行政院國家科學委員會補助專題研究計畫成果報告

行政院國家科學委員會補助專題研究計畫成果報告

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計畫類別：

þ

NSC- 89-2315-B-038-002-

執行期間： 89 年 8 月 1 日至 90 年 7 月 31 日

計畫主持人：

陳 盛

宣

本成果報告包括以下應繳交之附件：

□赴國外出差或研習心得報告一份

□赴大陸地區出差或研習心得報告一份

□出席國際學術會議心得報告及發表之論文各一份

□國際合作研究計畫國外研究報告書一份

執行單位：台北醫學大學醫學系

中 華 民 國 90 年 10 月 31 日

行政院國家科學委員會專題研究計劃成果報告

銀杏葉萃取物對酒精引發胃潰瘍的保護效用及機轉

The efficacy and mechanism of the ginkgo biloba extr act against ethanol-induced gastr ic ulcer s in r ats.

計劃編號: NSC- 89-2315-B-038-002-

執行期限: 89 年 8 月 1 日 至 90 年 7 月 31 日

主持人: 陳 盛

宣 副教授 台北醫學大學醫學系

中文摘要

本計畫探討銀杏萃取物是否可以改善酒精引起之胃潰瘍，我們以大鼠 做為實驗對象，發現 1 ml 之 50﹪酒精，經由胃管灌食至大鼠的胃中，可 引起大鼠明顯胃潰瘍現象。若先以血管注射銀杏萃取物 30 min, 則隨銀杏 萃取物劑量的增加，可防止酒精引起之潰瘍。進一步的實驗顯示銀杏萃取 物可防止酒精引起之過氧化作用，及防止胃黏膜壁的流失，然而銀杏萃取 物對胃液的分泌則無明顯的作用。此外，發現酒精可活化胃壁細胞 JNK 激

的活性，已知 JNK 的活化會促使胃壁細胞進行計畫性凋亡，然而銀杏萃 取物可防止酒精活化 JNK 激，這些結果顯示銀杏萃取物保護酒精引起之 潰瘍，是因為銀杏萃取物具有抗氧化能力及降低 JNK 激活化的能力。

關鍵詞: 銀杏萃取物, 酒精, 胃潰瘍, JNK

Abstr act

The effect of Ginkgo biloba extract (GBE) pretreatment on gastric mucosal injuries caused by ethanol was investigated in rats. Ethanol (50%, 1ml/rat) induced gastric lesions by gavage, and GBE pretreatment was found to provide a

dose-dependent protection against the ulcerogenic effect of ethanol. The pretreatment of GBE afforded a dose-dependent inhibition of ethanol-induced depletion of stomach wall mucus, NP-SH contents and an increase in the lipid peroxidation in gastric tissue.

GBE alone had an inhibitory effect on pylorus ligated accumulation of gastric

secretions. On the other hands, gastric ulcer induced by ethanol produced an increase in c-Jun kinase (JNK) activity in gastric mucosa. However, GBE significantly

suppressed the activation of JNK induced by ethanol. These results suggest that GBE

against ethanol-induced gastric lesions may be mediated through its free-radical

scavenging property, and decrease of the JNK activity.

Key Wor ds: ginkgo biloba extract, ethanol, gastric ulcer, c-jun kinase.

Intr oduction

Ethanol is a damaging agent of stomach in animal studies. Incubation of ethanol in rats usually induces gastric ulcer. The possible mechanisms of ethanol-induced gastric lesions are; i) Ethanol may directly damage the mucin layer or affect mucin synthesis (1). ii) Ethanol can increase gastric acid secretion (2). iii) Ethanol can be metabolized to acetaldehyde in stomach, and acetaldehyde-protein adduct may damage the mucosa (3). iv) Ethanol induces gastric cells’ apoptosis, which may contribute partially to the pathogenesis of gastric lesion (4). v) Ethanol may generate free radicals and damage the gastric mucosa (5, 6). Therefore, search of free-radical scavengers can be used for the prevention of ethanol-induced gastric damage.

Ginkgo biloba is a medicinal herb used for centuries as folk remedies. It enjoys a surge of public interest. The endurance of herbal medicines may be explained by the tendency of herbs to work slowly, often without toxic side effects, both on the illness and its symptoms. Ginkgo biloba is a member of the family Ginkgoaceae. It was cultivated in China in the mid 1700s (7). Originating in southeastern China some 200 million years ago, it lives to an age of 1,000 or more years and is the last remaining member of its order. The ginkgo tree was introduced to Europe in the early 18

century, and is now grows in temperate regions throughout the world (8). ‘Ginkgo’ is derived from the Japanese word ginkyo, meaning ‘silver apricot’, referring to the seed.

Biloba translates as ‘two-lobed’, referring to the bilobed appearance caused by the split in the middle of some of its fan-shaped leaf blades.

Because the fresh herbs are often neither accessible nor convenient, various preservation methods have evolved. One of the most common is drying, or made as an infusion or decoction. The most concentrated form of herbal medicines are the

tinctures and extracts made by soaking herbs in alcohol or apple cider vinegar (9). The

standardized ginkgo biloba leaf extract was generically known as EGb761, contains

ginkgo flavone glycosides and terpene lactones. The ginkgo flavone glycosides

(quercetin, kaempferol and isorhamnetin) act as antioxidants and mild inhibitors of

platelet aggregation. Terpene lactones (ginkgolides A, B, C, and bilobalide) provide

neuroprotection by enhancing utilization of oxygen and glucose, improving perfusion

and rheologic properties of blood, inhibiting platelet activating factor (PAF) (10,11),

and generally stimulating cognitive function (9). Animal studies indicate an increase

in the density of muscarinic cholinergic receptors, β-adrenoceptors. Increases in

acetylcholine turnover and dopamine synthesis have been studied. Free-radical

scavenging (12) and prolongation of endothelium derived relaxing factor (NO) have

also been demonstrated in vitro (9).

Pharmacological research on ginkgo began in the 1950s. Since then, many studies have been published on the use of ginkgo biloba to ameliorate cerebrovascular insufficiency (11, 12), and enhance mental performance (13). These studies indicate that ginkgo’s effects are related to improved central cognitive function (14), cerebral blood, and glucose consumption. Improved vigilance, sociability, mood and behavior have also been observed. Recent studies found statistically improvement of

Alzheimer’s disease (15) in the treated compared to placebo group. Additionally, peripheral vascular disease and geriatric depression resistant to standard

antidepressent therapy have been shown to improve significantly with ginkgo therapy (9). Because of its free-radical scavenging (12,10) activities and improvement in the rheologic properties of blood (10,11), which is probably beneficial to mucosal blood flow, ginkgo biloba extract can also be used for the prevention of ethanol-induced gastric injury.

c-Jun N-terminal kinases (JNKs) is a member of the family of mammalian mitogen-activated protein (MAP) kinase, known as stress-activated protein kinases (SAPKs), that mediates intracellular signals originated from diverse extracellular stimuli, including growth factors, cytokines, UV light, heat shock, and a variety of chemotherapeutic drugs (17). The activation of the JNK cascade in cells leading to a wide range of cellular functions including inflammation, cell growth, and cell death.

Recently, several reports have demonstrated that activation of JNK play important role in process of cells apoptosis. Such as chemotherapeutic drug-doxorubicin, has been shown to activate JNK (18) and the drug being critical for the

apoptosis-triggering program for different cell lines. The expression of a

dominant-negative mutant of JNK prevented the induction of apoptotic cell death by UV or anticancer drugs (19). These observations suggest an essential role for JNK in the regulation of apoptosis induced by diversified stimuli.

In this study, GBE is very effective in the protection against ethanol-induced gastric lesions, and we want to pursue these investigations to understand the

mechanisms of GBE’s effect. These issues are very important for alcohol drinkers if they can use GBE before their drinking as a protective agent of their stomach.

Mater ials and Methods

Animals and exper imental design

Female Wistar albino rats (Animal Center of National Taiwan University,

Taipei, Taiwan), about 180-200 g were used for the study. The animals were

maintained in air conditional room with 14/10 hr light/dark cycles, fed with regular

chows and allowed free access to tap water. Before experimentation, the animals were

fasted but allowed to drink water for 36 hr to ensure an empty stomach. Ginkgo biloba extract (Cerenin ampule, 3.5 mg ginkgo biloba extract/ml) was purchased from DR.

Willmar Schwabe Karlsruhe F.R.G. (Germany). Ginkgo biloba extract were administered i.v. 30 min prior to the ulcerative challenge (50% ethanol, 1 ml) by gavage to the fasted rats. Animals were killed under ether anesthesia 45 min after treatment with ethanol, and their stomachs were removed. The stomachs were opened along the greater curvature and examined for lesions developed in the glandular portion under dissecting microscope (x10) (20). The number of ulcer lesions (U.No.) were calculated and expressed as the ulcer index (U.I.). The protective ratio (%) was calculated according to the following formula

Preventive ratio (%)=(a-b)/a x 100 a: The ulcer index of the control group b: The ulcer index of the experimental group

Deter mination of gastr ic wall mucus

The glandular stomach was removed and weighted. The glandular segments were transferred immediately to 0.1%Alcian blue solution in 0.16M sucrose solution with 0.05M sodium acetate to pH 5.0 and stained for 2 hr at room temperature. After rinsed with sucrose solution, the dye complexed with the gastric wall mucus was extracted with 0.05M magnesium chloride solutions. The aliquot of magnesium chloride solution (4 ml) was further extracted with equal volume of diethyl ether and centrifuged (3600 rpm, 10 min). The quantity of Alcian blue extracted/g (net) of glandular tissue was then calculated (21).

Estimation of non-pr otein sulfhydr yl gr oups (NP-SH) and malondialdehyde (MDA) contents

After rats were killed, the glandular stomach were opened and rinsed with ice-cold saline, and rapidly stored in a dry ice bath until analyzed. For the determined of NP-SH, the tissues were homogenized in ice cold 50 % (w/v) aqueous TCA and centrifuged. The NP-SH was determined by mixed with the supernatants and 5, 5’-dithiobis (2-nitrobenzoic acid) (DTNB) in phosphate buffer (pH 8.0). The absorbance (412 nm) was read after 5 min of incubation (22).

For determined of MDA, the supernatants were incubated with N-methyl-2-phenylindole and read absorbance at 586 nm according to the

manufacturer’s instructions (BIOXYTECH LPO-586 kits, OXIS International Inc.,

Portland, U.S.A.).

Deter mination of anti-secr etar y activity in Pylor us-ligated r ats

After the fasting period (36 hr), surgery was carried out under ether anesthesia. GBE was i.v. given 15 min before pylorus-ligation. Gastric juice was collected 3 hr after pylorus-ligation, they clarified by centrifugation (3000 rpm, 10 min) and the volume, and total acidity was determined. Acidity was determined by titration with 0.01 N NaOH to pH 7.0 and the volume was expressed in mEq/liter (23).

Wester n blotting and J NK kinase activity assay

Gastric mucosa was homogenized in Gold lysis buffer (40 mM Tris-NaOH pH 7.5, 500 mM NaCl, 0.1% NP-40, 6 mM EDTA, 6 mM EGTA, 10 mM

β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM sodium orthovanadate, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1mM DTT) and centrifuged. The supernatant was collected as total tissue lysate. Equal amounts of total tissue lysate (50 µg) were resolved by SDS-polyacrylamide gel electrophoresis (PAGE), and transferred onto Immobilon-P membrane (Millipore, Bedford, MA) as described previously (24). The membrane was then incubated with an anti-JNK1 antiserum (Transduction Laboratories, Lexington, KY). The membranes were subsequently probed with anti-mouse IgG antibody conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and visualized using enhanced

chemiluminescence’s kits (ECL, Amersham). For kinase assay, equal amounts of total tissue lysate (400 µg) were immunoprecipitated with JNK1 specific antibody and protein A/G-PLUS agarose for 15 h at 4℃. Kinase assay was carried out in 45 µl of kinase buffer (40 mM Tris-NaOH pH 7.5, 500 mM NaCl, 0.1% NP-40, 6 mM EDTA, 6 mM EGTA, 10 mM β-glycerophosphate, 10 mM NaF, 10 mM PNPP, 300 µM sodium orthovanadate, 1 mM benzamidine, 2 µM PMSF, 10 µg/ml aprotinin, 1 µg/ml leupeptin, and 1mM DTT) containing 5µM cold ATP, 10 µCi [γ-

P] ATP

(5000Ci/mmol, Amersham), and 1 µg GST-c-Jun fusion protein (Santa Cruz Biotechnology) as substrate, and incubated for 20 min at 25°C. Each sample was mixed with 8 µl of 5× Laemmli's loading buffer to stop the reaction, heated for 10 min at 100°C, and subjected to 8 % SDS-PAGE. The gels were dried, visualized by

autoradiography (24).

Statistical analysis

Results were expressed as mean ± S.E. for each study. Data were analyzed by Student's test and a p value of 0.05 or less was considered statistical1y significant.

Results

Effect of GBE on ethanol-induced gastr ic mucosal damage

All the rats that received 50 % ethanol (1ml) showed gastric mucosal damage.

GBE (8.75-26.25 mg/kg, i.v.) suppressed the formation of ethanol-induced gastric lesions including ulcer number (U No.) and the ulcer index (UI.) in a dose-dependent manner (Table 1). Normal saline did not have any protective effect and any gastric mucosal damage when given in the volume of 0.5~1.5 ml (i.v.) to rats. The maximal effect was obtained when GBE (26.25 mg/kg) was given 30 min prior to ethanol induction, resulting in a preventive ratio of 68.64% (p<0.05).

Effect of GBE on ethanol-induced gastr ic mucosal NP-SH and MDA contents Ethanol (50 %, 1 ml) treatment significantly reduced the NP-SH concentration in the gastric mucosa as compared with control. As shown in Table 2, GBE pretreatment significantly prevented the decrease in NP-SH concentrations in the dose of

8.75-26.25 mg/kg. However, GBE treatment alone was no change in the concentration of NP-SH in gastric mucosa. Moreover, pretreatment of GBE significantly prevented lipid peroxidation induced by ethanol, the MDA concentrations were 209.27 ± 10.48 or 118.82 ± 8.26 in the rat that treatment with ethanol alone or combined with GBE, respectively (Table 3).

Effect of GBE on ethanol-induced gastr ic wall mucus

There was a significant decrease in the gastric wall mucus after treatment with 1 ml, 50% ethanol. As shown in Table 4, pretreatment of GBE (8.75-26.25 mg/kg) significantly protected the decline in the gastric wall mucus levels of glandular gastric mucosa induced by ethanol.

Effect of GBE on pylor us ligation accumulated gastr ic acid secr etions and acidity GBE was i.v. administrated at dose of 8.75~26.25 mg/kg 15 min before pylorus ligation. Control rats received the same volume of saline (0.5~1.5 ml). As shown in Table 5, GBE slightly inhibited the gastric secretion and total acidity even at a dose of 26.25 mg/kg.

Effect of GBE on ethanol-induced J NK activity in gastr ic mucosa

Since JNK activation play an important role in the induction of cell apoptosis.

To explore whether the JNK signaling pathway is activated within gastric mucosa in

response to ethanol, we examined JNK activity by using an immunocomplex kinase

assay. As shown in Fig. 1, JNK activity was obviously increased after treatment with

ethanol (Top). However, GBE (26.25 mg/kg) significantly suppressed the induction of

JNK activity. Western blot analysis revealed that this JNK activation was not caused

by enhanced expression of JNK protein (bottom).

Discussion

The present study clearly demonstrated that GBE confers a dose-dependent protection against the gross damaging action of ethanol on gastric mucosa of rats.

Ethanol causes extensive ulceration in fasted animals. Such ulceration may be

attributed to stasis of gastric blood flow thereby leading to haemorrhage and necrosis.

In clinical therapy, GBE has been used to improve the rheologic properties of blood. It is possible that GBE appears to improve to mucosal blood flow and then decreased the gastric lesions in ethanol treatment of rats. Previous reports have also described that ulcerogenic process have relationship with the depletion of mucous and increase of gastric acid secretion. GBE has also found to inhibit the ethanol-induced changes in gastric wall mucus and pylorus ligation accumulated secretions.

Several studies have demonstrated that depletion of NP-SH concentrations and increase in contents of free radicals resulted in tissue injury and membrane damage (25, 26). In the present study, GBE pretreatment have inhibitory effect on

ethanol-induced depletion in the NP-SH concentrations and increase in the levels of MDA in gastric mucosa. Therefore, GBE blocked the loss of NP-SH and decreased the lipid peroxidation might have protection effect on mucosal tissue.

Other possible modes of action of protection by GBE may be due to its influence on the JNK activity, which result in the induction of cell apoptosis. Previous studies have reported that JNK kinase activity was elevated during the process of apoptosis and blocking of JNK activity was able to prevent cell apoptosis. In present study, ethanol significantly induced the JNK kinase activity in gastric mucosa and possibility further resulted in cell apoptosis and damage on mucosa. It appears that blocking of JNK activity may play an important role in the gastroprotection by GBE.

In conclusion, GBE has the ability to inhibit ethanol-induced gastric lesions and gastric secretion following pylorus-ligation. Thus the possible reasons of antiulcer effect of GBE may be due to: i) no loss of gastric mucus; ii) inhibition of lipid peroxidation; iii) blockade of JNK activity. Further studies are warranted to evaluate GBE at the pharmacologically effective dose before any consideration for clinical trials.

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Table 1. Effect of ginkgo biloba extract on the induction of gastric ulcers by 50%

ethanol in rats.

GBE, ginkgo biloba extract; EtOH, ethanol; U.No., ulcer number; U.I., ulcer index.

Each value repressed the mean ± S.E.

*Significantly different from vehicle with ethanol treatment (p<0.05).

Table 2. Effect of ginkgo biloba extract on the levels of NP-SH in the stomachs of rats treated by gavages with 50% ethanol.

Treatment and dose No. NP-SH concentrations (µg/100mg /wet tissue, mean ± SE)

Control (Normal saline) 5 12.21 ± 1.03

50% EtOH (1ml/rat) 5 2.82 ± 1.32

GBE (8.75 mg/kg) + 50% EtOH 5 11.32 ± 2.01

GBE (17.5 mg/kg) + 50% EtOH 5 5.66 ± 1. 82

GBE (26.25 mg/kg) + 50% EtOH 5 5.03 ± 1.25

GBE, ginkgo biloba extract; EtOH, ethanol; NP-SH, non-protein sulfhydryls.

*Significantly different from vehicle with ethanol treatment (p<0.05).

T Treatment and dose N No.

o.

Mucosal lesions P Preventive U.No. U.I.(mm

) ratio (%) Control (Normal saline) 5 5 0 0

50% EtOH (1ml/rat) 5 10 ± 4 20.31 ± 4.23

GBE (8.75 mg/kg) + 50% EtOH 5 8 ± 3 15.25 ± 3.21 24.91%

GBE (17.5 mg/kg) + 50% EtOH 5 3 ± 3

8.83 ± 3.59

56.52%

GBE (26.25 mg/kg) + 50% EtOH 5 2 ± 2

6.37 ± 4.22

68.64%

Table 3. Effect of ginkgo biloba extract on the lipid peroxidation in the stomachs of rats treated by gavages with 50% ethanol.

Treatment and dose No. Malondialdehyde concentration (nmol/g wet tissue, mean ± SE)

Control (Normal saline) 5 102.54 ± 15.32

50% Ethanol (1ml/rat) 5 209.27 ± 10.48

GBE (8.75 mg/kg) + 50% EtOH 5 212.32 ± 9.83

GBE (17.5 mg/kg) + 50% EtOH 5 125.48 ± 11.39

GBE (26.25 mg/kg) + 50% EtOH 5 118.82 ± 8.26

GBE, ginkgo biloba extract; EtOH, ethanol.

*Significantly different from vehicle with ethanol treatment (p<0.05).

Table 4. Effect of ginkgo biloba extract on the induction of changes in gastric wall mucus by gavages with 50% ethanol.

Treatment and dose No. Gastric wall mucus (µg Alcian blue /g wet tissue, mean ± SE)

Control (Normal saline) 5 442.21 ± 28.36

50% EtOH (1ml/rat) 5 356.87 ± 30.67

GBE (8.75 mg/kg) + 50% EtOH 5 348.87 ± 32.37

GBE (17.5 mg/kg) + 50% EtOH 5 395.27 ± 25.91

GBE (26.25 mg/kg) + 50% EtOH 5 410.82 ± 29.16

GBE, ginkgo biloba extract; EtOH, ethanol.

*Significantly different from vehicle with ethanol treatment (p<0.05).

Table 5. Effect of ginkgo biloba extract on the gastric secretions and acidity in pylorus ligation rats.

Treatment and dose No. Volume of gastric secretion (ml)

Titrable acidity mEq/liter

Control (Normal saline) 5 13.35 ± 0.98 63.82 ± 8.83

GBE (8.75 mg/kg) 5 12.67 ± 0.81 61.98 ± 7.34

GBE (17.5 mg/kg) 5 11.98 ± 0.53 58.14 ± 9.28

GBE (26.25 mg/kg) 5 10.78 ± 0.91 56.28 ± 8.91

GBE, ginkgo biloba extract; EtOH, ethanol.

Fig. 1.

Effect of GBE on ethanol-induced JNK activity in gastric mucosa of rats.

Rats were treated with various drugs as described in Materials and Methods. The gastric mucosa was homogenized and centrifuged, and then the supernatant was collected as total tissue lysate. Total tissue lysate (50 µg/lane) was separated on 10%

SDS-polyacrylamide gels and blotted with antibodies specific for JNK as described in

“Materials and Methods.”(Bottom). Total tissue lysate (400µg) were used for immunoprecipitation, the JNK kinase activity was assayed with Gst-c-Jun fusion protein as substrates. The experiment was performed as described in “Materials and Methods,” and

P-labeled Gst-c-Jun is shown. GBE, ginkgo biloba extract; EtOH, ethanol.

The End

JNK p-JNK

Cont EtOH

EtOH

+

GBE