Protection by Tetramethylpyrazine in Acute Absolute Ethanol-Induced Gastric Lesions
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Original Paper JoLm~lof Biomedical J Biomed Sci 2002;9:395-400 Received:January t0, 2002 Science Accepted:April 5, 2002 Protection by Tetramethylpyrazine in Acute Absolute Ethanol-Induced Gastric Lesions Chi-Feng Liu a,b Chun-Ching Lin c Lean-TeikNg d Song-Chow Lin b aNational Taipei College of Nursing, bDepartment of Pharmacology, Taipei Medical University, Taipei, eGraduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, dTajen Institute of Technology, Ping-Tung, Taiwan, ROC Key Words Previous studies have shown that gastric ulcers are Cytoprotection • Absolute ethanol • often associated with ingestion of aspirin [7, 13] and abso- Tetramethylpyrazine • Hemoglobin- Hematocrit • lute ethanol [25]. Acute absolute ethanol administration Malondialdehyde • Acute gastric damage often leads to tissue damage, especially of the gastric mucosa in the rat, and this model is useful for studying drugs with possible antiulcer action [25]. Tetramethylpy- Abstract razine (TMP) is a pharmacologically active constituent of Acute oral administration of absolute ethanol (1.0 ml/kg) Ligusticum wallichii French [12]. It not only blocks the to fasting rats produced extensive necrosis of the gastric entry of extracellular calcium through calcium channels mucosa within 1 h. Pretreatment 30 min before adminis- but also inhibits the release of intracellular stored calcium tration of ethanol with oral tetramethylpyrazine (TMP) in vascular smooth muscle cells [12]. TMP has been used prevented this necrosis. Gross examination of the gastric to treat ischemic heart disease [2]. Previous research has mucosa of rats that received TMP showed fewer gastric shown that TMP protects the myocardium [1] and has a lesions than that of rats who did not receive TMP. TMP protective effect on reserpine-induced acute hemorrhagic pretreatment in rats exhibited superoxide scavenging mucosal lesions in rats [19]. The aims of the present study activity in absolute ethanol-induced lipid peroxidation in were to investigate whether TMP administration per os gastric mucosal homogenates. TMP added in vitro to protects the gastric mucosa from absolute ethanol-in- gastric homogenates made from control rats also duced lesions in rats, and ifTMP does provide protection, showed scavenging activity. We conclude that the gas- what is the potential mechanism? tric protective mechanism of TMP could be attributed, at least in part, to its ability to inhibit lipid peroxidation and hence indirectly protect the gastric mucosa from oxida- Materials and Methods tive stress. Animals and Treatments Copyright© 2002 NationalScience Council, ROC and S. KargerAG,Basel Male Wistar rats (about 150-200 g) were purchased from the ani- mal center, College of Medicine, National Yang-Ming University. They were kept for at least I weekon commercial diets (Fu-So Co., © 2002 National Science Council, ROC Song-Chow Lin, PhD KARG E R. S. Karger AG, Basel Professor of Pharmacology Fax +41 6l 306 12 34 1021-7770/02/0095-0395518.50/0 Department of Medicine, Taipei Medical University E-Mail karger@karger, ch Accessible online at: 250, Wu-hsing Street, Taipei, 110 Taiwan (ROC) ~. karger.com www.karger.com/joumals/jbs Tel. +886 2 27361661, ext. 3196, E-Mail [email protected] Taipei, Taiwan) under controlled conditions (25 + 1 °C, 55 -+ 5% presence of various concentrations of TMP (50, 100 and 150 mg/kg). humidity) with free access to food and water. The rats were randomly To this mixture, 1.5 ml of 1.0% TBA and 1.5 ml of 20% acetic acid divided into five groups with 10 animals each. Group 1 (control) were added and the mixture was further incubated for I h in a 95 °C received saline (1.0 ml/kg, p.o.), group 2 received absolute ethanol water bath. After cooling, 5 mt of n-butanol was added and the mix- (1.0 ml/kg, p.o.) and groups 3-5 received TMP orally at doses of 50, ture was again shaken vigorously. The n-butanol layer was separated 100 and 150 mg/kg, respectively, 30 min before oral administration by centrifugation at 1,000 g for 10 rain, and the upper n-butanol layer of 1.0 ml/kg absolute ethanol. The animals were killed I h after abso- was collected to measure MDA production, in units of nmol/mg pro- lute ethanol administration. The gastric lesions were evaluated tein, at 532 nm on a spectrophometer [20]. according to an index described by Zhang et al. [25]. Scavenging Activity of Free Radicals: Cytochrome c Test Assessment oJNtomaeh Lesions The absolute ethanol-induced superoxide anion production in rat Blood samples were collected from the carotid artery, allowed to stomach was assayed according to the method described by McCord coagulate at room temperature for 1 h and then subjected to hemo- and Fridovich [10]. Xanthine oxidase converts xanthines to uric globin and hematocrit assays according to the method described by acid, simultaneously yielding its by-product superoxide anions, fol- Mock et al. [11] and Viau et al. [17], respectively. lowed by direct reduction of ferricytochrome c to ferrocytochrome c, Assessment of gastric lesions was also carried out by gross patho- which has a specific UV absorbance at a wavelength of 550 nm. logical examination of the stomach mucosa. Massive gastric hemor- When a compound shows superoxide scavenging activity, there is a rhage was observed in the rats' stomachs 1 h after oral administration decrease in the UV absorbance spectrum in the reduction of ferricy- of 1.0 ml/kg absolute ethanol. The stomachs were then dissected tochrome c. along the greater curvature, and the mucosa was examined for the number of absolute ethanol-induced mucosal lesions according to the Quantitative Evaluation of the Degree of Gastric Mucosal Injury method described by Gutierrez-Cabano [4] and Gutierrez-Cabano According to the method described by Umemoto et al. [ 16], the and Raynald [5]. Gutierrez-Cabano and Raynald [5] reported that photographs in figures 1 and 2 were obtained using a digital camera the intragastric administration of clarithromycin, a macrolide antibi- (Epson Photo 700). One representative digital picture was chosen otic, macroscopically protected the rat gastric mucosa from 96% of which showed a moderate degree of gastric mucosal injury and red ethanol-induced lesions. This protective effect was dose dependent, color. The digital picture was downloaded onto a computer and the the reduction of lesions being 92.3, 81.4, 52.2 and 5.4% at doses of gastric lesions were processed by image analysis with PhotoShop soft- 400, 200, 100 and 50 mg/kg, respectively [5]. Gutierrez-Cabano [4] ware, by means of which the red color was quantified (press 256 level also suggested that the protective effect afforded by intragastric poly- histogram) and expressed. ethylene glycol 400 against ethanol-induced gastric mucosal damage is partially mediated by alpha2-adrenoceptors, and a mucus-depen- Drugs and Chemicals dent mechanism may be involved. Absolute ethanol, hemoglobin and hematocrit kits, TBA, sodium dodeeyl sulfate, ferric chloride, n-butanol and cytochrome c were FeCl2-Stimulated Lipid Peroxidation in Rat Stomach purchased from Sigma Chemical Company (St. Louis, Mo., USA). Homogenate (in vitro) Acetic acid was obtained from Yon-chi Chemical Company in Tai- Rat stomach homogenate was prepared according to the method pei, Taiwan. TMP was a gift from the Institute of Chinese Materia described by Hongo et al. [6]. The inhibitory effect of TMP on FeC12- Medica, China Academy of Traditional Chinese Medicine, Beijing, induced lipid peroxidation in normal rat stomach homogenate was People's Republic of China. determined by the thiobarbituric acid (TBA)-malondialdehyde (MDA) adduct according to the modified method described by Yuda Data A naIysis et al. [23]. A mixture containing 0.5 ml of normal stomach homoge- Data were shown as mean + SE (n = 10). Statistical significance hate, 0.t ml of Tris-HC1 buffer (pH 7.2), 0.05 ml of 4 rrl)¢/FeC12 and was assessed by one-way analysis of variance coupled with Dunnett's 0.05 ml of 4 mM of various concentrations of TMP (0.01, 0.1 and test or Newman-Keuls test. The level of significance was p < 0.05. 1.0 mg/ml) was incubated for 1 h at 37 °C for homeostasis. After incubation, 9 ml &distilled water and 2 ml of 0.6% TBA were added to 0.5 ml of the incubated mixture and shaken vigorously. The mix- ture was then incubated for 30 rain in a 95 ° C water bath. After cool- Results ing, 5 ml of n-butanol was added and the mixture was again shaken vigorously. The n-butanol layer was separated by eentrifugation at Ej]ect of JMP on Absolute Ethanol-Induced Gastric 1,000 g for 10 rain, and the upper n-butanol layer was collected to Lesions measure MDA production, in units of pmol/mg protein, at 532 nm Normal rat stomach and the gastric lesions induced by on a spectrophometer [20]. oral administration of t.0 mt/kg absolute ethanol are Assessment of Lipid Peroxidation Activity (in vivo) shown in figures 1A and B, respectively. TMP was found In order to evaluate the inhibitory activity of TMP on the lipid to dose-dependently prevent the formation of gastric peroxidation generated assay system, portions of rat stomach tissue lesions induced by acute absolute ethanol administration dissected from groups 1-5 were sliced and homogenized (13,000 (fig. 2A-C). rpm, 3 min) with 25 rm~/Tris-HC1 buffer (pH 7.2; 10% w/v) [17]. In a glass test tube, 0.1 ml of stomach homogenate was incubated in a Severe gastric damage was observed from the outside shaking water bath for 1 h at 37 ° C in Tris-HCl buffer (pH 7.2), in the of the damaged stomach as indicated by the appearance of 396 J Biomed Sci 2002;9:395-400 Liu/Lin/Ng/Lin thick black or red lines.