英-GSPE 心肌细胞 氧化损伤

Pharmacological Research 47 (2003) 463–469 Grape seed proanthocyanidin extract attenuates oxidant injury in cardiomyocytes Zuo-Hui Shao a,b, Lance B. Becker a,b, Terry L. Vanden Hoek a,b, Paul T. Schumacker b, Chang-Qing Li a,b, Danhong Zhao a,b, Kim Wojcik b, Travis Anderson a,b, Yimin Qin b, Lucy Dey c,d, Chun-Su Yuan b,c,d,∗ a Department of Medicine, Section of Emergency Medicine, University of Chicago, 5841 S. Maryland Avenue, MC 5068, Chicago, IL 60637, USA b Emergency Resuscitation Center, University of Chicago, 5841 S. Maryland Avenue, MC 5068, Chicago, IL 60637, USA c Department of Anesthesia & Critical Care, The University of Chicago, 5841 S. Maryland Avenue, MC 4028 Chicago, IL 60637, USA d Tang Center for Herbal Medicine Research, University of Chicago, 5841 S. Maryland Avenue, MC 4028, Chicago, IL 60637, USA Accepted 30 December 2002 Abstract This study sought to test whether grape seed proanthocyanidin extract (GSPE) attenuates exogenous and endogenous oxidant stress induced in chick cardiomyocytes and whether this cytoprotection is mediated by PKC activation, mito KATP channel opening, NO produc- tion, oxidant scavenging, or iron chelating effects. Cells were exposed to hydrogen peroxide (H2O2) (exogenous oxidant stress, 0.5 mM) or antimycin A (endogenous oxidant stress, 100�M) for 2 h following pretreatment with GSPE at various concentrations for 2 h. Cells were also pretreated with GSPE or with inhibitors of PKC (chelerytherine), mito KATP channel (5-hydroxydecanoate), nitric oxide synthase (nitro-l-arginine methyl ester) for 2 h. Oxidant stress was measured by 2′,7′-dichlorofluorescin diacetate and cell viability was assessed using propidium iodide. Free radical scavenging and iron chelating ability was tested in vitro. GSPE dose-dependently attenuated oxidant formation and significantly improved cell survival and contractile function. However, inhibitors of PKC, mito KATP channel or NO synthase failed to abolish the protective action of GSPE during H2O2 or antimycin A exposure. In vitro studies suggested that GSPE scavenges H2O2, hydroxyl radical and superoxide, and may chelate iron. These results indicate that GSPE confers cardioprotection against exogenous H2O2- or antimycin A-induced oxidant injury. Its effect does not require PKC, mito KATP channel, or NO synthase, presumably because it acts by reactive oxygen species scavenging and iron chelating directly. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Grape seed proanthocyanidin extract; Hydrogen peroxide; Antimycin A; Cardiomyocytes; 2′,7′-Dichlorofluorescin diacetate; Dihydroethidium; Propidium iodide 1. Introduction Grape seed proanthocyanidins are a group of polyphenolic bioflavonoids which are known to possess broad pharma- cological activity and therapeutic potential [1]. Proantho- cyanidins, the major polyphenols found in red wine and grape seeds, have been reported to protect against oxidant injury during ischemia/reperfusion in rat heart [2–4]. Reactive oxygen species (ROS) have been implicated in the pathogenesis of stress-induced injury, including ischemia/reperfusion injury [5,6]. Our previous studies ∗ Corresponding author at Department of Anesthesia & Critical Care, The University of Chicago, 5841 S. Maryland Avenue, MC 4028, Chicago, IL 60637, USA. Tel.: +1-773-702-1916; fax: +1-773-834-0601. E-mail address: cyuan@midway.uchicago.edu (C.-S. Yuan). demonstrated that ROS generated intracellularly contribute to contractile dysfunction and cell death during simulated ischemia/reperfusion in a perfused cardiomyocyte model [7–9]. We also observed that some herbal extracts or ac- tive components of herbs exhibit antioxidant effects in that system [10–12], suggesting that the cultured, isolated and perfused cardiomyocyte model is appropriate for test- ing whether grape seed proanthocyanidin extract (GSPE) confers cardioprotection against oxidant injury. Hydrogen peroxide (H2O2) may be directly cytotoxic, or it may be converted to hydroxyl radical, which in turn could react with macromolecules, including DNA, proteins and lipids, potentially leading to cell death [13]. It has been reported that GSPE protected against H2O2-induced oxidative damage in cultured macrophages and neuroactive PC-12 cells [14,15] and in rat primary glial cultures [16]. 1043-6618/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S1043-6618(03)00041-0 464 Z.-H. Shao et al. / Pharmacological Research 47 (2003) 463–469 However, no study has investigated its ability to protect against endogenous or exogenous oxidant stress in car- diomyocytes. Myocardial mitochondria are an important source of ox- idant stress [8]. The mitochondrial electron transport chain (ETC) is capable of generating ROS if the ETC becomes highly reduced under conditions of reductive stress [17]. Mi- tochondrial ETC inhibition by the complex III inhibitor, an- timycin A, can cause similar oxidant damage by prolonging the lifetime of ubisemiquinone, the putative source of super- oxide generation [8]. The protective effect and mechanism of GSPE against antimycin A-induced endogenous oxidant stress therefore provide a measure of its ability to attenuate mitochondria-derived oxidant stress. This study aimed to test whether GSPE offers cardiopro- tection against exogenous H2O2-induced oxidant stress and antimycin A-induced endogenous oxidant stress in cultured cardiomyocytes. We also aimed to test whether activation of protein kinase C (PKC), opening of mitochondrial KATP (mito KATP) channel and production of nitric oxide (NO), and scavenging activity are involved in its protective effect. Our results show that GSPE possesses potent antioxidant activity and confers cardioprotective effects against both ex- ogenous H2O2- and antimycin A-induced oxidant stress. GSPE also scavenges H2O2, hydroxyl radical and superox- ide, and may chelate iron in vitro. The cardioprotective ef- fects may therefore be attributed to its scavenging ROS and chelating iron effects. 2. Materials and methods 2.1. GSPE and chemicals GSPE (batch no. AV 9090940) was kindly provided by InterHealth Nutrapeuticals Inc. (Concord, CA). Hydrogen peroxide (H2O2), antimycin A, chelerytherine (CHE) and nitro-l-arginine methyl ester (l-NAME) were obtained from Sigma Chemical Co. (St. Louis, MO). 5-Hydroxydecanoate (5-HD) was purchased from Biomol (Plymouth, PA). 2.2. Chick primary cardiomyocyte culture Chick embryonic ventricular myocytes were prepared as previously described [7]. Briefly, ventricles from 10-day-old chick embryonic hearts were separated, minced, and enzy- matically digested with 0.025% trypsin (Invitrogen, Grand Island, NY) for 4–5 cycles. Cell suspensions were collected and centrifuged, cell pellets were resuspended, and cells (0.7× 106) were plated onto glass coverslips and incubated under 5% CO2 at 37 ◦C. Fibroblast contamination was re- duced by preplating, and the purity of cardiomyocytes was confirmed by antimyosin heavy chain monoclonal antibodies [7]. All experiments were performed on 3–5-day culture, by which time synchronously contracting cells were observed. Viability exceeded 95%. 2.3. Video/fluorescent microscopy Cells were imaged with an Olympus IMT-2 inverted phase/epifluorescent microscope. Contractions were mon- itored by phase contrast Hoffman modulation optics and a CCD camera. Fluorescence intensity was measured us- ing a cooled slow-scanning PC-controlled camera (Hama- matsu, Hamamatsu City, Japan) coupled with Image pro Plus software for the quantification of fluorescent changes. 2.4. Measurement of intracellular ROS Intracellular oxidant stress was monitored by intracellular probe 2′,7′-dichlorofluorescin diacetate (DCFH/DA, 5�M, Molecular Probes, Eugene, OR) as previously described [7–9]. Upon entry, this dye is cleaved by cellular esterases to nonfluorescent 2′,7′-dichlorofluorescin (DCFH) and oxi- dized by ROS to a fluorescent product dichlorofluorescein (DCF). DCF fluorescence was measured using an excitation wavelength of 480 nm and a 520 nm band pass filter, and was expressed in arbitrary units. 2.5. Viability assay and contraction analysis Cell viability was assessed by the fluorochrome propidium iodide (PI, 5�M, Molecular probes, Eugene, OR). It is an exclusion fluorescent dye that binds to chromatin upon loss of cell membrane integrity. This method has been reported to evaluate the transition from reversible to irreversible cell injury in cultured cardiomyocytes [7,18]. At the end of each experiment, all cells in a field of approximately 500 cells were permeablized with digitonin (300�M). Percent cell death was expressed as the PI fluorescence relative to the maximal value seen after 1 h of digitonin exposure (100%). Cell contractions were assessed as previously reported [9]. A return of contraction was indicated as seen throughout the field of cells. 2.6. GSPE oxidant scavenging activity in vitro To test whether GSPE has the ability to scavenge H2O2 and/or hydroxyl radicals, we studied the effect of GSPE in increasing doses (10, 50, or 100�g/ml) on DCFH/DA (10�M) oxidation (measured with excitation 488 nm/emission 529 nm by fluorescent spectrophotome- ter) in the presence of H2O2 (1�M)/FeSO4 (50�M) in cuvettes. To further test whether GSPE has the ability to interfere with hydroxyl radical formation (from the interac- tion of H2O2 and iron) via metal chelating properties, the effect of GSPE (10�g/ml) in this cuvette system was com- pared to the metal chelators 1′,10′-phenanthroline (PHEN, 10�M), deferoxamine (DEF, 10�M) in the DCFH/DA and H2O2/FeSO4 system. H2O2 by itself was also tested in serial doses, but relatively high doses (up to 1 mM) were needed to achieve the same level of oxidation as with combinations Z.-H. Shao et al. / Pharmacological Research 47 (2003) 463–469 465 of low doses of H2O2 and FeSO4 (results not shown). Since these levels of H2O2 kill the cell almost immediately, we did not do further tests of GSPE using such nonphysiologic high doses of H2O2. To test whether GSPE can scavenge superoxide (O2•−), we measured the effect of increasing doses of GSPE on the oxidation of dihydroethidium (DHE) to fluorescent ethidium (Eth) (measured with excitation 475 nm/emission 610 nm) during the addition of xanthine (X) to a xanthine oxi- dase (XO) solution. Cuvettes contained DHE (100�M), X (0.4 mM), XO (0.02 U/ml) and DHE (100�M), X (0.4 mM), XO (0.02 U/ml) with increasing doses of GSPE (10, 50, or 100�g/ml). 2.7. Statistics An individual experiment (n) was observed in a field of approximately 500 cells on a coverslip. Duplicate ex- periments were performed on separate coverslips. Results are reported as mean + S.E.M. and two-tailed unpaired t-tests were performed, with P < 0.05 considered signi- ficant. 3. Results 3.1. Effect of GSPE on DCFH oxidation and cell viability in H2O2-exposed cardiomyocytes Chick cardiomyocytes were loaded with DCFH/DA (10�M) and exposed to H2O2 (0.5 mM) for 2 h or pre- treated with GSPE (10, 50, or 100�g/ml) for 2 h prior to H2O2 exposure. Oxidant stress was measured by oxida- tion of DCFH. As shown in Fig. 1(A), cells exposed to H2O2 (0.5 mM) for 2 h showed a significant increase in DCF fluorescence. In cells pretreated with GSPE, DCF fluorescence was attenuated in dose-dependent manner. No evidence of increase in DCF fluorescence was observed in cells incubated with GSPE alone. These results suggest that GSPE attenuates oxidant stress induced by exogenous H2O2. Cells were incubated with PI (5�M) and exposed to H2O2 (0.5 mM) for 2 h, or pretreated with GSPE for 2 h prior to H2O2 exposure. As seen in Fig. 1(B), cell death (PI uptake) significantly increased after 2 h of H2O2 exposure. In cells pretreated with GSPE (10, 50, or 100�g/ml), the cell death dose-dependently decreased from 60.6 ± 5.3% in H2O2 exposed cells to 39.1 ± 6.7% (P < 0.01, n = 10), 27.5±5.3% (P < 0.001, n = 10), 25.0±4.5% (P < 0.001, n = 10), respectively. No significant increase in PI fluores- cence was seen in cells incubated with GSPE alone. Cell contraction recovered in GSPE (10, 50, or 100�g/ml) pre- treated cells (3/10, 5/10, 5/10) but was not observed in cells exposed to H2O2 alone (0/10). These results indicate that GSPE improves cell survival and enhances the return of contraction. Fig. 1. (A) Effect of GSPE on DCFH oxidation during H2O2 exposure. Cardiomyocytes were loaded with DCFH/DA (10�M) and exposed to H2O2 for 2 h or pretreated with GSPE (10, 50, or 100�g/ml) for 2 h. The DCF fluorescence increased in H2O2 exposed cells but decreased in GSPE pretreated cells. ∗P < 0.01, ∗∗P < 0.001 compared to H2O2 alone. (B) Effect of GSPE on cell viability during H2O2 exposure. Cardiomy- ocytes were incubated with PI (5�M) and exposed to H2O2 for 2 h or pretreated with GSPE (10, 50, or 100�g/ml) for 2 h. PI uptake increased in H2O2 exposed cells and significantly decreased in GSPE pretreated cells. ∗P < 0.01, ∗∗P < 0.001 compared to H2O2 alone. 3.2. Effect of antimycin A on DCF fluorescence and cell viability in cardiomyocytes Cell damage induced by exogenous oxidants may differ from that induced by endogenous oxidant production, due to the site-specific nature of oxidative damage. To deter- mine whether GSPE confers protection against endogenous oxidants, antimycin A, a mitochondrial ETC complex III inhibitor, was used to enhance the generation of O2•− at site III [19]. Fig. 2(A) shows that antimycin A (100�M) exposed to cells for 2 h increased DCF fluorescence as ex- pected. Compared to antimycin A alone, DCF fluorescence was attenuated in cells exposed to antimycin A and treated with GSPE (10, 50, or 100�g/ml). Fig. 2(B) shows that cell death was also significantly increased at the end of 2 h of antimycin A exposure, but that cells exposed to antimycin A and treated with GSPE (10, 50, or 100�g/ml) were sig- 466 Z.-H. Shao et al. / Pharmacological Research 47 (2003) 463–469 Fig. 2. (A) Effect of GSPE on DCF fluorescence during antimycin A exposure. DCF fluorescence increased after 2 h of antimycin A (100�M) exposure, but decreased in GSPE (10, 50, or 100�g/ml) pretreated cells. ∗P < 0.01, ∗∗P < 0.001 compared to antimycin A alone. (B). Effect of GSPE on cell viability during antimycin A exposure. Cell death increased in antimycin A exposed cells compared with controls, but this was less in GSPE (10, 50, or 100�g/ml) pretreated cells. ∗P < 0.01, ∗∗P < 0.001 compared to antimycin A alone. nificantly protected from 52.0 ± 3.5% in antimycin A ex- posed cells to 35.1±3.8% (P < 0.01, n = 10), 32.3±4.0% (P < 0.01, n = 10), 26.6 ± 3.6% (P < 0.001, n = 10), re- spectively. Again, cell contraction recovered in GSPE (10, 50, or 100�g/ml) pretreated cells (3/10, 4/10, 5/10) but was not observed in cells exposed to antimycin A alone (0/10). These data indicated that GSPE attenuates endogenous ox- idant injury and confers significant protection against the associated cell death. 3.3. PKC, mito KATP channel, and NO had no effect on GSPE action PKC and mitoKATP channel, and NO have been impli- cated in the cardioprotection against cellular injury induced by ischemia and reperfusion [20–22]. To determine if the protection conferred by GSPE occurred through a similar mechanism, cells were incubated with PI (5�M) and pre- Table 1 Effects of PKC, mito KATP channels or NO on reducing cell death by GSPE during H2O2 exposure Pretreatment PI uptake (%) H2O2 (0.5 mM) 56.2 ± 4.9 GSPE (10�g/ml) + H2O2 (0.5 mM) 36.8 ± 3.4∗ CHE (10�M) + GSPE (10�g/ml) + H2O2 (0.5 mM) 33.2 ± 6.1∗∗ 5-HD (500�M) + GSPE (10�g/ml) + H2O2 (0.5 mM) 40.5 ± 7.3∗ l-NAME (200�M) + GSPE (10�g/ml) + H2O2 (0.5 mM) 39.2 ± 5.7∗ Cell death increased in H2O2 (0.5 mM) exposed cells, but this re- sponse was attenuated in cells pretreated with GSPE (10�g/ml) alone. GSPE (10�g/ml) with chelerythrine (CHE, 10�M), GSPE (10�g/ml) with 5-hydroxydecanoate (5-HD, 500�M) or GSPE (10�g/ml) with nitro-l-arginine methyl ester (l-NAME, 200�M) failed to abolish the protective effect of GSPE. n = 10 for each group. ∗ P < 0.01. ∗∗ P < 0.001 compared to H2O2 alone exposed cells. Table 2 Effects of PKC, mito KATP channels or NO on reducing cell death by GSPE during antimycin A exposure Pretreatment PI uptake (%) Antimycin A (100�M) 50.6 ± 6.2 GSPE (10�g/ml) + Antimycin A (100�M) 34.2 ± 4.6∗ CHE (10�M) + GSPE (10�g/ml) + antimycin A (100�M) 38.5 ± 6.5∗ 5-HD (500�M) + GSPE (10�g/ml) + antimycin A (100�M) 36.7 ± 7.3∗ l-NAME (200�M) + GSPE (10�g/ml) + antimycin A (100�M) 37.3 ± 3.9∗ Cell death increased in antimycin A (100�M) exposed cells, but this re- sponse was attenuated in cells pretreated with GSPE (10�g/ml) alone. GSPE (10�g/ml) with chelerythrine (CHE, 10�M), GSPE (10�g/ml) with 5-hydroxydecanoate (5-HD, 500�M) or GSPE (10�g/ml) with nitro-l-arginine methyl ester (l-NAME, 200�M) failed to abolish the protective effect of GSPE. n = 10 for each group. ∗ P < 0.01 compared to antimycin A alone exposed cells. treated with GSPE (10�g/ml) alone or GSPE (10�g/ml) with CHE (2�M, a PKC inhibitor) or 5-HD (500�M, a mito KATP channel inhibitor), or l-NAME (200�M, a NO synthase inhibitor) for 2 h, then exposed to H2O2 (0.5 mM) or antimycin A (100�M) for 2 h. Compared to GSPE (10�g/ml) alone pretreated cells, there were no significant differences in GSPE (10�g/ml) treated cells also given CHE or 5-HD, or l-NAME (Tables 1and 2). Thus, CHE, 5-HD or l-NAME failed to abolish the protective effects of GSPE during H2O2 or antimycin A exposure. 3.4. GSPE scavenging ROS and chelating iron in vitro Fig. 3 shows that the addition of H2O2 (1�M)/FeSO4 (50�M) produced an increase in DCF fluorescence over 15 min, indicating the presence of an oxidant process. Addition of GSPE (10, 50, or 100�g/ml) caused a concentration-dependent attenuation of the increases in Z.-H. Shao et al. / Pharmacological Research 47 (2003) 463–469 467 Fig. 3. Effect of GSPE on DCF fluorescence in H2O2/FeSO4 system. An increase in DCF fluorescence was seen after addition of H2O2 (1�M)/FeSO4 (50�M) to DCFH/DA (10�M) in solution. An attenua- tion of this response was observed with addition of GSPE (10, 50, or 100�g/ml). The symbols: ( ) H2O2/FeSO4, ( ) H2O2/FeSO4 with GSPE (10�g/ml), ( ) H2O2/FeSO4 with GSPE (50�g/ml), and ( ) H2O2/FeSO4 with GSPE (100�g/ml). DCF fluorescence, suggesting that GSPE directly scav- enges H2O2 and/or hydroxyl radicals. Fig. 4 shows that the addition of GSPE (10�M), 1′10′-phenanthroline (PHEN, 10�M) or deferoxamine (DEF, 10�M) significantly attenu- ates DCFH/DA oxidation in the H2O2/FeSO4 system. GSPE attenuated this oxidation more than either PHEN or DEF, suggesting that GSPE may act in part as an iron chelator. Fig. 5 shows that the addition of X (0.4 mM)/XO (0.02 U/ml) induced an increase in Eth fluorescence over 15 min, in- dicating the formation of O2•−. After addition of GSPE Fig. 4. Iron chelating ability of GSPE in H2O2/FeSO4 system. Addition of H2O2 (1�M)/FeSO4 (50�M) in cuvettes containing DCFH/DA (10�M) solution led to an increase in DCFH oxidation. An attenuation of DCF flu- orescence was observed with addition of phenanthroline (PHEN, 10�M), deferoxamine (DEF, 10�M) or GSPE (10�g/ml). A greater less in DCF fluorescence was seen when addition of GSPE (10�g/ml). The symbols: ( ) H2O2/FeSO4, ( ) H2O2/FeSO4 with PHEN (10�M), ( ) H2O2/FeSO4 with DEF (10�M), and ( ) H2O2/FeSO4 with GSPE (10�g/ml). Fig. 5. Effect of GSPE on Eth fluorescence in xanthine/xanthine oxidase system. An increase in Eth fluorescence was observed after addition of X (0.4 mM)/XO (0.02 U/ml) to DHE (100�M) in solution. A dose-related attenuation of this response was seen after addition of GSPE (10, 50, or 100�g/ml). The symbols: ( ) X/XO, ( ) X/XO with GSPE (10�g/ml), ( ) X/XO with GSPE (50�g/ml), and ( ) X/XO with GSPE (100�g/ml). (10, 50, or 100�g/ml), Eth fluorescence dose-dependently attenuated, suggesting GSPE directly scavenges O2•−. 4. Discussion Grape seeds contain