Preventive Effect of the Flavonoid, Wogonin, Against Ethanol-Induced Gastric Mucosal Damage in Rats
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P1: KEG PL252-ddas-486385 DDAS.cls March 18, 2004 17:55 Digestive Diseases and Sciences, Vol. 49, No. 3 (March 2004), pp. 384–394 (C 2004) Preventive Effect of the Flavonoid, Wogonin, Against Ethanol-Induced Gastric Mucosal Damage in Rats SOOJIN PARK, PhD,*† KI-BAIK HAHM, MD, PhD,† TAE-YOUNG OH, MS,† JOO-HYUN JIN, BS,† and RYOWON CHOUE, PhD* Whether wogonin (5,7-dihydroxy-8-methoxyflavone), a flavonoid originated from the root of Scutel- laria baicalensis Georgi, which has been shown to have antiinflammatory and antitumor activities in various cell types, possesses a gastric cytoprotective effect was investigated in an ethanol-induced gastric damage model in rats. Ethanol administration alone induced evident gastric damage including gastric hemorrhages and edema, while this gastric damage was significantly attenuated by wogonin pretreatment (30 mg/kg B.W.) 1 hr before ethanol administration. As major protective mechanisms of wogonin on ethanol-induced gastric damage, we found that wogonin showed either antiinflamma- tory effects through dual actions on arachidonic acid metabolism, i.e., induction of prostaglandin D2 and suppression of 5S-hydroxyeicosatetraenoic acid (5S-HETE), or preventive induction of profuse apoptosis in the stomach. Conclusively, the flavonoid wogonin could be used as a preventive agent of alcohol-induced gastropathy, whose actions were proven to be strong antiinflammation and apoptosis induction. KEY WORDS: gastric mucosal damage; ethanol; wogonin; COX; PGD2; apoptosis. Alcohol is an etiological factor that has a close relationship tective or a detrimental role in the alcohol injury model with gastric mucosal damage including gastritis and peptic (1, 4). ulcer diseases (1). This alcohol-induced gastric mucosal Flavonoids are naturally occurring polyphenolic com- damage occurs both directly and indirectly through vari- pounds which are widely distributed in plants. Various ous mediators such as cyclooxygenase (COX), lipoxyge- flavonoids such as quercetin, naringin, and saponins have nase (LOX), cytokines, and oxygen-derived free radicals been used as a remedy for inflammatory diseases in folk (1–3). Interestingly, alcohol injury is usually independent medicine around the world (5, 6). Several gastroprotec- of gastric acid secretion and luminal pH and there is a tive mechanisms of flavonoids have been suggested in- growing body of evidence that inflammatory mediators cluding increasing endogenous PG, decreasing histamine and expression of COX-2 mRNA are induced by alcohol secretion, inhibiting Helicobactor pylori, and scaveng- (1) and that prostaglandin (PG) plays either a cytopro- ing oxygen-derived free radicals. The modes of action of flavonoids are quite different depending on the disease (5). Wogonin, 5,7-dihydroxy-8-methoxyflavone, originat- Manuscript received September 17, 2003; accepted January 16, 2004. ing from the root of Scutellaria baicalensis Georgi, has From the *Department of Medical Nutrition, Graduate School of East- West Medical Science, Kyung Hee University, Seoul, and †Genomic Re- been used as a traditional phytochemical for inflammation- search Center for Gastroenterology, Ajou University School of Medicine, related diseases (7). This flavonoid has been shown to exert Suwon, Korea. antiinflammatory effects via selective inhibition of both Address for reprint requests: Prof. Ryowon Choue, Graduate School of East-West Medical Science, Kyung Hee University, 130-701 COX-2 (7) and inducible nitric oxide synthase (iNOS) in Dongdaemun-ku Hoeki-dong, Seoul, Korea; [email protected]. macrophages (7, 8) or induction of apoptosis via activation 384 Digestive Diseases and Sciences, Vol. 49, No. 3 (March 2004) 0163-2116/04/0300-0384/0 C 2004 Plenum Publishing Corporation P1: KEG PL252-ddas-486385 DDAS.cls March 18, 2004 17:55 PREVENTIVE EFFECT OF THE FLAVONID WOGONIN ON GASTROPATHY (3, 10, 30 mg/kg B.W.). One hour later, the rats were all killed by cervical dislocation. The stomachs were rapidly removed and opened along the greater curvature, extended on a plastic board, and photographed. The total areas of macroscopic hemorrhages and erosions were assessed by planimetry gastric lesion index (expressed as square millimeters). For histological assessment, the glandular stomach was fixed Fig 1. Chemical structure of wogonin (5,7-dihydroxy-8- in 10% neutral buffered formaldehyde solution, sectioned, and methoxyflavone). embedded in paraffin. Four-micrometer-thick sections were de- paraffinized, stained with hematoxylin and eosin, and then ex- amined under a light microscope. The specimens were assessed of caspase-3 in HL-60 cells (9). However, the effects of according to the criteria of Laine et al. (10). In brief, a 1-cm length wogonin in gastric physiology or pathophysiology in an of each histological section was assessed for epithelial cell loss (a score of 0 to 3), edema in the upper mucosa (a score of 0 to 4), in vivo system have not been reported. hemorrhagic damage (a score of 0 to 4), and inflammatory cells Based on known antiinflammatory and apoptosis induc- (a score of 0 to 3). Each section was evaluated on a cumulative ing activities of wogonin, we investigated whether wogo- basis to give the mean histological score, the maximum score nin can prevent alcohol-induced gastritis in rats and com- thus being 14. pared the preventive effects of wogonin against alcohol The stomach was rapidly removed and opened along the greater curvature and rinsed with 0.9% ice-cold saline, and the stomach injury with the efficacy of rebamipide, a well- remaining gastric mucosa, after pathologic examination, was known drug prescribed clinically for the treatment of gas- scraped with glass slides. Specimens were immediately frozen tritis and gastric ulcer (10, 31). in liquid N2 and stored at 70 C for biochemical assay. Western Blot Analysis. Each frozen gastric mucosa was ho- mogenized in ice-cold 20 mM Tris–HCl buffer, pH 7.5, contain- MATERIALS AND METHODS ing 2 mM EDTA, 0.5 mM EGTA, 300 mM sucrose, and 2 mM phenylmethyl sulfonyl fluoride (PMSF) with a tissue homoge- Agents and Chemicals. 5, 7-Dihydroxy-8-methoxyflavone nizer at 4C. Protein concentrations were determined by Bio-Rad (wogonin; Figure 1) was purchased from Wako Co. (Osaka, reagent (Bio-Rad, Hercules, CA) using bovine serum albumin Japan). Wogonin was suspended in 0.5% carboxymethylcellu- as standard. Ten micrograms of protein from the homogenates lose (CMC; Sigma-Aldrich, St. Louis, MO). Rebamipide (Lot. was denatured in the sample buffer, subjected to electrophore- No 100110 of pure chemical) was generously provided by Ot- sis on an 8% Tris–glycine gel, and transferred onto polyvinyli- suka Pharmaceutical Co. (Tokushima, Japan). 14C-Arachidonic dene difluoride (PVDF) membranes. The blots were pretreated acid (14C-AA; 50 mCi/mmol) was purchased from Perkin Elmer with Tris-buffered saline containing 5% nonfat dry milk, 1% (Boston, MA). Standards for eicosanoid metabolites, PGD2, albumin, and 0.1% Tween 20 and then incubated overnight at PGE2, PGF2, 15S-hydroxyeicosatetraenoic acid (HETE), 12S- 4 C with antibodies for COX-1 (SC1754; Santa Cruz Biotech, HETE, and 5S-HETE were all purchased from Cayman Chemi- Santa Cruz, CA), COX-2 (SC1756; Santa Cruz Biotech), HSP 70 cal Co. (Ann Arbor, MI). All chemicals used in the experiment (Santa Cruz Biotech and StressGene), and -tubulin. Filters were were reagent grade. washed three times and incubated with a horseradish peroxidase- Animals. Male Spraque Dawley rats (200- to 250-g body conjugated secondary antibody against goat IgG, developed us- weight [B.W.]) were obtained from Samtaco (Seoul, Korea). The ing a commercial enhanced chemiluminescence (ECL) system, animals were maintained at 23 1C and 40–60% relative hu- and exposed to films. midity, and were acclimatized for 7 days before the experiment, Biosynthesis of PGs from 14C-AA via COX. The COX ac- fed standard pellet chow, and given fresh water ad libitum. The tivity was determined by the method established in a previous rats were food-deprived for 24 hr but allowed water ad libitum study (12). Each gastric mucosa specimen was homogenized in until 1 hr prior to the experiments in wire mesh bottomed indi- 20 ml of ice-cold 20 mM Tris–HCl buffer, pH 7.5, containing vidual cages. 2 mM EDTA, 0.5 mM EGTA, 300 mM sucrose, and 2 mM PMSF Experimental Design. A total of 72 rats were randomly di- with a polytron (Brinkman Instruments, Westbury, NY) at 4C. vided into six groups of 12 animals. All animals were adminis- To remove cell debris and nuclei, the homogenates were cen- tered absolute ethanol (0.5 ml/100 g B.W.)intragastrically except trifuged at 800g at 4C for 20 min and the resultant supernatant normal control groups, which were administered vehicle, 0.5% was subjected to ultracentrifugation at 105,000g and 4C for CMC (0.5 ml/100 g B.W.). In the experimental groups, wogo- 60 min (Beckman l5-50E) to separate subcellular fractions. The nin was administered as a suspension with vehicle (0.5% CMC, microsomal fraction was resuspended with buffer which con- 0.5 ml/100 g B.W.) and pretreated with different concentrations tained 100 mM Tris–HCl, pH 8.0, with 5 mM EDTA, 2 mM of 3, 10, and 30 mg/kg B.W. respectively, 1 hr prior to ethanol glutathione, 50 mM L-tryptophan, and 2 M hemoglobin. Protein administration. Rebamipide (10 mg/kg B.W.), a drug known to concentrations of each fraction were determined using Bio-Rad be effective against alcohol-induced gastritis (10), was used as a reagent, with bovine serum albumin as standard. One hundred control to compare the effects of wogonin. micrograms of microsomal protein was mixed with COX buffer Assessment of Ethanol-Induced Gastric Mucosal Damage. and preincubated at room temperature for 5 min, while 14C- After a 24-hr fast, gastric damage was produced by introduc- AA containing 0.01 Ci from a stock solution in ethanol was ing absolute ethanol (0.5 ml/100 g B.W.) intragastrically in the aliquoted into another assay tube and then dried down under presence or absence of various doses of wogonin pretreatment N2.