Effect of chronic administration of Ginkgo biloba extract or Ginkgolide on the hypothalamic-pituitary-adrenal axis in the rat

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EFFECT OF CHRONIC ADMINISTRATION OF GINKGO BILOBA EXTRACT OR GINKGOLIDE ON THE HYPOTHALAMIC-PITUITARY-ADRENAL

AXIS IN THE RAT

A. Marcilhacl, N. Dakinel , N. Bourhiml-21 V. Guillaumel , M. Grinol, K. Drieu3, C.

Oliver1

1 - Laboratoire de Neuroendocrinologie Experimentale, INSERM U 297, lnstitut Jean Roche, Faculte de Mddecine Secteur Nord, Boulevard Pierre Dramard, 13916

Marseille Cedex 20

2 - Laboratoire de Biochimie, Biologie Cellulaire et Mol&ulaire, Faculte des Sciences Ain Chock, BP 5366, Casablanca, Maroc

3- lnstitut Henri-Beaufour - IPSEN, 35, Rue Spontini 75116 Paris (Received in final form March 30,1998)

Summary

The hypersecretion of glucocorticoids during exposure to various stressors may induce or worsen pathological states in predisposed subjects. Therefore it is of interest to evaluate drugs able to reduce glucocorticoid secretion. It has recently been shown that chronic administration of a Ginkgo biloba extract (EGb 761) inhibits stress- induced corticosterone hypersecretion through a reduction in the number of adrenal peripheral benzodiazepine receptors. The present study was designed to analyze the effect of EGb 761 and one of its components, Ginkgolide B on the biosynthesis and secretion of CRH and AVP, the hypothalamic neurohormones that regulate the pituitary- adrenal axis. Chronic administration of EGb 761 (50 or 100 mg/kg p.o.

daily for 14 days) reduced basal corticosterone secretion and the subsequent increase in CRH and AVP gene expression. Under the same conditions, surgically-induced increase in CRH secretion was attenuated while the activation of CRH gene expression, ACTH and corticosterone secretion following insulin-induced hypoglycemia remained unchanged.

Chronic i.p. injection of Ginkgolide B reduced basal corticosterone secretion without alteration in the subsequent CRH and AVP increase.

However, the stimulation of CRH gene expression by insulin- induced hypoglycemia was attenuated by Ginkgolide B. These data confirm that the administration of EGb 761 and Ginkgolide B reduces corticosterone secretion. In addition, these substances act also at the hypothalamic level and are able to reduce CRH expression and secretion. However the latter effect appears to be complex and may depend upon both the nature of stress and substance (Ginkgolide B or other compounds of EGb 761).

Key Wonis: wib 761, giakgolide B, stress, CRH, arginine vampresin, corticu&erone, hypophysial portal blood, in sihr hybridization

Corresponding author: C. Oliver, MD, Laboratoire de Neuroendocrinologie

Experimentale, INSERM U 297, Facultb de Mddecine Secteur Nord, Boulevard Pierre

Dramard, 13916 Marseille Cedex 20, France, Phone 4 91 65 43 11, Fax 4 91 69 87 12

2330 Ginkgo B&ha Extracts and HPA Axis Vol. 62, No. 25,1998

Glucocorticoids exert their actions in most tissues of the body and play a crucial physiological role in maintaining metabolic, hydroelectrolytic, and vascular functions.

These functions are severely altered after adrenalectomy and rapidly lead to death.

Following exposure to stress, glucocorticoids secretion increases, It has long been proposed that this increase is essential for the adaptation of the body to stress. The mechanisms of action of glucocorticoids during exposure to stress are still discussed:

permissive effects on the metabolic and vascular actions of other substances such as catecholamines, angiotensine II and/or inhibition of several body reactions to the stress situations that are potentially deleterious (1). However, the physiological importance of the increased glucocorticoids secretion during stress has recently been questioned.

Indeed, it has been reported that the tolerance to surgical stress was similar in adrenalectomized monkeys replaced with a regular or with an excessive (1%fold) glucocorticoid substitutive treatment (2). Then, basal secretion of glucocorticoids may be sufficient for the adaptation of the organism to stress situations. The adverse effects of prolonged exposure to high levels of glucocorticoids (which include decreased immunological defenses, hypertension, metabolic dysfunctions, neurotoxicity) may induce or worsen pathological states in predisposed subjects. Therefore, pharmacological agents able to inhibit or to reduce glucocorticoid hypersecretion during stress may be useful.

The regulation of glucocorticoids secretion depends upon ACTH and hypothalamic neurohormones. Among them, corticotropin-releasing hormone (CRH) and arginine vasopressin (AVP) are now recognized as the most important physiological substances involved in basal and stress-induced ACTH and glucocorticoid secretion. The secretion of both neurohormones is controlled by a variety of brain monoamines and peptides such as catecholamines, serotonin, NPY... (3). Recently, an extract of Ginkgo biloba (EGb 761), a widely used medicinal plant product, has been shown to inhibit corticosterone secretion in rats (4). It has been demonstrated that EGb 761 acts directly at the level of the adrenal gland through a diminution of the number of peripheral benzodiazepine receptors (5). The EGb 761-induced decrease in plasma glucocorticoids concentrations is expected to stimulate CRH and AVP expression. In addition, EGb 761 may also have a direct hypothalamic or suprahypothalamic action. In the following paper, we have analyzed the effect of chronic administration of EGb 761 or Ginkgolide B, one of the components of EGb 761, on corticosterone secretion and CRH and AVP gene expression under basal conditions and after exposure to stress.

The influence of chronic treatment with EGb 761 on CRH release into hypophysial portal blood (HPB) has also been tested.

Animals

Methods

Male Sprague-Dawley rats (225250 g b.w.) from Depre (St Doulchard, France) were used in all experiments. Food and water were provided ad libitum. The rats were housed (6 animals/cage) under controlled lights (lights on from 6 a.m. to 8 p.m.).

Experimental design

All experimental protocols were reviewed and approved by the Ethical Committee on

Animal Studies of the University of Aix-Marseille II. Drugs were administered either

orally or by i.p. injections to obtain a maximal bioavailability. EGb 761 (50 or 100 mg/kg

in 2 ml distilled water) or the same volume of vehicle were administered orally every

day for 14 days and the animals were studied 24 h later. Ginkgolide (2 mg/kg) was

administered i.p. every day for a total of 14 davs. All studies were oerformed between 9

and 12 a.m.

Vol. 62, No. 25,1!I98 Ghk,goBibbaExtracbandHPAhis 2331

First protocol: this study was conducted with EGb 761 chronically-treated rats. The animals were anesthetized with pentobarbital (40 mg/kg b.w. i. p.). A femoral artery was cannulated. Then, the ventral hypothalamus and pituitary gland were exposed by a parapharyngeal approach as described by Porter and Smith (6). Surgery lasted for approximately 30 min. After an additional 30-min rest period, a blood sample was collected from the femoral artery; the pituitary stalk was cut and the pituitary gland was removed; then, pituitary stalk blood was collected by aspiration with a polystyrene pipette every 10 s during 60 min, and immediately put into chilled polystyrene tubes maintained in an ice bath throughout the experiment, as previously described (7).

Second protocol: this study was conducted with EGb 761 or Ginkolide B chronically treated rats. Twenty four hours after either the last administrations, the rats of each group were separated into two subgroups which were i.p. injected either with regular insulin (4 IU/lOO g b.w.) (Umulin, Lilly) or with vehicle. They were were sacrificed by decapitation 180 min later. Trunk blood was collected into chilled polystyrene tubes containing 400 ul of a 4% EDTA solution and centrifuged for 20 min at 4°C. The resulting plasma was stored at -20°C until cotticosterone assay. Brains were rapidly removed, frozen on dry ice and kept at -70°C.

In situ hybridization of CRH and AVP mRNA

In situ hybridization: sixteen consecutive coronal sections (12 j.tm) through the hypothalamic paraventricular nucleus between levels A 5660 p and A 5340 p of the atlas of K&rig & Klippel (8) were cut in a cryostat microtome at -20” C. The sections were thaw-mounted onto twice gelatin-coated slides, dried on a slide warmer, and kept at -70” C. They were divided in four groups of four consecutive sections. The first sections of each of the four groups were hybridized with the CRH probe and the second ones with the AVP probe. In situ hybridization was performed as previously described (9) with slight modifications. The sections were warmed at room temperature and fixed with 4% formaldehyde in PBS, pH 7.2, for 5 min. After two washes in PBS, they were placed in 0.25% acetic anhydride in 0.1 M triethanolamine-0.9% NaCI, pH 8.0, for 10 min and delipidated in ethanol and chloroform. They were hybridized at 37” C with 50

~1 of a buffer containing 4 X SSC (1 X SSC is 0.15 M NaCl and 0.015 M sodium citrate, pH 7.2) 50% (vol/vol) formamide, 10% (wt/vol) dextran sulphate, 50 fig/ml sheared single-stranded salmon sperm DNA, 25 j.tg/ml yeast tRNA, 1 X Denhardt’s solution, 0.1 M dithiothreitol and 1 x 10s dpm of probe under a parafilm coverslip. After a 20-h incubation at 37” C, the sections were washed in four 15-min rinses of 2 x SSC containing 50% formamide at 40” C, followed by two 30-min rinses of 1 x SSC at room temperature. The slides were exposed to X-ray films (BIOMAX MR, Kodak, Le Pontet, France), concomitantly with radioactive brain paste standards, for 1 hour (AVP hybridization) or 3 days (CRH hybridization). Sections were subsequently dipped in nuclear emulsion (1:l in water, K5, Ilford, Saint-Priest, France) and exposed for 12 hours (AVP hybridization) or 2 weeks (CRH hybridization). Synthetic oligonucleotides directed against bases 496-543 of the rat CRH mRNA (10) or bases 134-178 of the rat AVP mRNA (11) were used as probes. They were labelled using (35s) deoxyadenosine thiotriphosphate (1300 Ci/mmol, New England Nuclear, Paris, France) and terminal deoxynucleotidyl transferase (Boehringer Mannheim, Meylan, France) to a specific activity of about 9000 Ci/mmol. The specificity of the probes has already been described (9).

Quantification of the hybridization signal: four anatomically matched sections from each

animal were analyzed. The hybridization signal was quantified on the film

autoradiograms. The optical density of the hybridized areas was measured using a

2332 Ginkgo Biloba Extracts and HPA Axis Vol. 62, No. 25,19!%

Biocom 200 (Biocom, Les Ulis, France) image analysis system. Optical densities were converted to copies of probe per pm3 of tissue, using the radioactive brain paste standards, according to Young et al. (12).

Assays

CRH was measured by radioimmunoassay in acetone extracts of hypophysial portal plasma according to methods previously published (13). The limit of sensitivity of the assay was 10 pg/ml plasma. Corticosterone was measured by radioimmunoassay in peripheral plasma, as previously described (7). Norepinephrine and Epinephrine were assayed in 50 ~1 of plasma samples using a radioenzymatic assay kit commercially available from Amersham (Les Ulis, France) and previously used in our laboratory (14).

The limit of sensitivity of the method was 10 pg/ml for both catecholamines. Plasma glucose was measured using an enzymatic kit assay (Biotrol, France).

Statistical Analysis

The data are given as means + SE. All data were analysed for statistical significance using one-way analysis of variance followed by the Fisher’s test. Significance was defined at the 0.05 level.

Results First protocol

The determination of hormone levels was performed under conditions of pentobarbital anesthesia and intense surgical stress. In vehicle-treated animals, plasma ACTH and corticosterone concentrations were elevated (ACTH: 246 + 26 pg/ml, corticosterone:

26.5 f 0.9 pg/dl), as a consequence of the surgical stress. No significant change was observed in the group receiving EGb 761 at the dose of 50 mg/kg. However, a significant reduction in plasma ACTH (-59%, pcO.005) and corticosterone (-16.4%, pcO.005) was found in the group receiving EGb 761 at the dose of 100 mg/kg (Fig. 1).

q Control EGb 761 (50mg/kg) q EGb 761 (lOOmg/kg)

Fig. 1

Effect of chronic oral administration of EGb 761 (50 or 100 mg/kg) on hypophysial portal plasma CRH levels (A), plasma ACTH (B), or plasma corticosterone (C) concentrations in male rats. Values are mean f SE.

PcO.05 vs control.

VoL 62, No. 25, l!J!% Giio Bhba Extrads d HPA Axis 2333

The mean CRH levels in controls were 667 + 52 pg/ml; they were unaltered by EGb 761, 50 mg/kg and significantly reduced to 371 f 54 pg/ml (-45%; ~~0.005) in the group receiving EGb 761, 100 mg/kg (Fig. 1). In controls, the mean epinephrine and norepinephrine levels were 179 f 19 and 536 + 71 pg/ml, respectively. Epinephrine levels were significantly reduced after both doses of EGb 761, although the reduction of hormone concentrations was not dose-dependent. Norepinephrine levels were unchanged in the EGb 761, 50 mg/kg-treated group. However, they were significantly reduced (-30%. ~0.005) in the group of rats receiving EGb 761, at the dose of 100 mg/kg (Fig. 2). ’

=_

c 600

q Control q EGb 761 (50mg/kg) q EGb 761 (lOOmg/kg)

Fig. 2

Effect of chronic oral administration of EGb 761 (50 or 100 mg/kg) on epinephrine (A) or norepinephrine (6) levels in male rats. Values are mean k SE. * PcO.05 vs control.

Second protocol

Effect of chronic administration of EGb 761 (Fig. 3): EGb 761-treatment (at 50 or 100 mgkg) did not change plasma glucose concentrations in both saline- or insulin-injected animals. In the group of rats receiving saline, plasma corticosterone concentrations were lower when the rats were previously given EGb 761 daily for 14 days.

Corticosterone levels were even lower in rats treated with 50 mg/kg (5.1 + 1.8 ygldl) than in those receiving 100 mg/kg (9.5 f 2.5 pg/dl) as compared with controls (14.1 -I 3.9 pg/dl). After insulin-induced hypoglycemia, a large increase in corticosterone release was observed in all groups of rats and the amplitude of corticosterone response was similar in EGb 761 (50 or 100 mg/kg) and saline-treated animals. CRH mRNA levels increased after insulin-induced hypoglycemia as compared with saline- injected animals. This increased CRH gene expression was unaltered by chronic administration of EGb 761 at the two doses (Fig. 3C). AVP mRNA levels were not affected following treatment with EGb 761 (Fig. 3D).

Effect of chronic administration of Ginkgolide B (Figs. 4-6) : Chronic administration of

Gingkolide B was followed by a reduction in plasma corticosterone and a significant

increase in CRH and AVP mRNA levels, Following insulin-induced hypoglycemia, the

stimulation of cotticosterone release was similar in chronically Ginkgolide B- or saline-

treated rats. The stimulation of CRH mRNA levels was attenuated by Ginkgolide B

administration while the stimulation of AVP gene expression was not changed.

2334 Gbkgo Bilaba Extracts and HPA Axis Vol. 65 No. 25,1998

q Saline q Insulin

Control 50 100

EGb 761 (mg/kg) Fig. 3

Effect of chronic oral administration of EGb 761 (50 or 100 mg/kg) on plasma glucose

levels (A), plasma corticosterone concentrations (B), CRH mRNA levels (C), or AVP

mRNA levels (D) in male rats after an acute injection of insulin or saline. Values are

mean + SE.

P-z 0.05 vs saline-injected rats; # PcO.05 vs respective control.

Vol. 62, No. 25,1998 Giio Biloba Extra&s and HPA Axis 2335

q Saline m Insulin

Lz ⁵⁰ 1’:’ ^{l B}

Co&o1 Ginkgdlide B

Fig. 4

Effect of chronic i.p. administration of Ginkgolide B (2 mg/kg) on plasma glucose levels

(A), plasma corticosterone concentrations (B), CRH mRNA levels (C), or AVP mRNA

levels (C) in male rats after an acute injection of insulin or saline. Values are mean f

SE. * P< 0.05 vs saline-injected rats; # P<O.O5 vs respective control.

2336 Ghkgo Biloba Extracts and HPA Axis Vol. 62, No. 25,19!%

Discussion

Significant and complex modifications of the hypothalamic-pituitary-adrenal (HPA) axis have been observed during chronic treatment with EGb 761 or Ginkgolide B. These changes may be due to an action of these compounds at two sites: the adrenal gland and the hypothalamus. Indeed, a direct effect at the adrenal level is likely since both compounds decreased basal corticosterone secretion. This observation confirms previous reports on the inhibitory action of Ginkgo biloba extract on the secretion of corticosterone. Amri et al. (5) have shown that, in the rat, a 8-day treatment with EGb 761 at 100 mg/kg resulted in a 50% reduction in plasma corticosterone. Similar data have been obtained with chronic administration of Ginkgolide A and B. These authors have studied the mechanisms of action of EGb 761 and ginkgolides on the adrenal gland. They have. observed a reduction of the ligand binding capacity, protein levels, and messenger RNA expression of the adrenocortical mitochondrial peripheral-type benzodiazepine receptor (PBR). PBR concentrations are very high in the adrenal fasciculata and they have been shown to play a key role in the regulation of cholesterol transport from cytosol to mitochondria and corticosteroid synthesis (15). Concomitantly, serum aldosterone and testosterone levels were unaffected suggesting that the effect was specific for the adrenal fasciculata cells.

In addition to the adrenal site of action, EGb 761 and Ginkgolide B could also affect the HPA axis at the hypothalamic level. Under basal conditions EGb 761 prevented the expected rise in CRH or AVP mRNA levels in face of the reduced glucocorticoid inhibitory tone. This finding is consistent with the lack of change in plasma ACTH levels after chronic EGb 761 treatment (5). During the stressful surgical procedure needed for HPB collection, animals treated with EGb 761 showed reduced CRH secretion together with a decrease in plasma epinephrine and norepinephrine levels. Rapin et al. (4) have reported that 20 days of oral treatment with EGb 761 (50 or 100 mg/kg/day) suppressed alterations in both discrimination learning and plasma hormones (corticosterone, norepinephrine and epinephrine) induced by an auditory stress. The decrease in CRH secretion found in our study does not seem to be related to the changes in plasma epinephrine and norepinephrine concentrations since it is established that peripheral catecholamines do not cross the blood brain barrier (16). During a strong metabolic stress such as insulin-induced hypoglycemia, EGb 761 treatment was unable to modify the increase in CRH or AVP gene expression. The reasons for the lack of effect of EGb 761 on this type of stress is undetermined yet. The activation of the HPA axis by insulin- induced hypoglycemia may occur mainly at the ventromedial hypothalamic nucleus (17) while the regulation of basal and surgically-induced ACTH and corticosterone secretion involves extrahypothalamic structures. Alternatively, EGb 761 may modulate type I glucocorticoid receptors, at the hypothalamic level. Indeed, it is established that type I receptors mediate tonic inhibition of baseline glucocorticoids on the HPA axis, while type II receptors only come into play when glucocorticoids concentrations reach high levels after stress (18). This hypothesis could explain the reduction of CRH mRNA or CRH secretion by EGb 761 when glucocorticoids concentrations were not (controls) or moderately elevated (rats prepared for surgery) and the lack of effect of this drug when glucocorticoids secretion was maximally stimulated (during insulin-induced hypoglycemia). Further experiments will be helpful in understanding this phenomenon.

The extract EGb 761 contains flavonol heterosides (24%), terpenoids (3.1%

ginkgolides, 2.9% bilobalide) and also proanthocyanidine and organic acids (19). The

identification of the EGb 761 component acting at the hypothalamic level is now under

investigation. Although the precise mechanisms involved in the hypothalamic action of

Ginkgolide B are yet unknown, they may involve PBR. Indeed, high concentrations of

PBR have been found in the hypothalamus (20) and Calogero et al. (21) have reported

in vivo and in vitro studies showing that a PBR agonist 4-chloro-diazepam (Ro-4864)

Vol. 62, No. 25,1998 Giio Bi Extracb and HPA Axis 2337

Saline Insulin -

Fig. 5

Darkfield view of the effect of chronic i.p. administration of Ginkgolide 6 (2

mg/kg) on CRH mRNA in the hypothalamic paraventricular nucleus. Bar

equals 100 Km.

2338 Ginkgo Biloba Extracts and HPA Axis Vol. 62, No. 25, 1998

Saline Insulin -

Fig. 6

Darkfield view of the effect of chronic i.p. administration of Ginkgolide B (2

mg/kg) on AVP mRNA in the hypothalamic paraventricular nucleus. Bar

equals 100 pm.

Vol. 62, No. 2S,l!W8 Ginkgo Biloba Extracts and HPA Axis 2339

stimulates ACTH and corticosterone secretion through an increased CRH release. So far, the action of EGb 761 on the expression of PBR at the hypothalamic level has not been investigated. Such a mechanism may be complex since PBR in the central nervous tissue has been localized in glial cells and not in neurons (22). It must be noticed that another component of EGb 761 may play a role on hypothalamic neurons since EGb 761, unlike Ginkgolide B, did not reduce CRH gene expression during insulin-induced hypoglycemia.

The effect of Ginkgolide B at the central nervous system level may be different from those of EGb 761. Indeed, under basal conditions, the decrease in plasma corticosterone concentrations induced by chronic Ginkgolide B treatment resulted in an increase in both CRH and AVP mRNA levels, suggesting that Ginkgolide B does not act directly at the paraventricular nucleus level. This observation is supported by the demonstration that chronic Ginkgolide A and B treatment increased circulating ACTH levels (5). However, the insulin-induced hypoglycemia-induced stimulation of CRH gene expression was significantly blunted in rats treated with Ginkgolide B. The regulation of CRH synthesis and secretion under stressful conditions is not completely understood. It is known that central catecholamines and serotonin have a stimulatory effect on CRH release (23, 24) and that hypothalamic norepinephrine increases during insulin-induced hypoglycemia (23). Although hypothalamic catecholamines and serotonin concentrations have not been measured in this study, this suggests that Ginkgolide B may modulate the monoaminergic inputs to the CRH synthesizing cell bodies.

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