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. 2001 Sep;49(3):364–371. doi: 10.1136/gut.49.3.364

Oxidative stress is more important than acid in the pathogenesis of reflux oesophagitis in rats

T Oh 1, J Lee 1, B Ahn 1, H Cho 1, W Kim 1, Y Kim 1, Y Surh 1, S Cho 1, K Lee 1, K Hahm 1
PMCID: PMC1728432  PMID: 11511558

Abstract

BACKGROUND—Although antisecretory medications such as histamine type II receptor antagonists or proton pump inhibitors have been used to treat reflux oesophagitis, a considerable number of patients do not achieve complete mucosal healing or suffer from either sustained symptoms or ensuing complications, suggesting other damaging factors or impaired mucosal resistance are also involved in the pathogenesis of reflux oesophagitis.
AIMS—The present study was designed to evaluate oxidative stress as the major pathogenic factor of reflux oesophagitis and to determine the usefulness of antioxidants in the treatment of reflux oesophagitis.
MATERIALS AND METHODS—Reflux oesophagitis was induced by insertion of a 3 mm calibre ring into the duodenum, 1 cm distal to the ligament of Treitz, in Sprague-Dawley rats.
RESULTS—DA-9601, a novel antioxidant substance, significantly attenuated the gross and histopathological scores of reflux oesophagitis compared with those treated with ranitidine alone or reflux oesophagitis controls in a dose dependent manner. Only scattered erosions were observed in the antioxidant pretreated group but acid suppression by ranitidine was not effective in decreasing the severity of reflux oesophagitis. Significantly increased amounts of malondialdehyde (MDA), increased nuclear factor κB (NFκB) activation, and depletion of reduced glutathione (GSH) were observed in experimentally induced reflux oesophagitis. DA-9601 pretreatment attenuated the decrement in mucosal GSH levels and decreased MDA formation significantly. DA-9601 treatment caused significant reductions in activation of NFκB transcription factor, especially the p50 subunit, in accordance with the significantly higher levels of inhibitory protein of NFκB expression.
CONCLUSION—Reflux oesophagitis caused considerable levels of oxidative stress in the oesophageal mucosa and antioxidant treatment should be considered as supplementary therapy in the prevention or treatment of reflux oesophagitis with acid suppression.


Keywords: reflux oesophagitis; antioxidants; oxidative stress; rat

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Figure 1  .

Figure 1  

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(A) Generation of reflux oesophagitis in the rat oesophagus according to the surgical method. No significant reflux oesophagitis was observed in the group in whom a small calibre ring, 3 mm in diameter, was inserted into the duodenal bulb whereas a considerable degree of reflux oesophagitis was effectively generated in the group where the 3 mm small calibre ring was inserted into the duodenum, distal to the Treitz ligament, suggesting mixed reflux could provoke reflux oesophagitis rather than acid reflux alone in rats. (B, C) Gross appearance of the total oesophagus in each group. Multiple linear ulcerations were observed in the reflux oesophagitis control group (group II). In the DA-9601 pretreated groups (III and IV), only scattered haemorrhagic spots or focal erythematous lesions were observed in the oesophageal mucosa. Ranitidine alone (group V) was not effective in decreasing reflux oesophagitis. Group I, sham operated group. *p<0.05, **p<0.01 versus group II.

Figure 2  .

Figure 2  

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(A) Extent of oesophageal ulcers in the four groups (HAI, histological activity index). In the reflux oesophagitis control (group II) and ranitidine pretreated (group V) groups, oesophageal mucosa was almost denuded and moderate to severe degrees of inflammation replaced the submucosa of reflux oesophagitis. However, only scattered erosions or shallow ulcers were observed in groups III (pretreated with DA-9601 30 mg/kg) and IV (pretreated with DA-9601 100 mg/kg). A statistically significant decrease in the extent of oesophageal ulcer was noted in group IV (*p<0.05 compared with group II). (HAI ulcer: 0, none; 1, erosions; 2, multiple erosions; 3, ulcer; 4, large excavated ulcers.) (B) Degree of oesophageal inflammation in the four groups. In the reflux oesophagitis control and ranitidine pretreated groups, the oesophageal submucosa was almost replaced with inflammatory cell infiltrations in accordance with the occurrence of oesophageal ulcerations. DA-9601 pretreatment (groups III and IV) was excellent in decreasing oesophageal inflammation. A statistically significant decrease in the degree of oesophageal inflammation was noted in the DA-9601 100 mg/kg pretreated group (*p<0.05 compared with group II). (HAI inflammation: 0, none; 1, mild; 2, moderate; 3, severe.) (C) Oesophageal regenerating activities in the four groups. Any evidence of oesophageal regenerations was not seen in group II, with only scanty regeneration activities in group V, whereas significant oesophageal regeneration was observed in antioxidant pretreated group. Statistically significant increases in oesophageal regeneration were noted in groups III and IV (*p<0.05 compared with group II). (HAI regeneration: 0, none; 1, a little; 2, some; 3, many.)    

Figure 3  .

Figure 3  

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Changes in histological activity index (HAI) and representative pathological photographs. (A) Changes in HAI. DA-9601 treatment (groups III and IV) significantly decreased mean HAI scores for oesophageal ulcer and inflammation. *p<0.05, **p<0.01 versus group II. (B) Disappearance of oesophageal squamous epithelia with oesophageal inflammatory cell infiltration was noted after generation of reflux oesophagitis (group II). Excellent evidence of epithelial regeneration and attenuation of inflammatory cell infiltrations were noted in DA-9601 treated groups (groups III and IV). Treatment with antisecretory drug was not as effective in either decreasing the degree of oesophageal inflammation or imposing regenerating capacities (×100 original magnification). Group I, sham operated group; group V, ranitidine pretreated group.

Figure 4  .

Figure 4  

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Changes in malondialdehyde (MDA) levels, glutathione (GSH) content, and myeloperoxidase (MPO) activity in the five groups. (A) MDA levels in the reflux oesophagitis control group (group II) were significantly increased compared with those of normal controls (group I). DA-9601 pretreatment (groups II and III) significantly decreased the amount of MDA formation, which suggested inhibition of reflux oesophagitis associated lipid peroxidation by antioxidant treatment. Ranitidine (group V) was not effective in decreasing MDA formation. (B) Mucosal levels of GSH were significantly decreased in group II compared with the normal control group (group I) . However, DA-9601 pretreatment (groups II and III) preserved GSH levels significantly, which suggested supplementation of scavenging activities through DA-9601 treatment. (C) MPO activity was significantly increased after generations of reflux oesophagitis. DA-9601 treatment decreased mucosal MPO activity. **p<0.01, ***p<0.001 versus group I. 

Figure 5  .

Figure 5  

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Changes in nuclear factor κB (NFκB) transcription factor according to group. (A) Electrophoretic mobility shift assay (EMSA) for NFκB. The reflux oesophagitis control group (group II) showed high activity of NFκB in nuclear extracts of inflamed oesophageal mucosa. DA-9601 pretreatment (groups III and IV) caused decreases in the amounts of inflammation associated transcription factor NFκB in a dose dependent manner. (B) EMSA and supershift EMSA. EMSA, using various types of antibodies against the components of NFκB, showed binding of the p50 subunit of NFκB with nuclear extracts of inflamed oesophageal mucosa. (C) IκBα expression according to group. The reflux oesophagitis control group (group II) showed low level IκBα expression in cytoplasmic extracts of inflamed oesophageal mucosa whereas DA-9601 pretreatment (groups III and IV) caused preservation of significant amounts of IκBα, which suggested the possibility that reflux oesophagitis through mixed reflux caused increased proteosomic degradation of phosphorylated IκBα and activation of NFκB.

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