The Ginkgo biloba Extract (EGb 761) Protects and Rescues Hippocampal Cells Against Nitric Oxide-Induced Toxicity

Involvement of Its Flavonoid Constituents and Protein Kinase C

Authors


  • Lippincott Williams & Wilkins, Inc., Philadelphia

  • Abbreviations used: DCF, 2′,7′-dichlorofluorescein; D-MEM, Dulbecco’s modified Eagle’s medium; EGb 761, Ginkgo biloba extract; FBS, fetal bovine serum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; NO, nitric oxide; NR, neutral red (3-amino-7-dimethylamino-2-methylphenazine); OBAA, 3-[(4-octadecyl)benzoyl]acrylic acid; PKC, protein kinase C; SIN-1,3-morpholinosydnonimine; SOD, superoxide dismutase; SNP, sodium nitroprusside; U-73122, 1-[6-[(17β-3-methoxyestra-1,3,5(10)-trien-17-yl)-amino]hexyl]-1H-pyrrole-2,5-dione.

Address correspondence and reprint requests to Dr. R. Quirion at Douglas Hospital Research Centre, 6875 LaSalle Boulevard, Verdun, Québec, Canada H4H 1R3. E-mail: mcou@musica.mcgill.ca

Abstract

Abstract: An excess of the free radical nitric oxide (NO) is viewed as a deleterious factor involved in various CNS disorders. Numerous studies have shown that the Ginkgo biloba extract EGb 761 is a NO scavenger with neuroprotective properties. However, the mechanisms underlying its neuroprotective ability remain to be fully established. Thus, we investigated the effect of different constituents of EGb 761, i.e., flavonoids and terpenoids, against toxicity induced by NO generators on cells of the hippocampus, a brain area particularly susceptible to neurodegenerative damage. Exposure of rat primary mixed hippocampal cell cultures to either sodium nitroprusside (SNP; 100 μM) or 3-morpholinosydnonimine resulted in both a decrease in cell survival and an increase in free radical accumulation. These SNP-induced events were blocked by either EGb 761 (10-100 μg/ml) or its flavonoid fraction CP 205 (25 μg/ml), as well as by inhibitors of protein kinase C (PKC; chelerythrine) and L-type calcium channels (nitrendipine). In contrast, the terpenoid constituents of EGb 761, known as bilobalide and ginkgolide B, as well as inhibitors of phospholipases A [3-[(4-octadecyl)benzoyl]acrylic acid (OBAA)] and C (U-73122), failed to display any significant effects. Moreover, EGb 761 (50 μg/ml), CP 205 (25 μg/ml), and chelerythrine were also able to rescue hippocampal cells preexposed to SNP (up to 1 mM). Finally, EGb 761 (100 μg/ml) was shown to block the activation of PKC induced by SNP (100 μM). These data suggest that the protective and rescuing abilities of EGb 761 are not only attributable to the antioxidant properties of its flavonoid constituents but also via their ability to inhibit NO-stimulated PKC activity.

Nitric oxide (NO) is a neuronal messenger molecule whose overproduction can initiate neurotoxic events under pathological conditions (Dawson and Dawson, 1996). NO production has clearly been linked to neurodegeneration in animal models of ischemia (Huang et al., 1994; Kuppusamy et al., 1995; Iadecola, 1997; Maiese, 1998) and in vitro cultured cells (Maiese et al., 1993a, 1997). The final cellular pathways that lead from the generation of NO to neuronal death include the formation of the potent oxidant peroxynitrite (Radi et al., 1991), the release of intracellular Ca2+ (Maiese et al., 1994; Brorson et al., 1997), and the activation of protein kinase C (PKC) (Maiese et al., 1993b) and phospholipase C (Abbracchio et al., 1995). NO also mediates the neurotoxic effects of glutamate (Dawson et al., 1991) that are implicated in hypoxic/ischemic brain injury and possibly in Alzheimer’s disease (Meldrum and Garthwaite, 1990).

The Ginkgo biloba extract EGb 761 (Tanakan; IPSEN Laboratories, Paris, France) is a standardized mixture of active substances, including 24% flavonoid glycosides and 6% terpenoids (Drieu, 1986), obtained from green leaves of the G. biloba tree. EGb 761 is a polyvalent agent capable of scavenging free radicals such as NO (Marcocci et al., 1994a, b, reducing Ca2+ -stimulated intracellular events (Oyama et al., 1993, 1994), and modulating intracellular signal transduction events, including those involving phospholipases A and C (Bazan and Rodriguez de Turco, 1993) and PKC (Rodriguez de Turco et al., 1993; Rogue and Malviya, 1996). All of these signal transduction molecules are likely involved in brain ischemia and neurodegenerative diseases (Mattson, 1997).

It is interesting that in vivo experiments have revealed that a treatment with EGb 761 inhibits ischemia-induced activation of total PKC activity in rats (Rogue and Malviya, 1996) and decreases, partly via the inhibition of phospholipase C, the accumulation of hippocampal lipidderived second messengers in rats subjected to electroconvulsive shock (Bazan and Rodriguez de Turco, 1993; Rodriguez de Turco et al., 1993). In addition, systemic administration of bilobalide, one of the terpenoid constituents of EGb 761, protects hippocampal slices against hypoxia-induced phospholipid breakdown, presumably owing to an inhibition of phospholipase A (Klein et al., 1995, 1997). Moreover, ginkgolide B, another terpenoid constituent, has been reported to display an antagonistic activity against platelet-activating factor (Zablocka et al., 1995), a potent phospholipid derivative that is produced during cerebral ischemia through calcium-activated phospholipase A2 (Kramer et al., 1996). Such intracellular changes may contribute to the neuroprotective effects of EGb 761 in animal models of focal and global cerebral ischemia (Larsen et al., 1978; Spinnewyn et al., 1986; Seif-El-Nasr and El-Fattah, 1995), of retinal damage induced by ischemia (Droy-Lefaix et al., 1995; Szabo et al., 1997), and in hypoxia (Karcher et al., 1984; Oberpichler et al., 1988). Clinical trials support the potential therapeutic usefulness of EGb 761 in the treatment of cerebral insufficiency (Kleijnen and Knipschild, 1992) and mild cognitive impairments in elderly patients (Wesner et al., 1987; Rai et al., 1991), as well as in Alzheimer’s disease and vascular dementia (Hofferberth, 1994; Le Bars et al., 1997; Maurer et al., 1997).

The precise mechanisms underlying the neuroprotective effects of EGb 761, particularly with respect to ischemia, have yet to be clearly established, but they have been reported to be associated with either its terpenoid (Panetta et al., 1987; Oberpichler et al., 1990; Backhauss et al., 1992; Zablocka et al., 1995; Smith et al., 1996) and/or its flavonoid (Oyama et al., 1994; Smith et al., 1996) fractions. Thus, the aim of the present study was to investigate the mechanisms of neuroprotective effects of EGb 761 and of its different constituents on NO-related sodium nitroprusside (SNP)- and 3-morpholinosydnonimine (SIN-1)-induced toxicity (Maiese et al., 1997) in cultured hippocampal cells; these cells are particularly vulnerable to ischemic injury (Schmidt-Kastner and Freund, 1991) and in Alzheimer’s disease (Hyman et al., 1984).

MATERIALS AND METHODS

Materials and animals

G. biloba extract (EGb 761), its flavonoid fraction (CP 205), and the terpenes bilobalide (CP 160) and ginkgolide B (BN 52021) were supplied by the IPSEN Institute (Paris, France). Materials used for cell cultures were obtained from GibcoBRL (Burlington, Ontario, Canada). U-73122 {1-[6-[(17β-3-methoxyestra-1,3,5(10)-trien-17-yl)-amino]hexyl]-1H-pyrrole-2,5-dione} and 3-[(4-octadecyl)benzoyl]acrylic acid (OBAA) were obtained from Calbiochem (La Jolla, CA, U.S.A.). Unless stated otherwise, all other compounds were purchased from Sigma Chemical Co. (St. Louis, MO, U.S.A.).

Mixed hippocampal glial/neuronal cells were prepared from embryonic day 19 fetuses obtained from Sprague—Dawley rats (Charles River Canada, St.-Constant, Québec, Canada). Animal care was according to protocols and guidelines of the McGill University Animal Care Committee and the Canadian Council for Animal Care.

Hippocampal cell cultures

Hippocampal cell cultures were prepared as previously described (Bastianetto et al., 1999). In brief, following dissection, hippocampal cells were plated at day 0 at a density of ∼5 × 104 viable cells per well in 96-well plates coated with poly-D-lysine (10 μg/ml). Cells were grown in Dulbecco’s modified Eagle’s medium (D-MEM, high-glucose) containing 2 mM pyruvate, 25 mM KCl, 15 mM HEPES, and 10% (vol/vol) fetal bovine serum (FBS; ImmunoCorp, Montréal, Québec, Canada).

Mixed cell cultures were obtained by removing the original medium at day 3 and replacing it with medium of the same composition containing 10% (vol/vol) FBS. They were maintained at 37°C for 7 days in a humidified atmosphere of 5% CO2 and 95% air.

Enriched neuronal cell cultures were obtained by replacing the original culture medium at day 1 with serum-free medium of the same composition containing N2 supplement, with one-third of the medium supplemented with N2 being changed at day 4. Under such conditions, cultures were highly enriched (92-94%) with neurons at day 7 (Alonso et al., 1994).

Experimental treatments

On the day of the experiment, the medium was removed, and cells were gently washed once with HEPES-buffered high-glucose D-MEM (pH 7.4). Cells were then incubated for 20 h at 37°C in the same medium alone or in the presence of either SNP or SIN-1, with or without the different test compounds. After this incubation period, cell viability was determined using both the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and neutral red (NR; 3-amino-7-dimethyl-amino-2-methylphenazine hydrochloride) colorimetric assays (see below).

The rescuing effect of the different compounds was determined by pretreating hippocampal cells with HEPES-buffered high-glucose D-MEM containing SNP (1 mM) for a 2-h period. After the incubation period, the medium was removed and replaced with a similar one (without FBS) in the presence of different drugs. Cell viability was determined 24 h later using the MTT and NR colorimetric assays (see below).

All drugs were freshly prepared on the day of the experiment in a final concentration of 0.05% ethanol, and the solvent alone was found to have no effect on cell survival by itself (data not shown).

Assessment of cell viability: MTT and NR colorimetric assays

MTT is an indicator of the mitochondrial activity of living cells (Denizot and Lang, 1986) and represents a good marker of cell survival in numerous cell culture models of toxicity (see, for example, Doré et al., 1997). In the present study, cells were exposed to high-glucose D-MEM containing MTT (0.25 mg/ml). Following a 3-h incubation (37°C), MTT reduction into living cells was quantified at 570 nm using a microplate reader (Bio-Tek Instruments, Ville St.-Laurent, Québec, Canada).

Because oxidative stress may alter the enzymatic system involved in MTT reduction, the NR colorimetric assay was performed in parallel to assess cell survival. NR is a watersoluble, weakly cationic vital dye that permeates the intact plasma membrane and concentrates in lysosomes of viable cells (Draize et al., 1944). The NR colorimetric assay was originally described for measuring in vitro toxicity in fibroblasts (Draize et al., 1944) and was adapted for the determination of cell viability in rat primary hepatocyte cultures (Fautz et al., 1991). The culture medium was replaced by high-glucose D-MEM containing NR solution (25 μg/ml). After a 3-h incubation at 37°C, cultures were carefully washed once with high-glucose D-MEM to eliminate the excess of extracellular NR. The incorporated dye was then eluted from the cells by placing a solution of 50% aqueous ethanol supplemented with 1% acetic acid into each well. The optical density was automatically determined at 540 nm using a microplate reader as described above.

Assessment of intracellular oxidation

The accumulation of intracellular free radicals was quantified in parallel to the assessment of the protective and rescuing effects of EGb 761 using a 2′,7′-dichlorofluorescein (DCF) fluorescence assay, as previously described (Mattson et al., 1995). The cell-permeable 2,7-dichlorofluorescin diacetate (25 μM; Molecular Probes, Eugene, OR, U.S.A.) is readily converted into 2′,7′-dichlorofluorescin, which is able to interact with intracellular free radicals and peroxides to form the highly fluorescent DCF. The fluorescent dye was concomitantly applied at the beginning of the experiment with SNP (100 μM) and the different drugs. For the rescuing effects of EGb 761, 2,7-dichlorofluorescin diacetate was incubated for 2 h after preexposure to SNP (1 mM) in the presence of different drugs. Depending on the experiment, DCF fluorescence was quantified either 20 or 24 h later (excitation at 485 nm, emission at 530 nm) using a fluorescence multiwell plate reader.

Measurement of PKC activity

Seven-day-old mixed hippocampal cultured cells grown in six-well plates at a density of ∼5 × 106 viable cells per well were serum-starved for 3 h and then exposed to either vehicle or SNP (100 μM, 5 min) in the presence or absence of EGb 761 (100 μg/ml). Treated cells were immediately put on ice and rinsed with 0.5 ml of precooled Hanks’ buffer. Then 0.5 ml of precooled extraction buffer [25 mM Tris-HCl (pH 7.4), 0.5 mM EDTA, 0.5 mM EGTA, 0.05% Triton X-100, 10 mMβ-mercaptoethanol, 1 μg/ml leupeptin, and 1 μg/ml aprotinin] was added to each well, and the cells were scraped off and homogenized with 10 strokes on ice in a precooled Dounce homogenizer. Cell lysates were incubated on ice for 20 min and centrifuged at 14,000 g for 10 min at 4°C to remove cellular debris. The resulting supernatant was used to determine the activity of PKC by a commercial PKC assay kit (SignaTECT PKC Assay System; Promega, Madison, WI, U.S.A.) as suggested by Promega. Specific activity of PKC was obtained by subtracting the radioactivity of the reaction in the presence of 100 μM PKC peptide inhibitor (Promega) from that of the reaction without PKC peptide inhibitor. All the results were presented as percentages of the activity from cells exposed to vehicle (control).

Statistical analyses

Survival of vehicle-treated control groups not exposed to SNP, SIN-1, or the different drugs alone was defined as 100%, and the number of surviving cells in the treated groups was expressed as a percentage of control groups. One-way ANOVA followed by a Newman—Keuls multiple comparison test was used to compare control and treated groups with values of p < 0.05 being considered statistically significant. For the protein kinase assay, the statistical significance of differences between groups was analyzed using unpaired Student’s t test with values of p < 0.05 being considered statistically significant.

RESULTS

NO-induced toxicity

A 20-h exposure to SNP resulted in a concentration-dependent (50 μM-1 mM) decrease in cell survival as shown by MTT degradation and NR uptake (Fig. 1). Whereas exposure to 50 μM SNP damaged only ∼ 27% (MTT) to 34% (NR) of cells, a 96% (MTT) and 98% (NR) loss was measured in cells exposed to 1 mM SNP (Fig. 1). Both colorimetric assays gave similar results with respect to the efficiency of SNP-induced toxicity, with an apparent EC50 of 93 μM for MTT and 97 μM for NR (Fig. 1). Similarly, the other NO releaser, SIN-1 (5 mM), elicited cell injury [∼60% (MTT values) to 55% (NR values), p < 0.05 by unpaired Student’s t test], indicating that SNP-induced toxicity is triggered by NO (see Fig. 3).

Figure 1.

Assessment of the survival of rat hippocampal mixed cell cultures by MTT and NR colorimetric assays after a 20-h exposure to increasing concentrations of SNP. Data are mean ± SEM (bars) values of three separate experiments, each performed in quadruplicate. *p < 0.01 compared with vehicle-treated groups. CT, control.

Figure 3.

Neuroprotective effect of EGb 761 against toxicity induced by SIN-1 (5 mM) in rat hippocampal mixed cell cultures. Concentration-dependent protective effects of EGb 761 are observed against SIN-1-induced toxicity. Data are mean ± SEM (bars) values of three separate experiments, each performed in quadruplicate. *p < 0.05, **p < 0.01 compared with groups treated with SIN-1 alone. CT, control.

FIG. 1.

FIG. 3.

Protective effects of EGb 761 and its flavonoid constituents against NO-induced toxicity

The MTT assay indicated that the viability of hippocampal cell cultures was slightly, but significantly, increased when control groups were treated with 100 μg/ml EGb 761 compared with the vehicle-treated group [116 ± 2 vs. 100 ± 1%, p < 0.01]. However, a 20-h exposure to EGb 761 (100 μg/ml) in the initial medium (high-glucose D-MEM supplemented with FBS) did not significantly increase MTT values, suggesting that EGb 761 does not induce cell division and/or neurite out-growth by itself (104 ± 2 vs. 100 ± 2%).

As monitored using MTT and NR assays, the toxicity induced by either 100 or 500 μM SNP was strongly reduced, in a concentration-dependent manner, in the presence of EGb 761 (Fig. 2), whereas no protection was seen against a higher concentration of SNP (1 mM; data not shown). The protective effect was significant at 50 μg/ml EGb 761 and maximal at the highest concentration tested (100 μg/ml; Fig. 2).

Figure 2.

Neuroprotective effect of EGb 761 against toxicity induced by SNP (100-500 μM) as estimated by (A) MTT and (B) NR colorimetric assays in rat hippocampal mixed cell cultures. Concentration-dependent protective effects of EGb 761 are observed against SNP-induced toxicity. Data are mean ± SEM (bars) values of at least three separate experiments, each performed in quadruplicate. *p < 0.01 compared with groups treated with SNP alone, **p < 0.01 compared with vehicle-treated groups. CT, control.

FIG. 2.

Similar protective effects were observed when the cells were cotreated with EGb 761 (10-100 μg/ml) and SIN-1 (5 mM), with a maximal and complete effect at 50 μg/ml (Fig. 3).

We also investigated the effects of EGb 761 on enriched neuronal cell cultures that were cotreated with SNP (50 μM) for 24 h. The magnitude of the protective effect was as pronounced as that in mixed cells, with a significant effect observed at 50 μg/ml and a maximal potency at 100 μg/ml (Fig. 4).

Figure 4.

Neuroprotective effect of EGb 761 against toxicity induced by SNP (50 μM) in rat hippocampal enriched neuronal cell cultures. Concentration-dependent protective effects of EGb 761 are observed against SNP-induced toxicity. Data are mean ± SEM (bars) values of three separate experiments, each performed in quadruplicate. *p < 0.01 compared with groups treated with SNP alone, **p < 0.01 compared with vehicle-treated groups. CT, control.

FIG. 4.

We compared next the protective effects of EGb 761 with those of major constituents, taking into account their respective contents in the global extract. The flavonoid fraction CP 205 (25 μg/ml), which represents 24% of the total extract, significantly protected against toxicity induced by both SNP (100 μM) and SIN-1 (5 mM), as measured using MTT and NR assays (Table 1). In contrast, the terpenoid constituents bilobalide (CP 160, 1-5 μg/ml) and ginkgolide B (BN 52021, 1-5 μg/ml), which are present in EGb 761 in amounts of 2.9 and 1%, respectively, failed to protect against SNP-induced toxicity (Table 1).

Table 1. Effects of the flavonoid fraction (CP 205) and the terpenes bilobalide (CP 160) and ginkgolide B (BN 52021) against toxicity induced by either SNP or SIN-1 in rat hippocampal cell cultures
TreatmentMTT valuesNR values
  1. Data are mean ± SEM values of at least four separate experiments, each performed in quadruplicate.

  2. ap < 0.01 compared with groups treated with either SNP or SIN-1 alone.

Control100 ± 2100 ± 1
SNP (100 μM) 46 ± 250 ± 5
+ CP 205 (25 μg/ml) 73 ± 10a76 ± 6a
SIN-1 (5 mM) 40 ± 447 ± 5
+ CP 205 (25 μg/ml) 76 ± 8a73 ± 5a
SNP (100 μM) 63 ± 358 ± 3
+ CP 160 (1 μg/ml) 57 ± 271 ± 7
+ CP 160 (5 μg/ml) 57 ± 273 ± 8
+ BN 52021 (1 μg/ml) 59 ± 264 ± 7
+ BN 52021 (5 μg/ml) 62 ± 263 ± 6

TABLE 1.

Rescuing effects of EGb 761 and its flavonoid constituents against SNP-induced toxicity

Immediately following a 2-h exposure to SNP (1 mM), a decrease in cell survival (76 ± 8% of MTT values and 84 ± 7% of NR values) and an increase in DCF fluorescence (data not shown) were observed. This 2-h exposure period also significantly affected cell survival (40-46% of MTT values and 46-58% of NR values) when evaluated 24 h later using the two colorimetric assays (Fig. 5). Most interesting is that EGb 761 (10-100 μg/ml) strongly and dose-dependently increased cell survival when added to cultures up to 2 h post-SNP treatment (Fig. 5).

Figure 5.

Neurorescuing effect of EGb 761 against toxicity induced by SNP (1 mM) in rat hippocampal mixed cell cultures. Concentration-dependent protective effects of EGb 761 are observed against SNP-induced toxicity after a posttreatment period of 2 h. Data are mean ± SEM (bars) values of three separate experiments, each performed in quadruplicate. *p < 0.05, **p < 0.01 compared with groups treated with SNP alone. CT, control.

FIG. 5.

Moreover, the flavonoid fraction CP 205 (25 μg/ml) alone was also able to rescue hippocampal cells when applied 2 h after a preexposure to SNP (Table 2). In contrast, the terpenoids bilobalide (CP 160, 1-5 μg/ml) and ginkgolide B (BN 52021, 1-5 μg/ml) failed to rescue neurons against SNP-induced toxicity (Table 2).

Table 2. Effects of a posttreatment with the flavonoid fraction (CP 205) and the terpenes bilobalide (CP 160) and ginkgolide B (BN 52021) against toxicity induced by SNP in rat hippocampal cell cultures
TreatmentMTT valuesNR values
  1. Data are mean ± SEM values of at least three separate experiments, each performed in quadruplicate.

  2. ap < 0.01 compared with groups treated with SNP alone.

Control100 ± 4100 ± 2
SNP (1 mM) 40 ± 646 ± 6
+ CP 205 (25 μg/ml) 74 ± 8a69 ± 6a
SNP (1 mM) 36 ± 643 ± 5
+ CP 160 (1 μg/ml) 41 ± 742 ± 6
+ CP 160 (5 μg/ml) 39 ± 849 ± 7
+ BN 52021 (1 μg/ml) 37 ± 542 ± 6
+ BN 52021 (5 μg/ml) 36 ± 538 ± 4

TABLE 2.

Effects of various antioxidants, signal transduction inhibitors, and an L-type Ca2+ channel blocker against SNP-induced cell death

To investigate the possible mechanisms underlying NO-mediated toxicity, we tested various enzymatic and nonenzymatic antioxidants, as well as inhibitors of intracellular enzymes that are likely activated by NO, for their effects on SNP-induced toxicity. Ebselen (1 μM), a selenoorganic compound that displays peroxynitrite scavenging properties (Arteel et al., 1999), strongly attenuated SNP-induced toxicity (Table 3). These effects were shared by superoxide dismutase (SOD; 500 IU/ml), an enzymatic antioxidant that metabolizes anion superoxides to hydrogen peroxide, but not by catalase (100 IU/ml) (Table 3). Similarly, a cotreatment with the PKC inhibitor chelerythrine chloride (1 μM) or the L-type calcium channel blocker nitrendipine (25 μM) potently inhibited SNP-induced cell death, as assessed by both the MTT and NR assays (Table 3). However, U-73122 (1-5 μM) and OBAA (1-5 μM), inhibitors of phospholipase C and A, respectively, failed to protect against SNP-induced toxicity (Table 3).

Table 3. Effects of a cotreatment with antioxidants, an L-type Ca2+ channel blocker, and selective signal transduction inhibitors against toxicity induced by SNP in rat hippocampal cell cultures
TreatmentMTT values (%)NR values (%)
  1. Data are mean ± SEM values of three separate experiments, each performed in quadruplicate.

  2. ap < 0.01 compared with groups treated with SNP alone.

Control100 ± 2100 ± 1
SNP (100 μM) 23 ± 429 ± 4
+ Ebselen (1 μM) 87 ± 6a87 ± 3a
+ SOD (500 IU/ml)81 ± 8a96 ± 6a
+ Catalase (100 IU/ml)38 ± 1345 ± 10
SNP (100 μM) 37 ± 428 ± 3
+ Chelerythrine chloride (1 μM) 76 ± 5a77 ± 4a
+ Nitrendipine (25 μM) 69 ± 10a88 ± 10a
Control100 ± 2100 ± 2
SNP (100 μM) 43 ± 637 ± 6
+ U-73122 (1 μM) 50 ± 1038 ± 6
+ U-73122 (5 μM) 52 ± 636 ± 3
+ OBAA (1 μM) 40 ± 739 ± 7
+ OBAA (5 μM) 42 ± 1033 ± 6

TABLE 3.

Ebselen (1 μM), SOD (500 IU/ml), and chelerythrine chloride (1 μM) were also able to rescue markedly hippocampal cells after a preexposure to SNP (1 mM), whereas nitrendipine (25 μM), calatase (100 IU/ml), U-73122 (1-5 μM), and OBAA (1-5 μM) failed to demonstrate rescue properties (Table 4).

Table 4. Effects of a posttreatment with antioxidants, an L-type Ca2+ channel blocker, and selective signal transduction inhibitors against toxicity induced by SNP in rat hippocampal cell cultures
TreatmentMTT values (%)NR values (%)
  1. Data are mean ± SEM values of at least three separate experiments, each performed in quadruplicate.

  2. ap < 0.01 compared with groups treated with SNP alone.

Control100 ± 2100 ± 2
SNP (1 mM) 43 ± 340 ± 3
+ Ebselen (1 μM) 83 ± 6a71 ± 6a
+ SOD (500 IU/ml)94 ± 5a89 ± 5a
+ Catalase (100 IU/ml)55 ± 434 ± 2
SNP (1 mM) 35 ± 443 ± 4
+ Chelerythrine chloride (1 μM) 60 ± 7a66 ± 6a
+ Nitrendipine (25 μM) 33 ± 646 ± 5
SNP (1 mM) 37 ± 436 ± 4
+ U-73122 (1 μM) 36 ± 940 ± 6
+ U-73122 (5 μM) 28 ± 330 ± 3
+ OBAA (1 μM) 47 ± 837 ± 7
+ OBAA (5 μM) 45 ± 837 ± 7

TABLE 4.

Inhibitory effects of EGb 761 and its flavonoid constituents on NO-induced oxidative stress

It has been reported that NO can stimulate reactive oxygen species production (Bondy and Naderi, 1994). In the present study, we observed that a 20-h exposure to SNP (100 μM) induced significant increases (p < 0.01) in DCF fluorescence (532-583% above control values; Fig. 6A). The stimulatory effects of SNP were strongly and dose-dependently inhibited by a cotreatment with EGb 761 (10-100 μg/ml) with a maximal effect at the highest concentration tested (Fig. 6A). Similar, but slightly less potent, inhibitory effects were observed when the cells were cotreated with the flavonoid fraction CP 205 (25 μg/ml; Fig. 6A). In contrast, the terpenoids bilobalide (CP 160, 1 μg/ml) and ginkgolide (BN 52021, 1 μg/ml) did not decrease SNP-induced reactive oxygen species accumulation (356 ± 57 vs. 340 ± 54% and 344 ± 62 vs. 340 ± 54%, respectively). Moreover, reactive oxygen species accumulation in EGb 761 (10-100 μg/ml) and CP 205 (25 μg/ml) -treated control groups was dose-dependently decreased compared with vehicle-treated contrls, suggesting that EGb 761 and its flavonoid fraction can inhibit, by themselves, the production of free radicals (Fig. 6B).

Figure 6.

Effects of EGb 761 and CP 205 (25 μg/ml) on either (A) 100 μM SNP- or (B) vehicle-induced oxidative stress in rat hippocampal mixed cell cultures. EGb 761 or CP 205 was applied at the onset of SNP exposure (A) or in vehicle-treated controls (B). Oxidative stress was determined 20 h later by quantification of DCF fluorescence as described in Materials and Methods. Data are mean ± SEM (bars) values of at least three separate experiments, each performed in quadruplicate. *p < 0.05, **p < 0.01 compared with groups treated with either SNP or vehicle. CT, control.

FIG. 6.

Inhibitory effect of EGb 761 on SNP-stimulated PKC activity

As shown in Fig. 7A, SNP time-dependently stimulated PKC activity, with these effects being significant and maximal after a 5-min exposure. A similar increase was observed after an exposure to phorbol 12-myristate 13-acetate (400 nM), a well-known activator of PKC (252 ± 58 vs. 100 ± 2%, p < 0.01 by unpaired Student’s t test). Cotreatment with EGb 761 (100 μg/ml) significantly abolished SNP-induced activation of PKC activity but did not affect PKC activity in vehicle-treated cells (Fig. 7B).

Figure 7.

Time-dependent PKC activation induced by SNP (100 μM) in rat hippocampal mixed cell cultures (A) and effects of EGb 761 (100 μg/ml) on SNP (100 μM, 5 min)-induced PKC activity (B). PKC activity was measured using the protocol as described in Materials and Methods. Data are mean ± SEM (bars) values of three separate experiments, each performed in duplicate. *p < 0.01 compared with vehicle-treated group, **p < 0.05 compared with SNP-treated group. CT, control.

FIG. 7.

DISCUSSION

The present study indicates that EGb 761, acting principally via its flavonoid constituents, is able to protect and rescue hippocampal neuronal cells against NO-induced toxicity. This is consistent with earlier data reporting protective effects of EGb 761 in animal models of cerebral ischemia (Larsen et al., 1978; Spinnewyn et al., 1986; Droy-Lefaix et al., 1995; Seif-El-Nasr and El-Fattah, 1995; Szabo et al., 1997), in in vitro models of oxidative stress-induced toxicity (Oyama et al., 1993, 1994, 1996; Behar-Cohen et al., 1996), and, more recently, in a model of β-amyloid-induced toxicity (Bastianetto et al., 1998). This is of particular interest given that ischemic neurodegeneration is associated with a loss of hippocampal neurons and that this event is likely the result of an increased production of NO (Huang et al., 1994; Kuppusamy et al., 1995; Iadecola, 1997; Maiese, 1998). It is our contention that EGb 761 can inhibit the toxic events initiated by the production of excess NO.

The production of superoxide anions is one of the major factors involved in NO toxicity because superoxide anions can react with NO to form the highly toxic free radical peroxynitrite (Crow and Beckman, 1996). A pivotal role for superoxide anions in NO-related insults is emphasized by results showing that transgenic mice overexpressing SOD are resistant to brain ischemia (Kinouchi et al., 1991). We have shown in the present study that SOD can protect against SNP-induced toxicity, a finding that is in agreement with earlier studies (Dawson et al., 1993; Gonzalez-Zulueta et al., 1998). Thus, the superoxide scavenging properties of EGb 761 (Gardès-Albert et al., 1990; Marcocci et al., 1994a; Packer et al., 1995) are likely to explain, at least in part, its ability to block cell death and the increase in reactive oxygen species accumulation induced by the two NO donors used here, SNP and SIN-1.

EGb 761 might also directly scavenge peroxynitrites and inhibit lipid peroxidation because it has been reported both to block the cytotoxicity induced by peroxynitrites (Behar-Cohen et al., 1996) and to inhibit cyclosporin A-induced lipid peroxidation (Barth et al., 1991). This hypothesis is supported by the finding that the purported antiischemic agent ebselen completely protected and rescued hippocampal cells against SNP-induced toxicity. In contrast, the hydroxyl radical-scavenging properties of EGb 761 do not seem able to account for its protective effects because catalase failed to display any neuroprotective properties in our model.

Aside from decreases in reactive oxygen species accumulation, inhibition of PKC, levels of which are increased in the hippocampal CA1 region during cerebral ischemia (Cardell et al., 1990), has also been shown to block the toxic effects of NO (Maiese et al., 1993b) and to be linked to neuroprotection during global ischemia (Hara et al., 1990). In this respect, EGb 761 shares with the PKC inhibitor chelerythrine chloride, but not with phospholipase C (U-73122) or A2 (OBAA) inhibitors, the ability to block the toxic effects of SNP. These results are consistent with previous studies (Oyama et al., 1993; Rogue and Malviya, 1996) and suggest that the protective effects of EGb 761, together with its antioxidant properties, may be related to its inhibitory actions on PKC. The fact that EGb 761 was able to block SNP-activated PKC also supports this hypothesis. Nitrendipine protected against but failed to rescue from SNP-induced toxicity, suggesting that blockade of L-type voltage-dependent calcium influx is an early event that may inactivate PKC.

The rescuing effect of EGb 761 against NO-induced toxicity suggests that it can also inhibit active, but reversible, molecular pathways that are induced by NO and that lead to cell death. Among those, PKC is certainly a strong candidate because chelerythrine chloride mediated both the neuroprotective and neurorescuing effects of EGb 761 against NO-induced toxicity. In contrast, inhibitors of phospholipase C (U-73122) and A2 (OBAA) failed to mimic the actions of EGb 761, indicating that EGb 761 regulates PKC through phospholipase A2 and C-independent mechanisms.

EGb 761 contains two major groups of active substances—namely, flavonoids (24%), which are nearly exclusively flavonol-O-glycosides, and terpenoids (6%), including ginkgolides and bilobalides (Drieu, 1986). It is most likely that the protective and rescuing effects of EGb 761 against NO-induced toxicity are attributable to its flavonoid constituents. Indeed, we observed that the flavonoid fraction (CP 205) strongly inhibited both the toxicity and the free radical accumulation induced by SNP and/or SIN-1. This is in agreement with the purported free radical-scavenging and antioxidant actions of the flavonoid constituents of EGb 761 in various in vitro cell culture models (Ramassamy et al., 1993; Marcocci et al., 1994a,b; Oyama et al., 1994), as well as their ability to inhibit PKC (Middleton and Kandaswamy, 1993) and to reduce calcium-stimulated events (Oyama et al., 1993, 1994). On the other hand, the terpenoid fractions ginkgolide B (BN 52021) and bilobalide (CP 160) failed to protect against NO-related toxicity. Previous data postulated that ginkgolide B, on the basis of its platelet-activating factor antagonistic properties and its antioxidant activities, demonstrated protective actions in neuronal cultures (Krieglstein et al., 1995) and was responsible for the antiischemic effects of EGb 761 (Panetta et al., 1987; Oberpichler et al., 1990; Backhauss et al., 1992; Zablocka et al., 1995). The apparent discrepancy between these findings and our present results may be explained, at least in part, by the in vivo use of high doses of the ginkgolides, as well as the use of dimethyl sulfoxide as solvent to solubilize the ginkgolides. Indeed, dimethyl sulfoxide has been shown to possess, by itself, antiischemic and free radical-scavenging properties (Prehn and Krieglstein, 1993).

In summary, the results of the present study suggest that EGb 761 is able to protect completely and to rescue rat hippocampal cultured cells against NO-induced toxicity. These effects are mainly attributable to the polyvalent actions of the flavonoid constituents of EGb 761, including their ability to scavenge free radicals and to modulate signal transduction pathways, e.g., PKC, involved in cell death. The protective/rescuing properties of EGb 761 support its potential beneficial actions against ischemia-induced brain injury and related pathologies that are likely to be associated with the deleterious effects of an increased production of NO.

Acknowledgements

This work was supported by a research grant from the Medical Research Council of Canada. R.Q. holds a “Chercheur Boursier” salarial award from “le Fonds de la Recherche en Santé du Québec.” The authors would like to thank Dr. Yves Christen (IPSEN Institute) for providing EGb 761 and its different constituents.

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