Ginkgo biloba attenuates oxidative stress in macrophages and endothelial cells

Free Radical Biology & Medicine,Vol. 20, No. 1, pp. 121-127, 1996 Copyright © 1995 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/96 $15.00 + .00 ELSEVIER

0891-5849(95)02016-4

Brief Communication GINKGO BILOBA A T T E N U A T E S

O X I D A T I V E S T R E S S IN M A C R O P H A G E S AND ENDOTHELIAL CELLS

YONGQI RONG, ZHAOHUI GENG, and BENJAMIN H. S. LAU Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, CA, USA

(Received 25 April 1995; Revised 25 May 1995; Accepted 14 June 1995) A b s t r a c t The action of Ginkgo biloba extract (GBE) as an antioxidant was studied using various models of oxidative stress in macrophages and vascular endothelial cells. GBE was incubated with murine macrophages (J774) at 37°C and 5% CO2 for 1 h; oxidative burst was triggered by zymosan. The intensity of fluorescence was measured directly in 96-well plates using a computerized microplate fluorometer at 485 nm excitation and 530 nm emission. GBE exhibited both time- and concentrationdependent suppression of oxidative burst. Confluent monolayers of bovine pulmonary artery endothelial cells (PAEC) were preincubated with different concentrations of GBE for 16 h, washed, and then exposed to an organic oxidant tert-butyl hydroperoxide (tBHP) for 2 h. Lipid peroxidation products of PAEC were determined by measuring thiobarbituric acid-reactive substances (TBARS). Cell injury was assessed by measuring the release of intracellular lactate dehydrogenase (LDH), and cell viability was determined by the methylthiazol tetrazolium (MTF) assay, tBHP increased production of TBARS in PAEC. Preincubation with GBE inhibited the increase of TBARS induced by tBHP. GBE protected biomembranes from oxidative injury by decreasing intracellular LDH leakage from PAEC. MTT assay showed that GBE minimized loss of cell viability induced by oxidative injury. The extensive antioxidant effect of GBE may be valuable to the prevention and treatment of various disorders related to free radical-induced pathology.

Keywords---Ginkgo biloba extract, Macrophages, Pulmonary artery endothelial cells, Oxidative burst, Antioxidant effect, Antilipid peroxidation, Free radicals

cated in mediation of a variety of pathologic events, such as cancer, atherosclerosis, diabetes, liver disease, ischemia, and the aging process. 4'5 Oxidant injury of the endothelium is considered to be correlated with the early development of atherosclerosis. Accompanying acute inflammation, adherence of circulating neutrophils to vascular endothelium occurs. Subendothelial migration and localization of monocytes are earliest evidence in fatty-streak formation and thus in atherogenesis. The damage of endothelial cells may lead to cardiovascular and cerebrovascular diseases. Vascular endothelium is susceptible to injury by oxidants that increase with age. 6-8 This may be one explanation for the higher incidence of cardiovascular and cerebrovascular diseases in the elderly population. A native of southeastern China, and a popular ornamental tree in many parts of the world, Ginkgo biioba is a member of the Ginkgoaceae family. Ginkgo biloba (Chinese name: Pai-kuo, also Yin-hsing) has been a staple of Chinese medicine for thousands of years, being recommended for coughs, asthma, and acute allergic inflammations.9 In recent years, the extract of Ginkgo biloba has been researched extensively)°-t5

INTRODUCTION

Macrophages participate in immune response by presenting antigens, secreting chemical mediators, phagocytizing foreign invaders, and undergoing oxidative burst. Following phagocytosis, macrophages release a number of reactive oxygen species, including superoxide anion, hydrogen peroxide, hydroxyl radical, and singlet oxygen.~'2These oxygen radicals are believed to play an important role in microbicidal and tumoricidal activities. However, the generation of reactive oxygen species and their release to the extracellular milieu during oxidative burst also have potential for causing inflammation and tissue injury.3 Because macrophages are potent producers of oxygen radicals, they present themselves as an ideal model for studying modalities that may minimize the oxidative burst and the inflammatory response. Oxygen radicals have been impli-

Address correspondence to: Benjamin H. S. Lau, Department of Microbiology and Molecular Genetics, School of Medicine, Loma Linda University, Loma Linda, CA 92350, USA. E-mail address: [email protected] 121

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Duke et al. ~0purified a cyanide-insensitive superoxide dismutase from leaves of Ginkgo biloba. The purified enzyme exhibited a sensitivity to hydrogen peroxide and insensitivity to cyanide, which is typical of ironcontaining superoxide dismutases. This enzyme catalyses the disproportionation of superoxide radical according to the following equation: 202"- + 2H ÷ --' H202 + 02. Several studies have demonstrated the antioxidant activity of Ginkgo biloba extract in preventing isolated rat hearts from ischemia-reperfusion injury, ~' in reducing oxidative metabolism of rat brain neurons, ~2in minimizing oxidative degradation of rat liver microsomes, '3 and in reducing lipid peroxidation of human liver microsomes. 14 Using four diverse in vitro acellular assays, Marcocci et al.'5 recently reported that an extract known as EGb 761 exhibited strong freeradical scavenging activities. In our laboratory, we have studied several antioxidant substances using in vitro models. I6-2° In this study, the effect of Ginkgo biloba extract on oxidative stress in macrophages and vascular endothelial cells was determined. MATERIALS AND METHODS

Reagents Ginkgo biloba extract (GBE) used in this study was obtained from Japan Greenwave Ltd., Tokyo, Japan. The extract contains 24% flavonoids and 6% terpene lactones. Hanks' balanced salt solution (HBSS), zymosan A, N,N'-dimethyl formamide (DMF), 2-thiobarbituric acid (TBA), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), sodium dodecyl sulfate (SDS), tetraethoxypropane, and tert-butyl hydroperoxide (tBHP) were purchased from Sigma Chemical Co. (St Louis, MO). 2',7-dichlorofluorescein diacetate (DCFH-DA) was obtained from Molecular Probes, Inc. (Eugene, OR). 1-butanol was from Fisher Chemical (Fair Lawn, N J). CytoTox 96 T M Nonradioactive Cytotoxicity Assay Kit was supplied by Promega Co. (Madison, WI). Dulbecco's modified Eagle's medium (DMEM), Eagle's minimum essential medium (EMEM), trypsin-EDTA solution, and penicillin-streptomycin solution were from Mediatech Co. (Washington, DC). Fetal bovine serum (FBS) was obtained from Gemini Bioproducts (Calabasas, CA). Cell lines The murine macrophage cell line, J774A. 1, and the bovine pulmonary artery endothelial cell line (PAEC) were obtained from the American Type Culture Collection (ATCC, Rockville, MD). J774 cell line has been used as a model for studying oxidative burst. 2'-24 J774 cells and PAEC were grown in DMEM and EMEM,

respectively. The media were supplemented with 10% heat-inactivated FBS, 500 U/ml penicillin, and 0.5 mg/ ml streptomycin. Cultures were incubated at 37°C in a humidified 5% CO2 atmosphere for 3 - 4 days before being used for experiments.

Experimental design A macrophage cell line J774 was used to determine the effect of GBE on oxidative burst triggered by zymosan. PAEC were used as a model to study the effect of GBE on oxidative damage induced by an organic oxidant tBHP. Three assays were carried out: (1) lipid peroxidation; (2) lactate dehydrogenase release for cell injury; and (3) MTT assay for cell viability.

Oxidative burst assay J774 cells were harvested, washed once, and resuspended in HBSS. Assays were performed using 96well tissue culture plates (Falcon, 3072) as previously described. 25 The cell density was adjusted to 2 × 105/ ml, and 100 #1 of the cell suspension were pipetted into each well. Different concentrations of GBE in HBSS were added to wells. Plates were incubated for 1 h at 37°C in 5% COz. Following the removal of GBE from wells, zymosan and DCFH-DA diluted in HBSS were added. The plate was covered with a lid and incubated for various time intervals in a 37°C humidified incubator with 95% air and 5% CO2. The fluorescence intensity (relative fluorescence unit, RFU) was determined at 485 ___ 10 nm excitation and 530 ± 12.5 nm emission wavelengths using an automated florescence reader (Microplate Fluorometer Model 7610, Cambridge Technology, Watertown, MA) interfaced with a PC-compatible computer. Captured data were transferred to statistics and graphics programs directly for analysis without reentering.

Lipid peroxidation Various concentrations of GBE in HBSS were added to 24-well plates and incubated with PAEC (4 × 104 cells/well) for 16 h at 37°C in 5% CO2. Following the removal of GBE from wells, cells were washed with HBSS and then exposed to tBHP for 2 h. The supernatant in the wells was collected, and the extent of lipid peroxidation was determined by measuring thiobarbituric acid-reactive substances (TBARS) according to the method of Wey et al. 26 Trichloroacetic acid (12.5%, 0.2 ml) was added to an aliquot of the supernatant. TBA reagent (0.4 ml of 0.67% TBA and

Ginkgo and 1 mM EDTA) was added. The reaction mixture was heated at 95°C for 20 min in a water bath. After cooling with tap water, 3 ml of 1-butanol were added, and the mixture was shaken vigorously for 30 s. After centrifugation at 2,000 rpm for 10 min, the 1-butanol layer was removed for fluorometric measurement with excitation wavelength of 520 nm and emission wavelength of 553 nm, using LS-3 Fluorescence Spectrophotometer (Perkin-Elmer, Norwalk, CT). The value of fluorescence was calculated by comparing with standards prepared from tetraethoxypropane.

Lactate dehydrogenase (LDH) release PAEC (4 × 104 cells/well) in 24-well plates were preincubated with different concentrations of GBE for 16 h, washed, and then incubated with tBHP for 2 h. The supernatant was collected from each well and stored at 4°C. Cell monolayers were treated with lysis solution for 30 min at room temperature to lyse the cell membranes, and then lysate was collected. LDH activity was measured in both the supernatant and the cell lysate fractions by using CytoTox 96 Nonradioactive Cytotoxicity Assay Kit following the manufacturer's instruction. The assay is based on the conversion of a tetrazolium salt into a red formazan product. The intensity of color is proportional to LDH activity. The absorbance was determined at 492 nm with a 96-well plate ELISA reader (400 AT EIA, Whittaker Bioproducts, Walkersville, MD). The percent of LDH released from the cells was determined using the formula: Percent release = LDH activity in supernatant/(LDH activity in supernatant + LDH activity in cell lysate).

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test for honestly significant difference (HSD), and the results were expressed as the mean ± SE. A p value of less than 0.05 was considered significant. All statistical procedures were performed with Statgraphics software version 5.0 (STSC, Inc., Rockville, MD). RESULTS

Effect of GBE on oxidative burst of macrophages J774 cells were preincubated with various concentrations of GBE for 1 h. After removal of GBE, zymosan and DCFH-DA were added to each well. Fluorescence readings reflecting oxidative burst were assessed 1 h later by an automated micro-fluorometric assay. Figure 1 shows a concentration-dependent suppression of oxidative burst of J774 cells. Figure 2 shows a timedependent increase of fluorescence reflecting increase of oxidative burst. However, at each reading interval there was a concentration-dependent reduction of oxidative burst. The viability of these cells remained > 95% when evaluated with trypan blue exclusion, thus ruling out the possibility that the suppression might be due to GBE toxicity.

Protective effects of GBE against oxidative injury in PAEC tBHP, an organic oxidant, was used to induce lipid peroxidation and biomembrane damage resulting in a I

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MTT assay for cell viability The method of Mosmann 27 was used with some modifications. PAEC (7 × 103 cells/well) in 96-well plates were preincubated with different concentrations of GBE for 16 h. Following removal of GBE from wells, cells were washed with HBSS and then exposed to tBHP for 2 h. Cells in 96-well plates were rinsed with HBSS, and MTT (0.4 mg/ml, 100 #1) was added to each well. Following an additional 5 to 6 h incubation at 37°C, 100/zl of 10% SDS were added to dissolve the formazan crystals. Plates were incubated at 37°C overnight, and the absorbance was then measured at 620 nm using the EIA reader (Whittaker Bioproducts, Walkersville, MD).

Statistical analyses The data were analyzed by using one-way analysis of variance (ANOVA) followed by Tukey's multiple range

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Fig. 1. Effect of GBE on oxidative burst in J774 cells. Cells (2 × 105/ml) were preincubated with various concentrations of GBE (50, 100, 150, 200, and 400 #g/ml) at 37°C and 5% CO2 for 1 h. Following removal of GBE, zymosan and DCFH-DA were added. The fluorescence readings were taken after an additional 1 h of incubation at 37°C and 5% CO~. Bars represent SE of six samples. *Significant difference from control (p < 0.05).

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Fig. 2. Suppression of oxidative burst of J774 cells by GBE. J774 cells (2 × 1OS/ml) were preincubated with varying concentrations of GBE at 37°C and 5% CO2 for 1 h. Following removal of GBE, the oxidative burst was triggered by zymosan. The plates were incubated at 37°C, 5% CO2 and fluorescence readings were taken at intervals of 20 min up to 2 h. Each data point represents triplicate samples.

decrease o f e n z y m e activity and a disruption o f m e m brane integrity. T h e effect o f G B E on lipid p e r o x i d a d o n o f P A E C was m e a s u r e d b y the content o f T B A R S , products o f lipid peroxidation. N o significant change in T B A R S was noted when G B E alone was incubated with P A E C (Fig. 3). 2 0 0 / z M , 4 0 0 / z M , and 6 0 0 / z M t B H P i n d u c e d 19.9-fold, 22.4-fold, and 26.1-fold increases o f T B A R S , respectively. C o m p a r e d with control group without t B H P treatment, these elevations were significant (p < 0.05). W h e n cells were pretreated with 1 0 0 - 5 0 0 # g / m l o f GBE, a d o s e - d e p e n d e n t reduction o f T B A R S was noted (Fig. 3). A n M T T assay was used to m e a s u r e the viability o f P A E C . M T r is a p a l e y e l l o w substrate that p r o d u c e s a dark blue f o r m a z a n product w h e n incubated with living cells. The M T T ring is c l e a v e d b y m i t o c h o n d r i a d e h y d r o g e n a s e , and the reaction occurs only in living cells, reflecting a m i t o c h o n d r i a l function. G B E alone at dosages o f 1 0 0 - 5 0 0 # g / m l s h o w e d a slight increase o f cell viability, w h i c h is, however, not statistically significant (Fig. 4). t B H P at 100 /zM and 200 /zM caused a decrease o f cell viability b y 45.4% and 63.1%, respectively. T h e s e decreases were significant c o m p a r e d with the control without t B H P treatment (p < 0.05). Preincubation o f P A E C with G B E at d o s a g e o f 5 0 0 / z g / m l nullified the d a m a g e i n d u c e d b y 100 # M tBHP; and 400 and 500 # g / m l G B E also significantly protected the injury caused b y 200 # M t B H P (Fig. 4).

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Fig. 3. The effect of GBE on tBHP-induced lipid peroxidation of PAEC. Confluent PAEC monolayers in 24-well culture plates were incubated with GBE for 15 h, washed with HBSS twice, and then exposed to 200 /~M, 400 #M, 600 #M tBHP, or HBSS for 2 h. TBARS content in the supematant was determined by TBA fluorometric assay. Bars represent SE of triplicate samples. *Significant difference (p < 0.05) compared with respective control without GBE treatment.

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Fig. 4. Effect of GBE on tBHP-induced cell damage. PAEC monolayers in 96-well culture plates were preincubated with GBE for 16 h, washed, and then exposed to tBHP for 2 h. Cell viability was measured by the MTT assay. Bars represent SE of triplicate samples. • Significant difference from the respective control at "0" point (19 < 0.05).

Ginkgo and oxidative stress LDH is an intracellular enzyme that leaks into the culture medium when cell membranes are damaged. When GBE was preincubated with PAEC without subsequent exposure to tBHP, LDH release was maintained at a low level (Fig. 5). Two hundred micromoles, 400 #M, and 600 pM tBHP caused a significant increase of LDH leakage by 236.8%, 267.4%, and 295.4%, respectively. These increases were significant when compared with the control without tBHP treatment (p < 0.05). Preincubation of PAEC with GBE (100-500/zg/ml) for 16 h resulted in a dose-dependent decline of LDH release in tBHP-treated cells.

DISCUSSION

The salient feature of this study is that various models of oxidant stress were used to examine the antioxidant effect of GBE at different levels and from different angles. By using an oxidative burst model, we found that GBE decreased macrophage oxidative burst triggered by zymosan. The inhibitory effect of GBE on lipid peroxidation was shown in the oxidant model induced by tBHP. The protective action of GBE on biomembranes was manifested in different aspects: the mitochondrial dehydrogenase in M T r assay reflecting

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Fig. 5. Effect of GBE on tBHP-induced LDH release. Confluent PAEC monolayers in 24-well culture plates were preincubated with GBE for 16 h, washed, and then incubated with tBHP for 2 h. The reaction was stopped by removing the supernatant and adding lysis solution to each well. Both supernatant and cell lysate fraction were assayed for LDH activity. % release = LDH (supernatant)/LDH (supernatant + cell lysate). Bars depict SE of quadruplicate samples. *Significant difference (p < 0.05) compared with respective control without GBE treatment.

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the functional or biochemical changes, and intracellular LDH release embodying the membrane integrity and the final consequence of oxidant damage. Macrophages undergo an oxidative burst in response to phagocytic or membrane stimuli, with generation and release of a variety of reactive oxygen metabolites. During the oxidative burst, the cells generate a group of highly reactive oxygen species including superoxide anion, hydrogen peroxide, hydroxyl radicals, and singlet oxygen. 28-3° Although these oxygen radicals can be beneficial for mediating microbicidal and tumoricidal effects, they also have potential for damaging the adjacent normal tissues. This study demonstrates that GBE decreased macrophage oxidative burst. The suppression of oxidative burst by GBE may be mediated through a decrease of NADPH oxidase activity and/or an augmentation of the free radical scavenging systems. Using in vitro systems, Marcocci et al. have demonstrated that GBE exhibits free-radical scavenging activities. 15 In the presence of transition metals such as iron or copper, superoxide anion and hydrogen peroxide can form the highly reactive "OH through the metal-catalyzed Haber-Weiss or Fenton reactions. 3 Oxygen radicals can readily abstract a hydrogen atom from an unsaturated fatty acyl group, thus initiating the process of lipid peroxidation consisting of a set of chain reactions. Lipid peroxidation is regarded as one of the basic mechanisms of tissue damage mediated by free radicals. 3j In this study, tBHP, an organic oxidant, induced lipid peroxidation in PAEC. GBE effectively reduced lipid peroxidation in this model (Fig. 3). Whether GBE can enhance free radical scavengers such as glutathione, superoxide dismutase, and catalase is now under investigation. One of the serious consequences of lipid peroxidation is the damage of biomembranes such as mitochondrial and plasma membranes. After peroxidation of membrane fatty acids, the presence of short-chain fatty acids containing R-OOH, R-COOH, R-CHO, and ROH groups may severely affect membrane permeability and microviscosityY The maintenance of a constant membrane lipid fluidity (microviscosity) within precisely determined limits has been identified as an indispensable necessity for proper functioning of almost all types of cells. 33TBARS produced by lipid peroxidation can cause cross-linking and polymerization of membrane components. 34'35 This can alter intrinsic membrane properties such as deformability, ion transport, enzyme activity, and the aggregation state of cell surface determinants. Under extreme conditions, peroxidized membranes can lose their integrity. 36 In our experiments, tBHP induced a depression of mitochondria dehydrogenase activity with MTT assay and an in-

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crease of L D H leakage from PAEC. The protective action o f G B E on b i o m e m b r a n e s was clearly d e m o n strated in this study (Figs• 4, 5), which may be attributed to its antiperoxidative effect• One of the major biological targets of free radical oxidants is the cardiovascular system. Vascular cells are exposed to oxidation during inflammation, endotoxic shock, i s c h e m i a - r e p e r f u s i o n , and possibly hypertension. 37 There are four principal cells involved in a t h e r o s c l e r o s i s - - e n d o t h e l i u m , smooth muscle, platelet, and monocytes/macrophages. 38 Oxidant injury of the e n d o t h e l i u m is considered an early event in the • 39 d e v e l o p m e n t of atherosclerosls. A c c o m p a n y i n g acute inflammation, adherence of circulating neutrophils to vascular endothelium occurs. 4° Subendothelial migration and localization of monocytes are the earliest events in fatty-streak formation and thus in atherogenesis. Macrophages can injure n e i g h b o r i n g cells by forming toxic substances including superoxide anion. Macrophages could thus injure the overlying e n d o t h e l i u m and set the stage for events c u l m i n a t i n g in the proliferative lesions of atherosclerosis. Our present experiments demonstrated the antioxidant effect of G B E on P A E C and macrophages, suggesting that G B E m a y contribute to the prevention and treatment of atherosclerosis and other free radical-induced disorders. In conclusion, this study demonstrates that G B E decreased oxidative burst in m u r i n e macrophages. G B E also inhibited lipid peroxidation in PAEC. By using oxidative injury models, G B E was shown to reverse the decreased cell viability and to decrease L D H leakage from PAEC. In light of these antioxidant effects and preference of the general public for natural medicines, G B E possesses broad prospects for application and exploitation• Acknowledgements - - The technical assistance of Elsa Francis is

gratefully appreciated. This study was supported by the Chan Shun Research Fund for AIDS and Cancer (Chan Shun InternationalFoundation, Burlingame, CA, USA).

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ABBREVIATIONS

DCFH-DA-- 2',7-dichlorofluorescein diacetate DMEM--Dulbecco's modified Eagle's medium DMF--N,N'-dimethyl formamide EMEM--Eagle's minimum essential medium FBS--fetal bovine serum GBE--Ginkgo biloba extract HBSS--Hank's b a l a n c e d salt s o l u t i o n LDH--lactate dehydrogenase MTT--3-[4,5-dimethylthiazol-2-yl]-2,5diphenyltetrazolium bromide P A E C - - b o v i n e p u l m o n a r y artery e n d o t h e l i a l cells S D S - - s o d i u m d o d e c y l sulfate TBA-- 2-thiobarbituric acid TBARS--thiobarbituric acid-reactive substances tBHP--tert-butyl hydroperoxide