglycone baicalein are the major flavonoid components of the root of Scutellaria baicalensis. Recent studies have shown that they can attenuate oxidative stress in various in vitro models as they possess potent antioxidant activities. This study investigated alternative protective mechanisms of baicalein in a cardiomyocyte model.

Methods: Neonatal rat cardiomyocytes pretreated with the test compound were subjected to hypoxia/reoxygenation. The extent of cellular damage was accessed by the amount of released lactate dehydrogenase

Results: Pretreatment with baicalein up to 10 µM reduced lactate dehydrogenase release significantly (P<0.001), while pretreatment with baicalin up to 100 µM was ineffective. The cardioprotective effect of baicalein is not due to its antioxidant effect, because an adverse effect rather than a protective effect was observed when baicalein was present during hypoxia. Cotreatment with N-acetylcysteine attenuated the protective effect of baicalein and concomitantly increased intracellular reactive oxygen species level and the cytotoxic effect of baicalein, but N-acetylcysteine alone did not have such effects. In addition, cotreatment with catalase, but not superoxide dismutase or mannitol, reversed the cardioprotective effect of baicalein, suggesting the involvement of hydrogen peroxide in the cardioprotective mechanism. The NAD(P)H:quinone oxidoreductase inhibitors dicoumarol and chrysin also abolished the cardioprotective effect of baicalein. While pretreatment with baicalein did not increase antioxidant enzyme activities, it alleviated calcium accumulation in cardiomyocytes undergoing simulated ischemia.

Conclusion: These results highlight the important role of hydrogen peroxide produced during the auto-oxidation of baicalein in the cardioprotective effect of baicalein.

KEYWORDS baicalein; Prooxidant; Hydrogen peroxide; Cardioprotection; Hypoxia; Reoxygenation


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Baicalin and baicalein (Fig. 1) are flavonoids derived from the root of Scutellaria baicalensis Georgi. They exhibit superior free radical scavenging activities among the flavonoid components of the herb [1] and have been shown to attenuate oxidative stress in cardiomyocytes [2,3] and neuronal cells [4]. However, recent evidence indicates that the protective effects of flavonoids cannot be explained by their antioxidant properties alone [5,6]. Thus, more studies concerning the other protective mechanisms are necessary.


Figure 1
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  		Fig. 1 Structures of baicalein and baicalin.

 
In this study, hypoxia/reoxygenation (H/R) of the drug-pretreated cardiomyocytes was performed after washing away of the drug. Therefore, antioxidant effects of the drug were minimized and alternative protective mechanisms could be revealed. Paradoxically, our results demonstrated that the prooxidant rather than the antioxidant effects of baicalein governed the cardioprotective activity of baicalein in our model.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Animals
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85–23, revised 1996).

2.3. Chemicals and reagents
Baicalein, baicalin, chrysin, 2-deoxyglucose, dicoumarol (DIC), N,N'-dimethylthiourea (DMTU), N-acetylcysteine (NAC), mannitol, reduced glutathione (GSH), oxidized glutathione (GSSG), probenecid, propidium iodide (PI), DMEM base, M199, catalase (CAT), and superoxide dismutase (SOD) were purchased from Sigma-Aldrich, USA. Cell dissociation enzymes, DMEM with low glucose and other medium components were supplied by Gibco, USA.

2.2. Primary culture of cardiomyocytes
Cardiomyocytes were isolated from newborn Sprague–Dawley rats of both sexes as described [7]. After enriching the cardiomyocyte cultures for 2 days in a selective medium [8], the cultured cardiomyocytes were maintained in a low-serum medium (DMEM/M199 4:1 with 0.1% insulin–transferrin–selenium G supplement, 0.5% horse serum, and 0.3% penicillin–streptomycin) so as to inhibit the growth of nonmyocytes. Four-day cardiomyocyte cultures were used in subsequent experiments. The yield of cardiomyocytes was over 90% as determined by Mayer's haem alum-eosin Y staining method [9].

2.4. Hypoxia/reoxygenation treatment protocol
After drug pretreatment for 3 days, the cultured cardiomyocytes were washed with a solution (5 mM HEPES, 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1.5 mM CaCl2, pH 7.0). The cells were then incubated in a glucose-free DMEM base medium (pH 6.4) and then subjected to hypoxia to mimic the in vivo condition of myocardial ischemia. The cells were placed in a NAPCO2 INCUB 7101C-1 incubator (Precision Scientific, USA) at 37 °C. Nitrogen gas was then flushed into the incubator to bring the oxygen content down to 0.5% as monitored by an oxygen probe (Model no. KE-25, Japan Storage Battery, Japan). After 8 h of hypoxia, the cells were subjected to reoxygenation by changing the medium into a DMEM base medium with 5.5 mM glucose (pH 7.4) followed by incubation under normoxia for 1 h. The cells were then lysed by freeze–thawing in distilled water.

2.5. LDH assay
Lactate dehydrogenase (LDH) activities in the hypoxic and the reoxygenation media and in the cell lysates were determined as described [10] using a commercial enzyme prepared from rabbit muscle as standard (Roche Diagnostics #127884). The sum total of LDH activities in the hypoxic and reoxygenation media and the cell lysate obtained from the same well would give the total LDH activity. The percentage of LDH released during hypoxia or reoxygenation from the LDH activity in each of these media was standardized against the total LDH activity. The percentages of LDH released during hypoxia and reoxygenation would be combined to give the percentage of LDH released during H/R. By expressing the data in percentage term, the variation due to the differences in cell number in each well can be normalized.

2.6. Determination of cell viability
Cell viability was assessed by the exclusion of trypan blue [10] and PI [11] by the cells.

2.7. Assays for SOD, CAT, GPX, and GSH
Cardiomyocytes were washed twice with phosphate-buffered saline containing 0.05 mM EDTA and were sonicated at 4 °C. After centrifugation (800 x g, 5 min), the supernatants were immediately assayed for antioxidant enzyme activities. SOD activity was determined using a bovine erythrocyte SOD (Sigma S2515) as standard [12]. CAT activity was determined using a CAT from bovine liver (Sigma C9322) as standard [13]. Glutathione peroxidase (GPX) activity was assayed using cumene hydroperoxide as substrate [14]. Cellular GSSG and GSH were determined according to Anderson [15], calculated on the basis of a GSSG or GSH calibration curve. Protein content in the cell lysate was determined using bovine serum albumin (BSA) as standard [16].

2.8. Measurement of intracellular ROS
Intracellular reactive oxygen species (ROS) was assessed using the cell-permeable probe 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (cDCFH-DA; Fluka, Germany). Dye loading was performed by incubating the cardiomyocytes with 10 µM of cDCFH-DA at 37 °C for 30 min. The dissociated cells were analyzed by flow cytometry with FACSort cell sorter (Becton Dickinson). cDCF fluorescence was determined as the emission intensity at 530 nm.

2.9. Metabolic inhibition of cardiomyocytes and intracellular calcium level determination
The increase in intracellular calcium level or [Ca2+]i of cardiomyocytes undergoing metabolic inhibition (MI) was determined by the fluorescence of the calcium-sensitive dye fluo-4-AM (Molecular Probes) as described [17]. MI of the fluo-4-stained cells was carried out in a buffer (20 mM Mops, 125 mM NaCl, 4.4 mM KCl, 1.2 mM CaCl2, 2 mM NaCN, 20 mM 2-deoxyglucose, 1 mM probenecid, 1% BSA, pH 6.6) at 37 °C. PI was added to stain for nonviable cells before data acquisition. During flow cytometry, about 10,000 cells were analyzed in each sample. Data were collected for emission at 530 nm (FL1 channel) for fluo-4 and 670 nm (FL3 channel) for PI and were plotted simultaneously. Only those cells with low fluorescent intensity at 670 nm (PI-negative) were included in the comparative analysis of [Ca2+]i.

2.10. Statistical analyses
Student's t-test was used to compare the data between two groups, and ANOVA followed by t-test with Bonferroni's transformation for more than two groups of data. A P value of <0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Effects of pretreatment with baicalein or baicalin on H/R damage in cardiomyocytes
LDH leakage from cells is widely used as a marker of cellular injury and necrotic cell death [18]. Cardiomyocytes subjected to H/R insults had an increase in LDH leakage as compared with untreated cells (20% of the total LDH, data not shown). Pretreating the cardiomyocytes with baicalein reduced LDH leakage during H/R in a dose-dependent manner up to 10 µM (Fig. 2A, dark bars). No toxic effect of baicalein was observed at all the concentrations tested after the 3-day treatment as suggested by the insignificant differences in the total LDH activities of the baicalein-treated cardiomyocytes as compared with the control (Fig. 2A, white bars). In contrast, pretreatment with 10 µM baicalin did not produce a protective effect, and higher concentrations (30 and 100 µM) of baicalin increased H/R-induced LDH release (Fig. 2B, dark bars). This might be due to the toxic effect of baicalin at these concentrations, as suggested by the decrease in the total LDH activities of the baicalin-treated cardiomyocytes as compared with the control (Fig. 2B, white bars). Pretreatment with baicalein also increased the survival rate of cardiomyocytes undergoing H/R challenge, as shown in the trypan blue exclusion and PI exclusion results (Fig. 3A,B).Effect of baicalein during hypoxia on H/R damage in cardiomyocytes


Figure 2
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  		Fig. 2 Effects of baicalein and baicalin on cardiomyocytes subjected to H/R. Cardiomyocytes were pretreated for 3 days with (A) baicalein or (B) baicalin. After washing, the cells were subjected to H/R as described in Materials and methods. Total LDH activity (white bars) and percentage of LDH released during H/R (dark bars) were determined and expressed as the mean ± S.D. (n=6). **P<0.01, ***P<0.001 as compared with the drug-free control.

 

Figure 3
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  		Fig. 3 Pretreatment with baicalein increases the viability of cardiomyocytes undergoing H/R treatment. Control vehicle- or baicalein-pretreated cardiomyocytes were subjected to H/R before viability assessment by the exclusion of vital dyes: (A) trypan blue or (B) PI. Viability study was also performed on the vehicle-pretreated normoxic control (N) to demonstrate decrease in cell viability upon H/R treatment in C. Results are expressed as mean ± S.D. (n=6). *P<0.05, **P<0.01 as compared with C.

 
The presence of baicalein (10–30 µM) during hypoxic treatment augmented LDH release from the cardiomyocytes in a dose-dependent manner (Fig. 4). This implies that the cardioprotective effect observed with the pretreatment of baicalein (Fig. 2A) is not attributed to the carrying over of its antioxidant effects to reoxygenation.


Figure 4
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  		Fig. 4 The presence of baicalein during hypoxia increases H/R damage in cardiomyocytes. Cardiomyocytes were incubated with baicalein during hypoxia. After removing the hypoxic medium, they were subjected to reoxygenation in a drug-free medium. Results are expressed as the mean ± S.D. (n=6). ***P<0.001 as compared with the drug-free control.

 
3.3. NAC reverses the cardioprotective effect of baicalein and increases the cytotoxic effect of baicalein
Cotreatment with NAC reverted the protective effect of baicalein to an adverse effect (Fig. 5A). Total LDH activity of the baicalein-treated cardiomyocytes was reduced by 60% in the presence of NAC (Fig. 5B, dark bars), while control cells treated with NAC alone did not reveal such an effect (Fig. 5B, white bars).


Figure 5
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  		Fig. 5 NAC reverses the cardioprotective effect of baicalein and increases the cytotoxic effect of baicalein. Cardiomyocytes were pretreated with control vehicle or baicalein (10 µM) in the presence or absence of 1 mM NAC as indicated. (A) Percentage of LDH released during H/R and (B) total LDH activity were determined and presented. Results are expressed as the mean ± S.D. (n=6). **P<0.01, ***P<0.001 as compared with control in the same treatment group.

 
3.4. Cotreatment with baicalein and NAC increases intracellular ROS level in cardiomyocytes
To see whether increased cytotoxicity of baicalein in the presence of NAC was caused by an increase in intracellular ROS, the ROS contents of cardiomyocytes pretreated with baicalein alone and baicalein with NAC were compared. After 4 h of incubation, cDCF fluorescence of cells treated with 10 and 30 µM of baicalein did not show much difference as compared with that of the vehicle control (Fig. 6A). In contrast, baicalein increased cDCF fluorescence in a dose-dependent manner in the presence of NAC (Fig. 6B), implying that intracellular ROS level had increased.


Figure 6
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  		Fig. 6 Fluorescent distribution of cDCFH-DA-loaded cardiomyocytes pretreated with baicalein in the absence or presence of NAC. Cardiomyocytes were treated with 0 µM (grey area), 10 µM (solid line), and 30 µM (dotted line) of baicalein in the absence (A) or presence (B) of 1 mM NAC before determination of intracellular ROS.

 
3.5. Effects of antioxidants on the cardioprotective effect of baicalein
The increase in intracellular ROS level by cotreatment with baicalein and NAC may imply that baicalein could be autoxidized with concomitant generation of ROS, and in addition, NAC could amplify this effect. Hence, it is possible that the cardioprotective effect of baicalein might be mediated through ROS. Therefore, different antioxidants were employed to investigate the ROS dependence of the cardioprotective effect of baicalein. The results in Fig. 7A show that CAT reversed the protective effect of baicalein on cardiomyocytes against H/R, while SOD alone was ineffective. When both SOD and CAT were present, the cardioprotective effect of baicalein was also reversed to an intermediate level (Fig. 7A). Cotreatment with the SOD mimetic manganese(III) tetrakis(4-benzoic acid)porphyrin chloride (MnTBAP; A. G. Scientific, USA), which also contains a CAT activity [19], similarly, reversed the protective effect of baicalein (Fig. 7B). However, mannitol, which could act as an extracellular hydroxyl radical scavenger at the concentration used [18], had no effect on baicalein-induced protection against H/R (Fig. 7B). DMTU, a cell-permeable ROS scavenger with a higher specificity towards hydroxyl radical and a much lower one towards H2O2 at the concentration used [20], mildly reduced the cardioprotective effect of baicalein (Fig. 7B). No observable change in the total cellular LDH activity, as well as the cell viability, appeared in any of the cotreatment combinations (data not shown). These data suggest that the cardioprotective effect of baicalein is H2O2-dependent.


Figure 7
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  		Fig. 7 Effects of antioxidants on the cardioprotective effect of baicalein. Cardiomyocytes were pretreated for 3 days with control vehicle or baicalein (10 µM) in the presence or absence of (A) 500 U/ml SOD, 500 U/ml CAT, SOD+CAT (500 U/ml each); (B) 25 µM MnTBAP, 20 mM mannitol, 5 mM DMTU as indicated. Results are expressed as mean ± S.D. (n=6). **P<0.01, ***P<0.001 as compared with control in the same treatment group.

 
3.6. Dicoumarol and Chrysin reverse the cardioprotective effect of baicalein
H2O2 is generated during the auto-oxidation and redox cycling of quinones [21]. Flavonoids, particularly those with a catechol or a pyrogallol structure, are prone to oxidation by molecular oxygen with concomitant generation of superoxide radicals (O2n.–), H2O2, semiquinone radicals and quinones [22,23]. The quinone thus formed could be reduced intracellularly by the two-electron quinone reductase activities of NAD(P)H:quinone oxidoreductase (NQO [24]), therefore completing a redox cycle. NQO inhibitors, such as 3,3'-methylene-bis(4-hydroxycoumarin) (DIC) and chrysin (5,7-dihydroxyflavone), have been used to study the involvement of NQO in cell survival [25]. The results (Fig. 8A,B) that DIC and chrysin reversed the cardioprotective effect of baicalein in the absence of any observable toxic effect (data not shown) imply that NQO may participate in the cardioprotective mechanism of baicalein.


Figure 8
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  		Fig. 8 Dicoumarol and chrysin reverse the cardioprotective effect of baicalein. The effect of (A) DIC (30 µM for 30 min before drug administration) and (B) chrysin (2 µM as a cotreatment) on the cardioprotective effect of baicalein were studied. Results are expressed as mean ± S.D. (n=6). ***P<0.001 as compared with the control in the same treatment group.

 
3.7. Antioxidant status of cardiomyocytes treated with baicalein
A number of reports have shown that a low level of H2O2 can trigger cellular adaptive response against oxidative stress by inducing the antioxidant defense mechanisms [26,27]. To investigate further the role of H2O2 in baicalein-induced cardioprotection, the effects of baicalein on antioxidant enzyme activities and GSH level in cardiomyoyctes were studied. The results in Table 1 show that the SOD and GPX activities were not significantly affected by treatment with baicalein, while the CAT activity and the GSH level of the treated cardiomyocytes were slightly reduced by 19% and 17%, respectively. These data suggest that the cardioprotective effect of baicalein is not due to the induction of these antioxidant enzymes.


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  		Table 1 Antioxidant status in cardiomyocytes treated with baicalein

 
3.8. Effects of pretreatment with baicalein on [Ca2+]i of cardiomyocytes undergoing MI
In severely ischemic myocardium, subsequent reperfusion will lead to calcium overload due to the amplified sequestration of the elevated Ca2+ into the endoplasmic reticulum by Ca2+-ATPase as ATP synthesis begins to resume [28]. Therefore, reduction of [Ca2+]i during ischemia can greatly alleviate calcium overload during reperfusion. A commonly used MI-simulated ischemia model [29] was employed to investigate whether the cardioprotective effect of baicalein is related to a suppression of intracellular calcium accumulation. Fig. 9C shows that an increase in [Ca2+]i could be observed after 60 min of MI. During 20–60 min of MI, the increase in [Ca2+]i of the baicalein-pretreated cardiomyocytes was lower than that of the control cells (Fig. 9D) with statistical significance of P<0.05 at 20 min, P<0.01 at 40 min, and P<0.001 at 60 min. These results suggest that pretreatment with baicalein could decrease the elevation of [Ca2+]i in cardiomyocytes undergoing simulated ischemia.


Figure 9
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  		Fig. 9 Pretreatment with baicalein decreases the elevation of [Ca2+]i in cardiomyocytes undergoing MI. Cardiomyocytes were pretreated with control vehicle or 10 µM baicalein before dye loading and fluorescence measurement. (A) Dot plot of FL3 against FL1 fluorescence of cardiomyocytes without MI treatment. (B) Dot plot of FL3 against FL1 fluorescence of cardiomyocytes after 60 min of MI treatment. (C) Histogram plot of FL1 fluorescence of the selected cells in region R1: (i) without MI treatment (grey area); (ii) after 60 min of MI treatment (solid line indicated by an arrow). (D) Effects of pretreatment with baicalein on fluo-4 fluorescence after different duration of MI treatment. Results are expressed as mean ± S.D. (n=6). *P<0.05, **P<0.01, ***P<0.001 as compared with the corresponding control.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Both baicalein and baicalin can form stable semiquinone radicals, which is the basis for their potent antioxidant activities [1]. Baicalein semiquinone radical can be further oxidized into an ortho-quinone - 6,7-dehydrobaicalein [30]. It has been suggested that the auto-oxidation of flavonoids produces semiquinone radicals (.O–Ph–O–) and O2.– [22,23]. Although, the reaction of the deprotonated flavonoid (HO–Ph–O–) with molecular oxygen is slow (Eq. (1)), the O2.– generated can propagate the oxidation of the flavonoids (Eqs. (2) and (3)):



Formula 1



Formula 2



Formula 3

A recent study has reported the H2O2-generating ability of flavonoids [31]. Myricetin, being the most potent among the flavonoids, including baicalein, generated H2O2 at a maximal rate 4 h after incubation. Thus, the auto-oxidation of these flavonoids can take place in aqueous solution rather quickly by the alternate cycle of reactions of Eqs. (2) and (3) with the net production of H2O2. Such reactions could decrease the antioxidant effects of flavonoids or produce prooxidant effects.

In our culture system, cytotoxic effect of baicalein was not observed at 10 µM in the absence of NAC (Fig. 2A, white bars). Higher concentrations of baicalein could be cytotoxic to the cells (data not shown), probably due to the generation of ROS during auto-oxidation. We hypothesized that 6,7-dehydrobaicalein can be reduced back to baicalein or baicalein semiquinone radicals by NAC, and the resultant perpetuation of ROS production amplified the cytotoxic effect of baicalein (Fig. 5B, dark bars). The results that cotreatment with baicalein and NAC increased intracellular ROS of the cardiomyocytes (Fig. 6B) confirm this hypothesis. Consistent with our results, Thibodeau et al. [32] have reported that oxidative DNA damage induced by catechol–estrogen could be enhanced by NAC. The authors have suggested that NAC could regenerate the semiquinone form of catechol–estrogen from its quinone form. The redox cycling could increase the production of ROS, thereby increasing DNA damage.

These interesting findings further led us to speculate that the prooxidant effects of baicalein might be related to its cardioprotective effect. The fact that scavengers of H2O2, such as CAT, reversed the protective effect of baicalein (Fig. 7A,B) suggests that the cardioprotective effect of baicalein is mediated through H2O2. The H2O2 was most likely produced during the auto-oxidation of baicalein. The necessary involvement of H2O2 in the cardioprotective mechanism probably explains the ineffectiveness of baicalin (Fig. 2B, dark bars), because baicalin is resistant to oxidation. The 7-glucuronic acid apparently prevents further oxidation of the baicalin semiquinone radical into a quinone.

ROS generated during preconditioning cycles of ischemia/reperfusion have been implicated in mediating cardioprotection [33]. Using a chick cardiomyocyte model, Vanden Hoek et al. [34] have proposed that during hypoxic preconditioning, O2.– generated from the mitochondrial electron transport chain may be degraded by the cytosolic Cu,Zn–SOD to H2O2, which then activates subsequent mediators leading to preconditioning protection. In addition, they have also shown that exogenous H2O2 under normoxia produced a similar cardioprotective effect. Similar studies have shown that H2O2 concentrations ranging from 5 to 25 µM were effective [34–36]. In this investigation, 10 µM of baicalein was routinely used to induce cardioprotection, a concentration estimated to produce a similar level of H2O2 as reported in these cardiomyocyte models.

Up to now, there is no consensus about the site of H2O2 production, as well as the target of H2O2 in cardioprotection involving H2O2. Studies with the naphthoquinone menadione have revealed that H2O2 may be produced extracellularly [37,38] or within the mitochondria [37,39]. In the former case, menadione and its NQO-reduced counterpart have been suggested to shuttle across the plasma membrane in a redox cycle of H2O2 synthesis [37,38]. Concerning the reactivity of baicalein, we believe that the auto-oxidation of baicalein mainly take place extracellularly in our cell culture model as it happens in cell-free systems [31]. The fact that CAT cotreatment reversed the cardioprotective effect of baicalein (Fig. 7A) may implicate the importance of extracellular H2O2, although cellular uptake of exogenous CAT is also possible. In agreement with this hypothesis, some studies have shown that exogenous O2n.– or H2O2 could produce preconditioning-like protection in intact hearts or cardiomyocytes [35,40]. Others, however, have suggested that ROS generated at the mitochondria is sufficient to explain hypoxic preconditioning [34,36].

From our results (Fig. 8A,B), we also hypothesize that the auto-oxidation product 6,7-dehydrobaicalein will be reduced back to baicalein intracellularly by NQO activities. The resultant redox cycling may contribute to the amplified generation of H2O2 to attain an effective concentration. Metodiewa et al. [41] have shown that the cytotoxic effect of quercetin on CHO cells could be reduced by DIC. Likewise, the enhancement of the prooxidant effect of quercetin by NQO was implicated. However, baicalein, which is structurally similar to chrysin, has been reported to inhibit the activity of NQO for menadione [42]. While 6,7-dehydrobaicalein can still be a substrate for NQO, the results of the NQO inhibition study should be interpreted in caution due to the possible interaction of DIC and chrysin with other cellular processes.

The ROS signaling in ischemic preconditioning is not clear. The relative order of mitochondrial KATP channels, ROS and kinases, is still controversial [43]. Recent studies have also implicated the role of the oxidant-sensitive transcription factor NF-{kappa}B in preconditioning [44]. Although our data suggest that extracellular H2O2 may trigger the preconditioning-like protective effect of baicalein, unlike other studies [35,45], we could not demonstrate an increase in intracellular ROS (Fig. 6A). We have attempted to extend the pretreatment period with baicalein and found no change in the intracellular ROS of the treated cells at all the time points examined (data not shown). It seems that the small amount of H2O2 generated could not produce a cDCFH-detectable change. The effect of H2O2 may be restricted by CAT and GPX inside the cell. Nevertheless, the fact that treatment with baicalein slightly reduced cellular CAT and GSH (Table 1) indicates that the prooxidant effect of baicalein is still significant. This prooxidant effect may add to the adverse effect of the ROS produced during hypoxia [36] and may thus explain the increased LDH released in the presence of baicalein (Fig. 4). It also hints the possibility of the existence of intracellular targets of H2O2 generated from baicalein. Further investigations into the cardioprotective mechanism of baicalein are needed. Recently, Chou et al. [46] have reported that pretreatment with baicalein inhibited transforming growth factor-β1-induced apoptosis in hepatoma Hep3B cells via an increase in intracellular H2O2 formation and NF-{kappa}B activation, and that the baicalein-triggered activation of NF-{kappa}B is abolished by CAT. Their results, together with that of ours, may hint a common pathway, for instance, H2O2 formation and NF-{kappa}B activation, in the cytoprotective mechanism of baicalein.

Baicalein has been reported to attenuate the increase in [Ca2+]i induced by different pharmacological agents [47–49]. Pretreatment with baicalein has been shown to suppress the angiotensin II-stimulated increases in [Ca2+]i in vascular smooth muscle cells [50]. Our data in Fig. 9D demonstrate that pretreatment with baicalein can also produce similar effect on [Ca2+]i in cardiomyocytes. This indicates that alleviating calcium accumulation during myocardial ischemia and reperfusion may be one of the mechanisms of the cardioprotective effect of baicalein. How baicalein produces this effect remains to be elucidated.

In this report, we have demonstrate that pretreatment with baicalein can protect cardiomyoyctes against H/R, while an adverse effect was observed when baicalein was administered during hypoxia alone (Fig. 4). In contrast, in a chick cardiomyocyte perfusion system, treatment with baicalein during a transient hypoxic period alone, during reperfusion alone, or across the H/R period has been shown to attenuate oxidative stress [2,3]. The apparent discrepancy might be due to the difference in treatment regimens and the longer period of hypoxic treatment in this study, which might allow the prooxidant effects of baicalein to be revealed. The implication is that the time and the duration of treatment would be important in determining a beneficial or an adverse outcome if baicalein is to be used clinically in patients with ischemic conditions.

In conclusion, our data suggest that the protective effect of baicalein on cultured cardiomyocytes against H/R is mediated through an H2O2-dependent mechanism, and that the H2O2 is produced during the auto-oxidation of baicalein. The cardioprotective effect may involve the alleviation of calcium overload. This study demonstrates for the first time the importance of the prooxidant effect of flavonoids in cardioprotection. Further studies in this direction are highly warranted.


   Acknowledgements
 
We are grateful to The Chinese University of Hong Kong for the award of direct grants and for the provision of a graduate studentship to AYHW. We would like to thank Miss Jenny Cheung for technical assistance and Miss Cecy Kou for valuable advice.


   Notes
 
1 These authors contributed equally to the work. Back

Time for primary review 28 days


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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