JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY 2010, 61, 5, 581-591

www.jpp.krakow.pl

W. OPOKA1, D. ADAMEK2, M. PLONKA3,7, W. RECZYNSKI4, B. BAS4, D. DROZDOWICZ3,

P. JAGIELSKI5, Z. SLIWOWSKI3, P. ADAMSKI6, T. BRZOZOWSKI3

IMPORTANCE OF LUMINAL AND MUCOSAL ZINC IN THE MECHANISM

OF EXPERIMENTAL GASTRIC ULCER HEALING

Department of Inorganic and Analytical Chemistry, Faculty of Pharmacy, Jagiellonian University Medical College, Cracow, Poland;
1
2Department of Pathomorphology, Jagiellonian University Medical College, Cracow, Poland; 3Department of Physiology,

Jagiellonian University Medical College, Cracow, Poland; 4Department of Analytical Chemistry, Faculty of Material Science
and Ceramics, AGH University of Science and Technology, Cracow, Poland; 5Institute of Public Health, Faculty of Health Care
Faculty of Health Sciences, Jagiellonian University Medical College, Cracow, Poland; 6Institute of Nature Conservation Polish
Academy of Sciences, Cracow, Poland; 7Department of Physiotherapy, University School of Physical Education, Cracow, Poland

Zinc has been reported to exert a gastroprotective action against various experimental gastric lesions suggesting that this
trace element is involved in the integrity of the gastric mucosa. Compounds containing zinc, such as polaprezinc, were
developed in Japan and used as an antiulcer drugs in the treatment of human peptic ulcer disease. However, the precise
mechanism of Zn2+ containing compounds and their effects on mucosal integrity, gastroprotection and ulcer healing
remain unclear. We have determined the efficacy of zinc hydroaspartate, a compound containing Zn2+, in the mechanism
of gastric secretion and ulcer healing in rats with chronic gastric ulcers induced by acetic acid (initial ulcer area = 28 mm2).
Rats with gastric ulcers were randomized into two groups: A) with gastric fistulas (GF) and B) without gastric fistulas and
received a daily treatment with zinc hydroaspartate (32-130 mg/kg-d i.g.) for 3, 7 and 14 days. At the termination of each
treatment, the area of gastric ulcers were examined by planimetry, the gastric blood flow (GBF) at ulcer margin was
assessed by laser Doppler flowmetry and H2-gas clearance methods. The venous blood was withdrawn for a measurement
of plasma gastrin levels by radioimmunoassay (RIA). The concentration of Zn2+ in the gastric juice and mucosa at the
ulcer margin were determined by differential pulse anodic stripping voltammetry (DP ASV) and flame atomic absorption
spectrometry (FAAS) methods and the gastric biopsy samples were taken for histopathological assessment of the quality
of ulcer healing. The ulcers healed gradually, with the ulcer area in the vehicle control rats being diminished by 15%, 48%
and 78% upon ulcer induction at 3, 7 and 14 days, respectively. Zinc hydroaspartate dose-dependently inhibited the area
of gastric ulcer, the dose reducing this area by 50% (ID50) being about 60 mg/kg-d. The mucosal concentration of
Zn2+significantly was unchanged from the baseline immediately after ulcer induction (day 0) and at day 3 but then it rose
significantly at day 7 after ulcer induction. Treatment with zinc hydroaspartate (65 mg/kg-d i.g.), which significantly
raised the gastric luminal and mucosal levels of Zn2+, significantly accelerated ulcer healing at day 7 upon ulcer induction.
The GBF, which reached a significantly higher value at the ulcer margin than the ulcer bed, was significantly increased
in rats treated with zinc hydroaspartate compared with vehicle-controls. The gastric acid output was significantly inhibited
in GF rats with gastric ulcer at day 3 then restored at day 14 followed by a significant rise in the plasma gastrin levels.
Treatment with zinc hydroaspartate significantly inhibited gastric secretion and also significantly raised the plasma gastrin
level when compared to vehicle-control rats. We conclude that 1) trace micronutrients such as Zn2+ could be successfully
measured in the gastric juice and gastric mucosa during ulcer healing; 2) compounds chelating of Zn2+ can exert a
beneficial influence on the ulcer healing via Zn2+ mediated increase in gastric microcirculation, antisecretory activity and
gastrin release, which may enhance the cell proliferation and differentiation during ulcer healing, ultimately exerting a
trophic action on the ulcerated gastric mucosa.

K e y w o r d s : zinc, ulcer healing, ulcer margin, gastric acid secretion, gastric blood flow, gastric juice, gastrin

INTRODUCTION Both these enzymes are important components of cell antioxidant

enzyme activity. The mechanism of zinc-induced antioxidative
Zinc belongs to a class of microelements that are considered function remains unclear but this trace element slows down the
to play an important role in many vital biochemical reactions and oxidation processes and this may account for its potential
physiological processes, including the growth and development of antioxidant properties (5, 6). Recent evidence indicates that zinc
the cells (1, 2). This trace element has been also shown to may function as an indispensable element for optimal functioning
stimulate the gene transcription and cell proliferation and it is also of the human immune system (7, 8). Zinc compounds have long
responsible for activation of DNA and RNA polymerases (3, 4). been used in medicine as an antidepressant (9) and in various
582

forms of skin injury due to its efficacy in healing skin lesions and Institutional Animal Care and Use Committee of Jagiellonian
wounds (10, 11). University Medical College in Cracow and run in accordance to
The role of zinc in the mechanism of gastrointestinal integrity the statements of Helsinki Declaration regarding the handling of
has not been fully understood despite the fact that zinc has been experimental animals.
reported to have protective action against various experimental
gastric lesions (12, 13). Moreover, the clinical studies have Production of gastric ulcers
shown the antiulcer action of zinc in humans (14, 15). Previous
studies revealed that zinc acts as the essential element in the Gastric ulcers were produced in rats using our modification
physiology of the digestive system, accelerated the process of (29) of acetic acid method originally proposed by Okabe et al.
wound healing of various types of tissues including gastric ulcer (30). Animals were anesthetized with pentobarbital natrium, the
in experimental animals and in humans (16-18). abdomen was opened and the stomach was exposed. The volume
Furthermore, Helicobacter pylori (H. pylori)-infected of 75 µl of acetic acid was poured through the plastic mold (6
patients receiving standard anti-H. pylori therapy with mm diameter) onto the serosal surface of the anterior wall of the
amoxicillin, metronidazole, omeprazole and bismuth supported stomach just proximal to the antral gland area for 25 s. This
with capsules containing 220 mg of zinc sulphate (VI) every produced an immediate necrosis of the entire mucosa and
second day, exhibited a reduction in the ulcers size, which may submucosa (but not serosa) within the area where the acetic acid
reflect an acceleration of ulcer healing, that can be confirmed by was applied, i.e., about 28 mm2. The excess of acetic acid was
endoscopy (19). It is known that the development of the gastro- then removed and the serosa was gently washed out with saline.
duodenal ulcers may be influenced by many factors such as: H. Our previous studies documented that these ulcers became
pylori infection, longstanding stress and nonsteroidal anti- chronic within 2-3 days and healed completely within 2-3 weeks
inflammatory drugs (NSAID) treatment, excessive gastric without perforation or penetration to the surrounding organs as
secretion, alcohol, cigarette smoking or irritating constituents of described in our original technique (29, 31). After the application
the diet that lead to gastric or duodenal mucosal damage, always of acetic acid the animals were allowed to recover from
accompanied by inflammation (20). Zinc may be helpful both in anesthesia and received only water on the day of the operation
casual treatment due to its versatile activity and in palliative (day 0). Then, they were divided into various groups and
treatment facilitating damaged tissue regeneration. Zinc is a received normal chow and water ad libitum for the next 3, 7 and
component of about 300 enzymes that ensure correct cell 14 days, respectively.
metabolism (21). It has been shown that nutrients including zinc
have a positive impact on treatment of diseases such as celiac Effect of zinc hydroaspartate (HZnAsp) on gastric acid
disease, HIV/AIDS and inflammatory bowel disease (21). secretion, the rate of ulcer healing and gastric blood flow
The compound N-(3-aminopropionyl)-L-histidinato zinc in the ulcer area
(polaprezinc), a chelate of zinc and L-carnosine, is an antiulcer
agent developed in Japan (22). Polaprezinc was originally Three major series (A, B and C) of experiments were carried
designed to combine the beneficial effects of zinc with carnosine out in rats with chronic gastric ulcers. Series A was used to
but the question remains whether zinc or L-carnosine is the active determine the effects of twice daily intragastric (i.g.)
therapeutic ingredient of polaprenzinc. It is of interest that administration of vehicle (saline) or various doses of HZnAsp
carnosine itself increased the formation of granulation tissue and (Farmapol, Poland) ranging from 16.25 mg/kg-d up to 130
accelerated gastric ulcer healing in rats. Several reports have mg/kg-d. The chemical structure of HZnAsp bis[2-(amino-
shown the protective action of polaprezinc against experimental κN)aspartato(2-)-κO 1]zincate(2-) is presented in Fig. 1. In rats
gastric lesions induced by various noxious agents (23-25) and that of series B, the effects of HZnAsp (65 mg/kg-d i.g.) on gastric
polaprezinc accelerates gastric ulcer healing in humans (26). secretion was determined in separate group of 12 rats that were
Although the mechanism of gastroprotective and antiulcer actions equipped with gastric fistula (GF) in whom gastric ulcers were
of polaprezinc has not fully explained an increase in mucosal induced 1 month prior to the ulcer induction (31). Series C
secretions, the generation of endogenous prostaglandins (PGs), received saline (i.g. or i.p.) and served as control. HZnAsp was
the membrane-stabilizing effect, and antioxidant properties (23, dissolved in saline and given at graded doses or administered
26) were proposed to contribute to its beneficial effect. twice daily in the dose of 65 mg/kg-day i.g. that was selected
Our present study was designed to determine the role of zinc in based on the initial experiment with dose-dependent effect of
the process of gastric ulcer healing by monitoring the alterations in this compound. This dose of HZnAsp was administered to rats
the size of gastric ulcers during healing and the accompanying
changes in gastric blood flow (GBF) at the ulcer margin, ulcer bed
and non-ulcerated gastric mucosa. We measured the luminal as
well as the mucosal concentration of zinc in gastric juice and
mucosa by means of anodic stripping voltammetry (ASV) (27) and
flame atomic absorption spectroscopy (F-AAS) (28) methods.
Furthermore, we evaluated by histology the quality of healing of
the acetic acid ulcers without and with zinc administered in the
form of zinc hydroaspartate. In a separate group of conscious rats
equipped with gastric fistulas, the gastric acid secretion and plasma
gastrin were determined in order to assess the effect of zinc
hydroaspartate on these functional parameters during ulcer healing.

MATERIAL AND METHODS

Male Wistar rats, which weighed between 200-250 g and

fasted for 24 hours were used for gastric secretory studies and Fig. 1. The chemical structure of zinc hydroaspartate (HZnAsp),
the induction of gastric ulcers. This study was approved by the a compound containing zinc (Zn2+) ion.
 583

with gastric acetic acid ulcer throughout the period of 3, 7 and After the respective days from the onset of ulcer induction,
14 days. the control animals without ulcers and those with gastric ulcer
To evaluate the effects of HZnAsp administered were placed in special Bollman cages and the gastric fistulae
exogenously on the gastric blood flow (GBF), the animals were were opened in order to collect gastric juice. The gastric secretion
anesthetized with ether and the abdomen was opened with the was collected into calibrated tubes during six, thirty-minute
stomach exposed to assess the GBF at the ulcer margin, ulcer periods. In each thirty-minute fraction the volume of gastric juice
crater and in the contra-lateral intact mucosa using H2-gas in the vehicle-control animals and in those treated with HZnAsp
clearance technique or laser flowmetry (Model BPM 403A, were noted and the concentration of H+ in each sample was
blood perfusion monitor, Vasamedics Inc. St. Paul Minn., USA) measured by titration of the gastric juice with 0.1 N NaOH to
(32, 33). The gastric blood perfusion was measured by laser calculate gastric acid output. In addition, the Zn2+ ions have been
Doppler technique and expressed in ml/min/100 g of gastric determined by means of F-AAS method. The samples of the juice
tissue. The GBF was examined in three places: at the bottom of were thoroughly mixed before analysis due to their
an ulcer, at the ulcer margin and in the area not affected by ulcer. inhomogenity. The concentration of Zn2+ was measured using
The stomach was then removed and pinned open for the Perkin Elmer spectrometer Model 3110 (USA) in an air-acetylene
determination of the area of gastric ulcers by planimetry flame under standard conditions (wavelength 213.9 nm; slit 0.7
(Morphomat, Carl Zeiss, Berlin, German) by two investigators mm). In some experiments the gastric biopsy was excised,
under blinded conditions. Half of the stomach with gastric ulcer nitrogen shock frozen in Eppendorf tubes and kept in -80°C until
in rats with or without administration of vehicle (control) or the measurement of Zn2+ concentration in the gastric mucosa.
HZnAsp was taken during autopsy and immediately fixed in
10% formalin, embedded in paraffin and stained with Measurement of Zn2+ concentrations by the differential pulse
hematoxylin and eosin and alcian blue/periodic acid Schiff (AB- anodic stripping voltammetry (DP-ASV) procedure
PAS) methods for the histological assessment of the quality of
the ulcer healing. Coded specimens of mucosa stained with An electrochemical analyzer M161 (MTM-ANKO, Poland)
hematoxylin and eosin were evaluated at 260x magnification was used in this study as described before (27, 37). The classical
under blinded conditions. three-electrode quartz cell, volume 10 mL, consisting of a
control growth mercury drop electrode (CGMDE) type M164
Determination of plasma gastrin levels (MTM-ANKO, Poland) as a working electrode, used in the
hanging mercury drop electrode (HMDE) mode, Pt wire as the
At the termination of some experiments with exogenously auxiliary electrode, and a Ag/AgCl/3M KCl as the reference
administered vehicle or HZnAsp, the blood samples (about 3 ml) electrode. The pH was measurements were performed with
were taken from the vena cava (into tubes containing 2500 U laboratory pH-meter. All solutions used for analyses were
Trasylol, Bayer, FRG and 0.5 mg/ml of EDTA). For comparison, purged with argon. Experiments were carried out at room
intact rats fasted overnight and given only vehicle saline i.g., were temperature. The MTM-ANKO EAGRAPH software enabled
also anaesthetized with ether and the blood samples were collected electrochemical measurements, data acquisition and advanced
for the determination of control values of gastrin in the plasma. The processing of the results (38-40).
blood samples were stored at -20°C until radioimmunoassay (RIA) All solutions and the sample preparation were realized with
of gastrin using gastrin antiserum 4562 (kindly donated by quadrupled distilled water (last two stages from quartz). HNO3
Professor J.E. Rehfeld of Aarhus, Denmark) described in detail 65%, H2O2 30% and KNO3 (Merck, Suprapur®) were used for
previously (34, 35). The anti-gastrin antibody recognized gastrin- the preparation of samples and supporting electrolyte. Also
17 and gastrin-34 equally well. The RIA system for gastrin was Zn(II) standard stock solution (1000 mg·l–1, Merck) was applied.
sufficiently sensitive to detect approximately 2.5 pM/L plasma About 50 mg of dried sample of the gastric mucosa was weighed
equivalent to human gastrin-17 as described before (36). and transferred into a high pressure PTFE container and treated
with 4 ml of HNO3 and 2 ml of H2O2 (30%). The container was
Determination of gastric acid secretion and the luminal then placed into a microwave oven (Microwave digestion system
and mucosal concentration of Zn2+ in rats treated with vehicle Anton Paar Multiwave 3000). Digestion of the samples was
or HZnAsp carried out with the following program: 20 min under
microwave irradiation, a 45 min cooling time, and a 5 min
The alterations of gastric secretion during ulcer healing in waiting time. Digested samples were placed on a heated plate in
rats treated with vehicle (saline) and HZnAsp were tested in a order to evaporate the excess of reagents. The sample solutions
separate group of 30 starved rats with acetic acid ulcers, were cooled to room temperature and transferred quantitatively
surgically equipped with chronic gastric fistulas (GF). Control into volumetric flasks (5 ml) and filled up to the mark with four
sham-operated rats with GF were also included, but instead of times distilled water. Glassware was first immersed in 6 M nitric
acetic acid, 70 µl of saline was applied to the serosal surface of acid, and then rinsed repeatedly with distilled water.
the stomach for 20 s. Vehicle or HZnAsp (65 mg/kg) was applied The stripping was performed in the differential pulse (DP)
daily in a dose of 65 mg/kg i.g., to GF animals in a manner mode. The analysis was performed in a 5 mL aliquot containing
similar to that described above. After recovery from anesthesia 0.01 M KNO3 and 20-200 µL of a sample solution. The ASV
(day 0) or at day 1 to 3, and at day 7 and 14 after ulcer induction, procedure was performed with the following steps in an
GF rats with and those without gastric ulcers were placed in uninterrupted sequence: (a) a new drop generation for HMDE
individual Bollman cages to prevent coprophagy and to maintain electrode; (b) the pre-concentration step: Eacc=-1.15 V; 15 s „tacc”
the necessary restraint. Each GF was then opened, and the 60 s; (c) after a rest period of 5 s, a DP voltammogram was
stomach rinsed gently with 5-8 ml of tap water at 37°C. Basal recorded. Conditions for the DP mode were as follows: pulse
gastric secretion was collected for 120 min, during which time amplitude, 30 mV; potential step, 2 mV and 20 ms; potential
the rats received saline at a rate of 4 ml/h subcutaneously (s.c.). range, from -1.15 V to -0.75 V.
The gastric juice was collected every 30 min, the volume was Quantitative determination was performed using the
measured, and then the acid concentrations and output were standard addition method (27, 36) The voltammogram for the
determined and expressed as the output per 30 min as described blank solution demonstrated the electrochemical cell and
previously (34-36). supported the electrolyte purity.
584

Statistical analysis produced a significant rise in the GBF and this increase was not
significantly different from that obtained with HZnAsp given at the
All results are presented as mean ±SEM. Statistical analysis dose of 65 mg/kg-d (Fig. 3).
was conducted with the following methods; comparison between The time-course of gastric ulcer healing in rats treated with
two groups were done with t-Student’s test for unequal vehicle or the standard dose of HZnAsp (65 mg/kg-d i.g.)
variances, comparison between more than two group were done starting from the initial size recorded at day 0 to day 3, day 7 and
with the ANOVA, if the results shows statistical significance the day 14 is shown in Fig. 4. The acetic acid gastric ulcers healed
post-hoc Dunnett’s test. If the distribution of the data progressively in vehicle-control rats and the area of gastric ulcer
significantly differed from the normal distribution, ANOVA was was significantly reduced within 14 days after ulcer induction
replaced by nonparametric Kruscal Wallis test. Statistical but still about 65% of rats had the ulcer healing not completed.
analyses were carried out by using a computer program (SPSS At day 7 and day 14 upon the ulcer induction, the area of gastric
for Windows). All statistical analysis have been proceed with ulcers gradually decreased in vehicle-treated control rats by
SAS JMP 7.02 package (license nr GSE8SNJ0JT). Some results about 58% and 80%, respectively. In contrast, the area of ulcers
are presented by the box plots including mean lines, standard in rats treated with HZnAsp remained significantly smaller when
error boxes and standard deviation wickers. compared to the respective values in the vehicle-treated control
rats (Fig. 4). As shown in Fig. 5, the area of gastric ulceration
was significantly decreased in animals treated with HZnAsp at
RESULTS day 7 upon ulcer induction when compared to that measured in
the vehicle-control rats.
Effect of vehicle and HZnAsp on gastric ulcer healing and the
GBF at ulcer margin

Fig. 2 shows the macroscopic appearance of the acetic acid

gastric ulcer at day 7 upon ulcer induction in an animal treated
with vehicle (saline). The deep ulcer crater with clearly defined
ulcer margin and the ulcer bed is visible. The results of the 7 days
administration of vehicle or HZnAsp applied i.g. in graded doses
ranging from 16.25 mg/kg-d up to 130 mg/kg-d on the area of
gastric ulcers and the accompanying changes in GBF at the ulcer
margin were presented in Fig. 3. In rats treated with vehicle
throughout the period of 7 days, a significant reduction in the area
of these ulcers was observed from initial size 28 mm2 to 18.6±2.1
mm2. HZnAsp given in a dose 16.25 mg/kg-d i.g. failed to affect
significantly the area of gastric ulcers as compared to that
obtained in vehicle-control animals. With increasing the dose of
HZnAsp up to 32.5, 65 mg/kg and 130 mg/kg-d, a significant
decrease in the area of gastric ulcers was observed, by about 14%,
34%, 59% and 82%, respectively, as compared with that recorded
in the vehicle-treated animals (Fig. 3).
The GBF in the non-ulcerated mucosa of rats treated with
vehicle averaged 46±6 ml/min-100 g (taken as 100%). HZnAsp
applied i.g. in a dose of 16.25 mg/kg failed to affect the GBF at the
ulcer margin but a significant increase in the GBF at the ulcer
margin was recorded in the gastric mucosa of rats treated with Fig. 2. Gross appearance of the gastric ulcer induced in the rat
HZnAsp applied i.g. at higher doses ranging from 32.5 up to 130 stomach by the serosal application of acetic acid (for details, see
mg/kg-d (Fig. 3). HZnAsp administered at a dose of 130 mg/kg-d, Material and Methods).

Fig. 3. The area of gastric ulcers and

gastric blood flow (GBF) at ulcer
margin at day 7 in rats treated with
vehicle (Veh) and zinc hydroaspartate
applied i.g. in graded doses ranging
from 16.25-130 mg/kg-d i.g. Mean
±SEM of 6-8 rats. Asterisk indicates a
significant change as compared to the
value obtained in vehicle-treated gastric
mucosa at day 7 upon ulcer induction.
 585

Fig. 4. Mean area of gastric ulcers in rats

treated with vehicle (Veh, saline) and
HZnAsp (65 mg/kg-d i.g.) and
determined at day 0, 3, 7 and 14 upon
ulcer induction. Mean ±SEM of 6-8 rats.
Asterisk indicates a significant change
as compared to the value obtained at day
0 and day 3. Cross indicates significant
decrease below the values obtained in
vehicle-treated animals.

26

22
Ulcer area (mm2)

18

14

10
*
6 Fig. 5. The changes in the area of gastric
ulcers at day 7 upon ulcer induction in
rats treated daily with vehicle (saline)
2
and HZnAsp (65 mg/kg-d i.g.). Mean
Vehicle HZnAsp
±SEM of 8-10 rats. Asterisk indicates a
significant change as compared to the
mean ± SEM ± SD value obtained in vehicle-treated gastric
mucosa.

By histology, the non-ulcerated gastric mucosa showed the Fig. 8 shows the values in GBF in the intact gastric mucosa
normal gland architecture and an intact continuity of the surface and for comparison those recorded at the ulcer margin and the
epithelium and this histological appearance of gastric mucosa ulcer bed in rats treated for 7 days with vehicle or HZnAsp.
was similar in animals without ulcer induction who received i.g. The GBF in the non-ulcerated gastric mucosa was not
treatment with HZnAsp (Figs. 6A and 6B). At day 7, only 15% of significantly affected by the treatment with HZnAsp. In
control vehicle-treated rats showed almost complete healing of contrast, the GBF at the ulcer margin and ulcer bed reached
gastric ulcer as reflected by the remnant ulcer crater, but in those significantly lower values when compared to that in the non-
with macroscopically healed ulcer, microscopically the ulcer scar ulcerated gastric mucosa, though the GBF was significantly
exhibited marked gland dilation and an incomplete reconstruction increased at the ulcer margin than that in the ulcer bed. The
of mucosal cells at ulcer margin (Fig. 6C). In contrast, in the GBF at the ulcer margin and the ulcer bed was significantly
majority of rats treated with HZnAsp, the gastric ulcers were not higher in rats treated with HZnAsp when compared to the
observed both macroscopically or microscopically and the respective values of GBF at the ulcer margin and the ulcer bed
healing zone at the ulcer margin showed well developed recorded in vehicle-controls (Fig. 8).
restoration of surface epithelium, however the “healed” gastric
mucosa confirmed abnormal glandular appearance (Fig. 6D). Effect of HZnAsp on gastric acid secretion and plasma gastrin
The GBF at the ulcer margin was significantly decreased in during ulcer healing
the vehicle-control rats when compared to that in the intact non-
ulcerated gastric mucosa (Fig. 7). Administration of HZnAsp The results of the gastric secretory studies in conscious rats
significantly increased the GBF at the ulcer margin when equipped with gastric fistula with or without induction of
compared to the respective values of GBF at the ulcer margin gastric ulcers are presented in Table 1 and Fig. 9. In control rats
recorded in the vehicle-control animals (Fig. 7). without gastric ulcers, the basal acid output averaged 137±12
586

Fig. 6A-D. Histological appearance of

the intact gastric mucosa without
gastric ulcer. A: in rat treated with
vehicle for 7 days, the surface
epithelium is continued and the gastric
glands show normal architecture. B:
the non-ulcerated gastric mucosa of
rat treated with HZnAsp (65mg/kg-d
i.g.) shows the normal appearance not
significantly different from that
treated with vehicle. C: gastric ulcer in
rat treated with vehicle (control) at day
7 after ulcer induction. Note, that ulcer
crater is clearly visible, the healing
zone is poorly developed and non-
healed ulcer consists of inflammatory
cell reach exudate. D: gastric ulcer in
rat treated with HZnAsp at day 7 after
ulcer induction. Comparing to
vehicle-control rat, the gastric ulcer is
almost completely healed but the ulcer
scar is filled with poorly differentiated
epithelium forming dilated irregular
glands. So called "healed mucosa"
reflecting advanced healing process
still shows abnormal appearance.
H&E, magnification x260.

50
Gastric blood flow at ulcer margin

48
(ml/min/100 g tissue)

46

44
*+
42 Fig. 7. The gastric blood flow (GBF) in
gastric mucosa of intact rats and those
40
* with acetic acid induced gastric ulcer
treated throughout the period of 7 days
with vehicle (saline) or HZnAsp (65
38 mg/kg-d i.g.). Mean ±SEM of 6-8 rats.
Intact Vehicle HZnAsp Asterisk indicates a significant change
with gastric ulcer as compared to the value obtained in
intact animals. Asterisk and cross
indicate a significant change as
mean ± SEM ± SD compared to the value obtained in
vehicle-control rats.

µmol/30 min and plasma gastrin level reached the value of also significantly increased the plasma gastrin concentration
38±3 pM/L. Immediately after induction of gastric ulcers, the comparing to the values obtained in animals treated with
gastric acid output was significantly reduced by about 55% and vehicle. After 7 days, the values of gastric acid output in
the plasma gastrin level was significantly increased by about vehicle-control rats were still significantly lower when
15% as compared with the respective values in control rats compared to those obtained in intact rats without ulcer (Fig. 9).
without ulcer induction. Treatment with HZnAsp (65 mg/kg-d In rats treated with HZnAsp, a significant decrease in the
i.g.) resulted in a further significant decrease in gastric acid gastric acid output was still observed on day 7 when compared
outputs and the significant rise in plasma gastrin as compared to respective vehicle-controls (Table 1, Fig. 9). On day 14, the
to those treated with vehicle (saline) (Table 1). On day 3, the gastric acid outputs in the vehicle-treated animals reached the
gastric secretion tended to increase but still significant value similar to those attained in rats without ulcer induction
inhibition of gastric acid output was observed in vehicle- but, in contrast, the decrease in the gastric acid output was still
treated animals when compared to those in rats without gastric observed in HZnAsp-treated rats when compared to those
ulcers. HZnAsp significantly reduced gastric acid output and treated with vehicle (Table 1).
 587

50

(ml/min/100 g tissue) 48
Gastric blood flow

*
46

44

42

40
*
Fig. 8. The alterations in gastric blood
flow (GBF) in non-ulcerated gastric
38
mucosa, at ulcer margin and ulcer bed
Non-ulcerated Ulcer margin Ulcer bed in rats treated throughout the period of
7 days with vehicle (saline) or HZnAsp
(65 mg/kg-d i.g.). Mean ±SEM of 6-8
mean ± SEM ± SD
rats. Asterisk indicates a significant
change as compared to the value
Vehicle HZnAsp
obtained in vehicle-treated animals.

Fig. 9. The gastric acid output in rats

without or with gastric ulcer equipped
with gastric fistulas and treated with
vehicle (saline) and HZnAsp (65
mg/kg-d i.g. for 7 days). Mean ±SEM
of 6-8 rats. Asterisk indicates a
significant change as compared to the
value obtained in vehicle-treated
animals. Cross indicates a significant
change as compared to the value
obtained in rats without gastric ulcer.
Asterisk and cross indicate a significant
change as compared to the value
obtained in rats with gastric ulcer
without HZnAsp treatment.

Effect of vehicle or HZnAsp administration on the concentration

of Zn2+ in gastric juice and gastric mucosa in rats without or DISCUSSION
with gastric ulcer
This study demonstrated that certain trace microelements,
Fig. 10 shows the time sequence of the effect of vehicle or such as Zn2+ ion could be important for the gastric ulcer healing
HZnAsp administration on the luminal Zn2+ content in intact rats process. The supplementation of the stomach with
and those treated with vehicle of HZnAsp (65 mg/kg-d i.g.) as experimentally induced gastric ulceration with zinc-containing
determined on day 1, day 3 and day 7 upon ulcer induction. Fig. 11 compound HZnAsp, which increased the Zn2+ content in the
shows the effect of 7 days administration of vehicle and HZnAsp ulcer area and its luminal concentration in gastric juice,
on the concentration of Zn2+ in the gastric mucosa at ulcer margin. markedly accelerated the ulcer healing and enhanced the gastric
The luminal Zn2+ contents were similar in intact non-ulcerated microcirculation around the ulcer. One of the major finding of
gastric mucosa and in vehicle-control rats with gastric ulcer and our present study is the direct quantitative evidence for Zn2+
those treated with HZnAsp on day 1 and day 3 but in contrast, at deficiency in the ulcerated gastric mucosa, suggesting that the
day 7 the significant rise in the gastric luminal Zn2+ contents was endogenous Zn2+ content plays an important role in the process
observed when compared to the respective values measured in the of ulcer healing with respect to zinc supplementation treatment
vehicle-control rats. In rats with gastric ulcer, a significant decrease modalities in peptic ulcer disease. The importance of this finding
in the luminal Zn2+ ion concentration on day 7 was recorded in rats is supported by the fact that this depletion of Zn2+ content in the
with gastric ulcer comparing to respective controls without gastric gastric mucosa at the ulcer margin was abolished in animals
ulcer (Fig. 10). In contrast, the treatment with HZnAsp supplemented with HZnAsp.
significantly increased the luminal and mucosal Zn2+ contents at Our present study demonstrates that exogenous
day 7 upon ulcer induction (Fig. 10 and 11). Zn2+administered in the form of HZnAsp, dose-dependently
588

accelerated the healing of chronic gastric ulcers and that this ulcer both the ulcer area and in gastric juice. This information could
healing action was accompanied by the rise in gastric blood flow significantly influence the natural course of ulcer healing. The
at the ulcer margin, the increase in luminal and mucosal mechanism of the Zn2+ -induced gastric healing process is not fully
Zn2+content and the enhancement in plasma gastrin levels. The explained in this study and further studies are definitely needed to
importance of endogenous Zn2+ in the healing of gastric ulcer is determine whether potent gastroprotective and ulcer healing
emphasized by the fact that the Zn2+ concentration at the ulcer mediators such as endogenous PG and NO could be involved in
margin was diminished during ulcer healing and this effect was the ulcer healing action of zinc-containing compounds.
abolished by the supplementation with HZnAsp of animals with Our study showed for the first time that the ulcer healing
gastric ulcer. It is of interest to note that the luminal Zn2+ effects of Zn2+ were accompanied by a notable increase in the
concentration at the ulcer margin was already significantly plasma gastrin levels indicating that endogenous gastrin may not
decreased during the first days of ulcer induction. This fact only contribute to the to the spontaneous process of ulcer healing
suggests that the early fall in the concentration of this as originally proposed (41) but could participate in the
microelement resulted in the depletion of the ion concentration in acceleration of this healing by zinc-containing compounds. The
increase in plasma gastrin observed in rats treated with HZnAsp
could contribute to the acceleration of ulcer healing by zinc,
Table 1. Effect of seven days administration of vehicle (saline) because the prolonged hypergastrinemia induced by the
and HZnAsp (65 mg/kg-d i.g.) on gastric acid secretion in rats administration of exogenous gastrin-17 and cholecystokinin
with gastric ulcers equipped with gastric fistula. Results are (CCK) throughout the period of 5-15 days, increased cell
means ±SEM of 10-12 rats. Asterisk indicates a significant proliferation at the ulcer margin and accelerated healing of the
change as compared with the value obtained in intact animals. acetic acid ulcers (36, 40). The mechanism of this increase in
Cross indicates a significant increase as compared to the value plasma gastrin increments in HZnAsp-treated rats is not fully
obtained in vehicle control animals at respective days upon ulcer understood but could be attributed to the inhibitory action of
induction. Asterisk and cross indicate a significant change as HZnAsp on gastric acid secretion as demonstrated in our present
compared to the value obtained in vehicle control animals at study in rats with gastric ulcer that were surgically equipped
respective days upon ulcer induction. with gastric fistulas. Interestingly, the treatment with HZnAsp
inhibited not only basal gastric acid secretion in animals without
Type of test Gastric acid output Plasma gastrin
gastric ulcer, but also found to be inhibited in rats with gastric
(µmol/30 min) (pM/L) ulcer induction using the acetic acid technique. Furthermore, we
Intact (Control) 137 ± 12 38 ± 3 revealed, for the first time, that plasma gastrin level was
Day 0 significantly elevated in animals with chronic gastric ulcers
treated with this zinc-containing formulation. This suggest that
Vehicle 56 ± 8* 41 ± 2 zinc may contribute to the ulcer healing action via an increase in
HZnAsp 51 ± 2* + 46 ± 2*+ the plasma level of gastrin, possibly due to its inhibitory effect
Day 3 on gastric secretion. This notion is supported by our previous
Vehicle 65 ± 6* 48 ± 5* evidence (31) and present observations that gastric secretion was
suppressed during the initial phase of ulcer healing and that
HZnAsp 45 ± 5* + 68 ± 6*+ following the spontaneous healing of ulcers in the vehicle-
Day 7 controlled animals, the gastric secretion returned progressively
Vehicle 88 ± 7* 54± 6* to the level recorded in intact animals. In contrast, treatment with
HZnAsp resulted in greater inhibition of gastric secretion on day
HZnAsp 53 ± 4* + 76 ± 7+
7 upon ulcer induction when compared to that in vehicle-treated
Day 14 animals with this inhibition persisting up to the end of the study
Vehicle 135 ± 9 58 ± 9 period which was on day 14, with the values still being
HZnAsp 78 ± 8* + 68 ± 6*+

Fig. 10. The changes in the gastric

luminal concentration of Zn2+ assessed
by DP-ASV method in gastric juice
collected from intact rats and those with
gastric ulcer treated for 7 days with
vehicle (saline) and HZnAsp (65
mg/kg-d i.g.). Mean ±SEM of 8-10 rats.
Asterisk indicates significant change as
compared to the value obtained in intact
rats and those with gastric ulcer treated
with vehicle. Asterisk and cross indicate
a significant change as compared to the
values recorded at day 3 in rats with
gastric ulcer treated with HZnAsp.
 589

Fig. 11. The changes in the gastric

mucosal concentration of Zn2+ assessed
by DP-ASV method in gastric mucosa
of intact rats and those with gastric
ulcer treated for 7 days with vehicle
(saline) and HZnAsp (65 mg/kg-d i.g.).
Mean ±SEM of 8-10 rats. Asterisk
indicates significant change as
compared to the value obtained in intact
rats. Cross indicates a significant
change as compared to the values
recorded in rats without gastric ulcer.

significantly lower than that measured in vehicle treated suppression of NF-κB-mediated inflammation (46). These
animals. Thus, this increase in the plasma gastrin observed in findings indicate that Zn(II)-curcumin complex prevents
HZnAsp-treated animals could be secondary to the decrease in pylorus-ligation-induced damage by a mechanisms, which
luminal acidity, due to the reduction in acid secretion caused by involves the reduction of NF-κB activation and subsequent
this compound. The importance of gastrin in the mechanism of production of pro-inflammatory cytokines (46). Previous studies
ulcer healing was recently emphasized by the fact that transient revealed that muco-protective zinc-containing compounds such
hypergastrinemia that caused a trophic response in the gastric as polaprenzinc exhibit a potent reactive oxygen species (ROS)
mucosa, accelerated healing of gastric ulcers induced by acetic quenching effects (47). These compounds exerted various other
acid and that this acceleration was reversed by administration of pharmacological actions, such as induction of the expression of
anti-gastrin antibodies (41). It is also possible that zinc- certain HSP proteins including HSP-27, HSP-72 and HO-1 (47).
containing compounds could influence directly the G-cells to Both, HSP and HO-1 were shown to display cytoprotective and
stimulate production of gastrin, but this hypothesis needs to be anti-inflammatory effects via the inhibition of adhesion
further explored. molecules on polymorphonuclear leukocytes, or they were noted
Zinc complexes were originally shown to have antiulcer to inhibit proinflammatory cytokine production by the gastric
activity. Zinc-carnosine is an antiulcer drug commonly used in epithelial cells. Polaprenzinc significantly inhibited the
the treatment of gastric ulcers in Japan (22, 23). Of note, the indomethacin-induced apoptosis of RIE-1 cells and the zinc
zinc-indomethacin and the zinc-naproxen complexes more component, rather than L-carnosine, contributed to the inhibition
significantly attenuated these ulcerogenic effects, when of the indomethacin-induced apoptosis (47). The gastro-
compared with the parent NSAID, without affecting their protective and ulcer healing properties of zinc is supported by
therapeutic action (42, 43). Mucosal protection by zinc- scientific evidence suggesting that zinc inhibited apoptotic
containing drug, polaprenzinc was attributed to the stimulation proteins such as caspase-1 and caspase-3 signaling, mainly via
of mucus production (24), antioxidant activity (22), membrane- ROS reduction in an ethanol-induced HepG2 cell (a human
stabilizing action (13), and the induction of heat shock protein hepatocellular liver carcinoma cell line) injury model, and also
(HSP) 70 (44). Besides HSP70, involvement of HSP32, also in RIE-1 cells (47).
regarded as heme oxygenase (HO)-1, the potent protective factor Zinc could inhibit oxidative stress via protection of cellular
against gastric damage and gastrointestinal disorders was membranes and macromolecules, by its stabilizing effects of
reported (43). HO-1 is believed to act as the inducible isoform of lipids and proteins, and preservation of sulfhydryl groups for
HO, which catalyzes the first and rate-limiting step in heme proteins, preventing oxidation by forming strong thiolate
degradation to produce equimolar quantities of biliverdin, complexes. Moreover, these protective and anti-apoptotic
carbon monoxide (CO), and free iron. Biliverdin is subsequently actions of zinc were shown to be mediated by the smac/DIABLO
converted to bilirubin via the action of biliverdin reductase, and signaling in indomethacin-induced apoptosis of RIE-1 cells (47).
free iron is promptly sequestered into ferritin. CO and other Our study concurs with these past reports by demonstrating
toxic agents at low concentrations exert distinctly different that intragastric supplementation with zinc in animals with
effects on physiological and cellular functions; however, the gastric ulcers led to a considerable enhancement in the mucosal
major effect is vasodilatation. Besides vasodilatation CO leads and luminal Zn2+content, ultimately leading to the acceleration of
to an inhibition of platelet aggregation (45). Interestingly, the ulcer healing. Furthermore, we found that the healing effect of
addition of Zn (II) to curcumin resulted in the protection against zinc hydroaspartate involves hyperemia at the ulcer margin, and
gastric lesions induced by pylorus ligation by suppressing the this microcirculatory effects could be attributed to zinc. However,
expression of mRNA for NF-κB, TGF-1 and IL-8 (46). It was the hypothesis that zinc induced an increase in the gastric blood
concluded that the antiulcer effect of zinc preparation against flow at the ulcer area and in the ulcer bed which could be
experimental pylorus-induced gastric lesions might be mediated by others vasoactive mediators such as NO originating
attributable to its ability to inhibit gastric acid secretion, and to from the vascular endothelium, gastric epithelium or from the
enhance the mucosal defense mechanism through the capsaicin-sensitive nerve endings could not be ruled out.
590

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