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1、11 Yonago Acta medica 2011;54:1119 Abbreviations: IL, interleukin; iNOS, inducible form of NOS; LPS, lipopolysaccharide; NIH, National Institute of Health; NOS, NO synthase; NSAID, nonsteroidal anti-inflammatory drug; PGE2, prostaglandin E2; TNF, tumor necrosis factor Protective Effects of Ginger ag
2、ainst Aspirin-Induced Gastric Ulcers in Rats Zhongzhi Wang, Junichi Hasegawa, Xinhui Wang, Akiko Matsuda, Takahiro Tokuda, Norimasa Miura and Tatsuo Watanabe* Division of Pharmacotherapeutics, Department of Pathophysiological and Therapeutic Science, and *Division of Integrative Physiology, Departme
3、nt of Functional, Morphological and Regulatory Science, School of Medicine, Tottori University Faculty of Medicine, Yonago 683-8503, Japan We investigated the mechanism underlying the protective effects of ginger against gastric damage induced by aspirin in rats. Gastric mucosal lesions were produce
4、d by orally ad- ministering 200 mg/kg aspirin suspended in 1% carboxymethylcellulose solution to pylo- ric-ligated male Wistar rats. Ginger powder (200 mg/kg) markedly reduced the aspirin- induced gastric hemorrhagic ulcer area. The total acidity of gastric juice was not signifi- cantly influenced b
5、y aspirin or ginger. Ginger powder did not affect the aspirin-induced reduction in mucosal prostaglandin E2 (PGE2) content; however, it did ameliorate the aspirin-induced increases in mucosal activity of the inducible form of NO synthase (iNOS) and plasma tumor necrosis factor (TNF)- and interleukin
6、 (IL)-1 levels. In the next ex- periment, high and low doses of 6-gingerol and 6-shogaol were used instead of ginger pow- der in the same experimental model to examine their roles in the anti-ulcer mechanism of ginger. Both 6-gingerol and 6-shogaol reduced aspirin induced ulcer formation, mucosal iN
7、OS and plasma TNF- and IL-1 levels. In conclusion, ginger powder prevents the as- pirin induced gastric ulcer formation by reducing mucosal iNOS activity and the plasma levels of inflammatory cytokines but does not affect gastric juice or acid production or mu- cosal PGE2 content. This protective ef
8、fect of ginger powder against gastric ulcers may be attributable to both gingerol and shogaol. Key words: aspirin; gastric damage; ginger powder; inflammatory cytokine; inducible form of NO synthase activity Aspirin is a potent nonsteroidal anti-inflammatory drug (NSAID) that is used for the treatme
9、nt of rheumatoid arthritis and related diseases as well as the prevention of cardiovascular thrombotic diseas- es. Gastric ulcer associated with the use of aspirin is a major problem. Many factors such as gastric acid and pepsin secretion, gastric microcircula- tion, prostaglandin E2 (PGE2) content
10、(Laine et al., 2008), and proinflammatory cytokines interleukin (IL)-1 and tumor necrosis factor (TNF)- (Santucci et al., 1995; Appleyard et al., 1996) play important roles in the genesis of gastric mucosal damage, and its subsequent development (Wang et al., 2007; Wallace, 2008). It has been report
11、ed that increases in NO synthase (NOS) activity is involved in the gastrointestinal mucosal defense and also in the pathogenesis of mucosal damage (Muskara et al., 1999; Wallace et al., 2000). Ginger (Zingiber officinale) has been used as a spice and an ingredient of Chinese traditional 12 Z. Wang e
12、t al. stomach medicine for thousands of years. In tra- ditional medicine, ginger has been used to treat many inflammatory conditions and associated pain (Altman and Marcussen, 2001). The major pungent constituents of ginger, 6-gingerol and 6-shogaol, have been shown to have many interesting phar- ma
13、cological effects, such as anti-oxidant, anti- tumor promoting and anti-inflammatory effects (Surh, 2002; Kim et al., 2005; Young et al., 2005). However, the mechanism underlying the protective effects of ginger against gastric damage is unclear. Here, we investigated the antiulcer effects of ginger
14、 in aspirin-induced gastric ulcer model rats. Materials and Methods Animal experiment using ginger powder The experimental protocol was approved by the ethics committee on animal experiments in Tottori University, and the experiments were carried out in accordance with the guidelines for animal expe
15、ri- ments in the same facility. Male Wistar rats at 8 weeks of age (weigh- ing 300350 g) were purchased from Shimizu Laboratory Supplies (Kyoto, Japan). They were acclimated in an air-conditioned room at 25C and 55% humidity and given standard chaw for a few days. Before surgery, the rats were faste
16、d for 24 h and allowed free access to drinking water. Then, the animals were anesthetized using pentobarbi- tal (30 mg/kg body weight, intraperitoneally), the abdomen was opened, and the pyloric end of stom- ach was ligated without causing any damage to its blood supply. The stomach was then replace
17、d, and the abdominal wall was closed in two layers with sutures. After they had fully recovered from the anes- thesia, the animals were divided into 4 groups of five animals each. Group 1 orally received 3 mL of 1% carboxymethylcellulose in water (vehicle) by gavage. Group 2 orally received ginger p
18、owder (200 mg/kg body weight) suspended in 3 mL of 1% carboxymethylcellulose in water. Group 3 orally received aspirin (200 mg/kg body weight) suspend- ed in 3 mL of 1% carboxymethylcellulose in water. Group 4 orally received aspirin together with gin- ger powder suspended in 3 mL of 1% carboxym- et
19、hylcellulose in water. At 4 h after the test drug administration, the animals were anesthetized, and then blood was collected from the heart, and the stomach was removed after the esophageal end had been tied (Jainu and Devi, 2006; Jainu et al., 2006). The stomach was cut open along the greater curv
20、a- ture, and the contents were collected in tubes and centrifuged at 200 g for 10 min. The resultant supernatant was used for the estimation of acid and gastric juice output. The stomach was then washed with warm saline, and the inner surface was photo- graphed to allow the measurement of the area c
21、ov- ered by hemorrhagic ulceration. Next, the gastric mucosal tissues were removed, frozen in liquid ni- trogen and stored at 80C. The total acid content of the gastric juice was determined by titrating it with 0.01 N NaOH, using phenolphthalein as indi- cator, and was expressed as mEq/4 h/100 g (Ja
22、inu et al., 2006; Wang et al., 2007). The acidity of gas- tric juice was calculated as total acid content/gastric juice volume in mEq/mL (Khushtar et al., 2009). In animal experiments using 6-gingerol and 6-shogaol, the same procedures as were used in the first experiment to produce the pylorus liga
23、ted rats were repeated using high dose (2 mg/kg) and low dose (1 mg/kg) 6-gingerol, and high dose (1 mg/kg) and low dose (0.5 mg/kg) 6-shogaol, in- stead of ginger powder suspended in 3 mL of 1% carboxymethylcellulose in water. The doses of 6-gingerol and 6-shogaol used in the experiment were determ
24、ined by those found in normal ginger (Schwertner and Rios, 2007). Quantification of the gastric hemorrhagic ulcer area using NIH image The photographs of the stomach were digitized and converted to binary images through gray scale im- aging (Khan, 2004). Using the National Institute of Health (NIH)
25、image-J software, the area of gas- tric hemorrhagic ulcers (mm2) was calculated. 13 Protection against gastric ulcers by ginger Histological studies Gastric tissue samples from each group were fixed in 10% formalin for 24 h. The specimens were then embedded in paraffin, sectioned and stained with he
26、matoxylin and eosin, before being evaluated by light microscopy. Measurement of mucosal PGE2 Frozen gastric mucosal tissue (1 g) was added to 5 mL homogenization buffer (0.1 M phosphate (pH 7.4), containing 1 mM EDTA and 10 M indo- methacin) and homogenized. The lysate was then centrifuged in a micr
27、ocentrifuge at 16,000 g for 15 min at 2C to 8C. The supernatant was trans- ferred to a new tube, and its total protein content was analyzed using the advanced protein assay. PGE2 concentrations were investigated using the PGE2 ELISA Kit (R Bender MedSystems, San Diego, CA). Reagents Aspirin was purc
28、hased from Sigma Chemical (St. Louis, MO). Carboxymethylcellulose sodium salt, 6-gingerol and 6-shogaol were purchased from Wako Pure Chemical Industries (Osaka, Japan). The ginger powder was a generous gift from Ya- wata Bussan (Yonago, Japan). It has been con- firmed that 100 g of ginger powder co
29、ntains 0.68 g of 6-gingerol (Japan Food Research Laboratories, Tokyo, Japan). Statistical analysis All values are expressed as the mean SEM. All results were analysed by one way analysis of vari- ance followed by the proper post-hoc test by SPSS 11.0 J (SPSS Japan, Tokyo). P 0.05 was consid- ered st
30、atistically significant. Results Aspirin-induced gastric hemorrhagic ulcer formation The macroscopic findings of the opened stomach are shown in Fig. 1a. Hemorrhagic gastric ulcers covered with coagulated blood were more apparent in the aspirin administered group (Aspirin, Group 3) than the control
31、(Control, Group 1). Ginger powder alone (Ginger, Group 2) had no effects on the stomach, and the coadministration of ginger powder with aspirin inhibited aspirin-induced ulcer formation (Aspirin + Ginger, Group 4). Figure 1b shows the mean area of gastric hemorrhagic ulcers in each group. Even in th
32、e control conditions, pin- point ulcers were sometimes seen. Aspirin-induced ulcer formation was completely inhibited by the coadministration of ginger powder (P 0.01). Histopathological findings of gastric mu- cosa The histopathological findings of the gastric mu- cosa are shown in Fig. 2. The gast
33、ric mucosa obtained from the control (Group 1) rats showed 14 Z. Wang et al. an intact cellular architecture (not shown). Ulcers combined with distorted gastric glands, a dam- aged mucosal epithelium, inflammatory exudates and cellular debris were found in the stomachs of the aspirin-treated rats (F
34、ig. 2a, Aspirin). The protection against these histopathological changes induced by the coadministration of ginger powder Fig. 1. Effects of ginger powder against aspirin-induced gastric ulcers in rats. a: Photographs of the gastric mucosa in the control group and the ginger, aspirin and aspirin wit
35、h ginger treatment groups are shown. b: The hemorrhagic ulcer area (cm2) measured in each condition. In this and the following figures, values are shown as the mean SEM. *P 0.01 com- pared with the control rats. #P 0.01 compared with the aspirin-treated rats. Fig. 2. Histological examinations of gas
36、tric mucosal tissue. Ulcer formation with distorted gastric glands, a damaged mucosal epithelium and cell debris are shown in a (Aspirin); however, the coadministration of ginger with aspirin pro- tected against these changes, as shown in b (Aspirin + Ginger) (hematoxylin and eosin stain). Bar = 500
37、 m. to rats resulted in the maintenance of glandular organization and the structure of the muscularis mucosa (Fig. 2b, Aspirin + Ginger). The histologi- cal examination of the ginger powder alone-treated rats showed a normal cellular architecture in the gastric mucosa with no pathological changes (n
38、ot shown). a Aspirin + GingerAspirinGingerControl Aspirin + GingerAspirin ba 15 Protection against gastric ulcers by ginger Gastric juice and acid production Both ginger and aspirin reduced gastric juice pro- duction /100 g body weight. However this effects disappeared by coadministration of ginger
39、and as- pirin. The total acidity showed no significant dif- ferences among these conditions (Table 1). Mucosal PGE2 levels The gastric mucosal PGE2 contents measured in each condition are compared in Fig. 3. Aspirin reduced the mucosal PGE2 content, and the coad- ministration of ginger powder failed
40、 to inhibit the aspirin-induced reduction of PGE2 content. Mucosal iNOS expression Aspirin administration led to a significant increase in gastric mucosal iNOS activity in the ulcerated rats as compared to that measured in the control or ginger administered rats (Fig. 4). The coadmin- istration of g
41、inger powder resulted in a significant decrease in gastric mucosal iNOS activity com- pared with that observed in the aspirin alone group (P 0.05). No differences were seen in iNOS lev- els between the ginger powder treated Group 2 and Group 1 (control). Expression of TNF- and IL-1 in plasma The pla
42、sma concentrations of TNF- and IL-1 in the rats are shown in Fig. 5. The concentrations of TNF- and IL-1 were significantly increased after the administration of aspirin compared with those seen in the control rats (Aspirin, Figs. 5a and b). Ginger powder attenuated these increases in the plasma lev
43、els of TNF- and IL-1 , even after the coadministration of aspirin in rats (Ginger + Aspi- rin, Figs. 5a and b). Table 1. Effects of ginger powder on gastric mucosal factors in experimental gastric ulcer model rats Group Parameter Volume of gastric juice Acidity (mL/100 g) (mEq/mL) Control 6.15 1.3 5
44、2.54 11.54 Ginger 3.03 0.27* 47.85 12.40 Aspirin 3.80 0.42* 46.71 0.37 Aspirin + Ginger 6.20 0.72# 50.25 7.02 Values are shown as the mean SEM. * P 0.05 compared with the control rats. # P 0.05 compared with the aspirin group. Fig. 3. Effects of ginger powder on the PGE2 level of the gastric mucosal
45、 tissue. Aspirin administration reduced the PGE2 level, and the coadministration of ginger powder (200 mg/kg) for 4 h failed to inhibit the reduction in the PEG2 level. *P 0.01. NS, not significant; PGE2, pros- taglandin E2 (pg/mL). Fig. 4. Effects of ginger powder in iNOS activity in the gastric mu
46、cosa. The coadministration of ginger powder (200 mg/kg) inhibited the increase in iNOS activity in- duced by aspirin alone. *P 0.01 compared with the control rats. #P 0.05 compared with the aspirin-treated rats. iNOS, inducible form of NO synthase (dpm/10 mg). 16 Z. Wang et al. Fig. 5. Effects of gi
47、nger powder on the plasma levels of TNF- and IL-1 . The aspirin-administered rats showed marked increases in their plasma TNF- and IL-1 levels. The coadministration of ginger powder completely inhib- ited these marked increases in TNF- (a) and IL-1 (b) levels. *P 0.05, *P 0.01compared with the contr
48、ol rats. #P 0.05, #P 0.01 compared with the aspirin administered rats. TNF- , tumor necrosis factor- ; IL-1 , interleukin-1 . Fig. 6. Effects of 6-gingerol and 6-shogaol on the gastric mucosal lesion formation induced by aspirin in rats. Both 6-gingerol and 6-shogaol inhibited the gastric ulcer form
49、a- tion induced by aspirin. The animals were treated alone with 2 mg/kg 6-gingerol (Gingerol), 1 mg/kg 6-shogaol (Shogaol), 200 mg/kg aspirin (Aspirin); and treated combinedly with Aspirin and 1 mg/kg 6-gingerol (lowG), 2 mg/kg 6-gingerol (highG), 0.5 mg/kg 6-shogaol (lowS), 1 mg/kg 6-shogaol (highS). *P 0.01 compared with the control. #P 0.05 compared with the aspirin-induced rats. Effects of 6-gingerol and 6-shogaol in the presence of aspirin High and low doses of 6-gingerol and 6-shogaol, the major constituents of ginger powder, had no