Gastric ulcers are considered a modern epidemic and reportedly affect at least 10% of the world population [
The gastric mucosal defense involves secretion of luminal factors such as mucus and bicarbonate, epithelial barriers, and continuous blood flow through mucosal microvessels. These factors are modulated by secondary components of the defense system including prostaglandins (PGs) and nitric oxide (NO) [
Therefore, the aim of this study was to investigate the effects of
GC-7101 is the standardized extract of the bud of
Male Sprague-Dawley rats weighing 180–200 g (Orient Bio, Inc., Gapyeong, Korea) were housed in standardized conditions at
Gastric ulcers were induced by HCl/EtOH treatment according to previous reports [ inhibition (%) = (1 − lesion index of test animal/lesion index of vehicle-treated HCl/EtOH animal) × 100.
Gastric ulcers were induced by indomethacin treatment according to previous reports [
Rats were fasted for 24 h before experiments but given free access to tap water. Fasted rats (
Gastric ulcers were induced by acetic acid treatment according to the method described by Okabe and Pfeiffer [
Gastric wall mucus content was determined as described by Kitagawa et al. [
GC-7101 at 50 mg/kg was selected as the optimal effective dose for evaluating the molecular mechanisms of GC-7101 against acute gastric lesions with HCl/EtOH treatment.
The steady-state level of malondialdehyde (MDA), the end-product of lipid peroxidation, was analyzed in stomach tissue homogenates by spectrophotometric measurement of the level of thiobarbituric acid-reactive substances at 535 nm according to the method described by Buege and Aust [
Catalase (CAT) activity was measured as follows: 150
Superoxide dismutase (SOD) activity was measured based on the ability of the enzyme to inhibit the process of pyrogallol autoxidation. Briefly, stomach tissues were homogenized in 50 mmol/L phosphate buffer (pH 7.8). The homogenate was centrifuged at 1600 g for 15 min. To various concentrations of tissue supernatants, 20
A commercial PGE2 enzyme-linked immunosorbent assay (ELISA) kit (Assay Designs, Ann Arbor, MI, USA) was used for quantification of the gastric mucosal PGE2 concentration.
A commercial nitrate/nitrite colorimetric assay kit (Cayman Chemical, Ann Arbor, MI, USA) was used for quantification of the gastric mucosal NO content.
Myeloperoxidase (MPO) activity in stomach tissue was measured using commercially available MPO colorimetric activity assay kit (BioVision, Milpitas, CA, USA) according to the manufacturer’s protocol.
Isolated stomach tissue was homogenized in PRO-PREP Protein Extraction Solution (iNtRON Biotechnology Inc., Seongnam, Korea) in a microcentrifuge tube. After standing in a cold ice-bath for 30 min, whole homogenates were centrifuged at 13000 g for 5 min. The supernatant was collected, and the protein concentrations of whole homogenates were determined using the BCA Protein Assay kit (Pierce Biotechnology, Rockford, IL, USA).
NE-PER (Pierce Biotechnology) was used for extraction of nuclear and cytosolic fractions according to the manufacturer’s instructions. Briefly, isolated stomach tissue was homogenized in cold Cytoplasmic Extraction Reagent (CER)1 (with protease inhibitor cocktail set III; Calbiochem, La Jolla, CA, USA). After incubation for 10 min, CER2 was added to break down the cytoplasmic membrane. After centrifuging (16000 g for 5 min), the cytoplasmic extract was collected. Nuclear Extract Reagent with protease inhibitor cocktail set III (Calbiochem) was added to the remaining nuclear pellet and, after a 40 min incubation and centrifugation (16000 g for 10 min), the nuclear extract was harvested. Protein concentrations were determined using the BCA Protein Assay kit (Pierce Biotechnology).
Protein samples were loaded on 10–15% polyacrylamide gels and were then separated by SDS/PAGE and transferred to polyvinylidene fluoride membranes (Millipore, Billerica, MA, USA) using the Semi-Dry Trans-Blot Cell (Biorad Laboratories, Hercules, CA, USA). After transfer, the membranes were washed with 0.1% Tween-20 in 1× Tris-buffered saline (TBS/T) and blocked for 1 h at room temperature with 5% (w/v) skim milk powder in TBS/T. The blots were then incubated overnight at 4°C with primary antibodies. After washing three times for 5 min each in TBS/T, the membranes were incubated with appropriate secondary antibodies for 1 h at room temperature and detected using an ECL detection system (iNtRON Biotechnology Inc.), according to the manufacturer’s instructions. ImageQuant TL software (Amersham Biosciences/GE Healthcare, Piscataway, NJ, USA) was used for densitometric evaluation of visualized immunoreactive bands. Primary antibodies against NF-
Total RNA was extracted and first strand cDNA was synthesized by reverse transcription (RT) using oligo (dT) primers and SuperScript II RNase H-Reverse Transcriptase (Invitrogen Tech-Line, Carlsbad, CA, USA). PCR reactions were in 20
The overall significance of results was examined using one-way analysis of variance (ANOVA). Differences between groups were considered statistically significant at
Intragastric administration of acidified EtOH resulted in multiple hemorrhagic lesions in the glandular portion of the stomach, with a lesion index of
Antiulcerogenic activities of GC-7101 for acute and subchronic gastric ulcers.
Experimental models | Treatment | Dose | Gastric lesion | Inhibition (%) |
---|---|---|---|---|
HCl/EtOH | Vehicle | — | 81.8 ± 12.0 | — |
Ranitidine | 30 | 40.3 ± 7.9 |
50.7 | |
Rebamipide | 30 | 35.0 ± 8.3 |
57.2 | |
GC-7101 | 25 | 40.5 ± 6.8 |
50.5 | |
50 | 32.8 ± 4.4 |
60.0 | ||
100 | 41.9 ± 8.4 |
48.8 | ||
|
||||
Indomethacin | Vehicle | — | 35.6 ± 5.3 | — |
Ranitidine | 30 | 3.6 ± 0.9 |
89.8 | |
Rebamipide | 30 | 12.0 ± 2.1 |
66.4 | |
GC-7101 | 25 | 13.1 ± 2.1 |
63.3 | |
50 | 12.0 ± 2.1 |
66.4 | ||
100 | 9.6 ± 4.9 |
72.9 | ||
|
||||
Water immersion restraint stress | Vehicle | — | 21.1 ± 2.1 | — |
Ranitidine | 30 | 7.5 ± 1.1 |
64.4 | |
Rebamipide | 30 | 13.4 ± 1.5 |
36.6 | |
GC-7101 | 25 | 10.9 ± 1.4 |
48.3 | |
50 | 8.4 ± 1.1 |
60.2 | ||
100 | 8.3 ± 1.5 |
60.6 | ||
|
||||
Acetic acid | Vehicle | — | 20.0 ± 1.5 | — |
Ranitidine | 30 | 10.3 ± 1.1 |
48.4 | |
Rebamipide | 30 | 8.4 ± 1.5 |
57.7 | |
GC-7101 | 25 | 8.9 ± 1.7 |
55.6 | |
50 | 8.5 ± 1.9 |
57.6 | ||
100 | 5.3 ± 0.8 |
73.7 |
The values are represented as means ± SEM for eight to ten rats per group.
Rats receiving indomethacin had linear hemorrhagic lesions along the long axis of the stomach, with a lesion index of
WIRS induced linear and circular ulcers in the glandular portion of stomach with coagulated blood at the bases, with a lesion index of
Injection of acetic acid into the submucosal layer of the stomach induced round ulcer lesions at eight days after induction, with a lesion index of
Rats treated with HCl/EtOH showed a significant decrease in the Alcian blue binding capacity of the gastric wall mucus (
Effects of GC-7101 on gastric wall mucus content (a), mucosal PGE2 concentration (b), and mucosal NO content (c) in HCl/EtOH-induced gastric ulcer. The values are represented as means ± SEM for eight rats per group.
In vehicle-treated control rats, the mucosal PGE2 level remained at basal level (
In vehicle-treated control rats, the mucosal NO level was at basal levels (
HCl/EtOH treatment resulted in a significant increase in MPO activity to 308.3% of the vehicle-treated control group. The 50 mg/kg GC-7101-treated group showed a marked attenuation in this increase to 64.9% of the vehicle-treated HCl/EtOH group (Figure
Effects of GC-7101 on MPO activity (a), nuclear translocation of NF-κB (b), and inflammatory cytokines mRNA expression (c) in HCl/EtOH-induced gastric ulcer. Immunoblot and PCR shown are representatives of at least three experiments with similar results.
As shown in Figure
Rats receiving HCl/EtOH showed significant increases in inflammatory mediator mRNA expression compared to the vehicle-treated control group: 375.6% of the vehicle-treated control group for TNF-
In vehicle-treated control rats, MDA in stomach tissue was at basal levels (
Antioxidant properties of GC-7101 for HCl/EtOH-induced gastric ulcers.
Group | MDA (nmol/mg protein) | GSH/GSSG ratio | SOD (U/g protein) | Catalase (μmol/min/mg protein) |
---|---|---|---|---|
Vehicle-treated control | 0.4 ± 0.1 | 18.7 ± 1.0 | 97.0 ± 0.1 | 22.0 ± 2.1 |
HCl/EtOH | ||||
Vehicle | 1.4 ± 0.2 |
12.0 ± 0.7 |
65.6 ± 0.3 |
11.9 ± 1.3 |
GC-7101 50 mg/kg | 0.8 ±0.2+ | 21.1 ± 3.1++ | 102.0 ± 12.8+ | 18.9 ± 2.1+ |
The values are represented as means ± SEM for eight rats per group.
In vehicle-treated control rats, SOD activity remained at basal levels (
This study investigated the antiulcerogenic effect of GC-7101, an ethyl acetate fraction of
Major etiologic factors of peptic ulcers include irregular eating habits, alcohol consumption, excessive use of drugs such as nonsteroidal anti-inflammatory drugs, psychological and/or physiological stress, and
Human gastric ulcers are characterized by repeated recurrence or relapse and this is largely associated with the quality of healing of the preceding ulcer. Acetic-acid-induced gastric ulcers in rats closely resemble chronic ulcers in humans, particularly in the healing process [
Administration of EtOH, a necrotizing agent, not only causes direct insult to the stomach but also disrupts the balance in various factors of the gastric mucosa by increasing ROS formation and decreasing gastric mucus and prostaglandin production and gastric mucosal blood flow [
Emerging evidences support the notion that the enhancement in PGE2 and NO levels is a protective mechanism against gastric mucosal damage [
Gastric ulcers are considered to be the manifestation of an inflammatory response. Gastric inflammation increases leukocyte adherence to the endothelial surface of postcapillary venules and is characterized by the migration of macrophages and polymorphonuclear leukocytes in the ulcer area. Migrated macrophages release proinflammatory mediators such as TNF-
Oxidative stress is responsible for the loss of mucosal integrity caused by various aggressive factors including EtOH [
It is well documented that one of the sources of ROS in gastric mucosal injury is the activated neutrophils; MPO is a heme enzyme that uses the oxidizing potential of superoxide and hydrogen peroxide to convert chloride ion to hypochlorous acid and other ROS from neutrophils [
In conclusion, we demonstrated the antiulcerogenic effect of GC-7101, an ethyl acetate extract of
The authors declare that there is no conflict of interests regarding the publication of this paper.
This study was supported by Green Cross Corp. and Green Cross Health Science Co., Ltd., the manufacturer of GC-7101. The funding source had no role in the design and conduct of the study; in the collection, analysis, and interpretation of the data; or in the preparation, review, or approval of the paper.