The Effect And Mechanism Of Aspirin On The Recurrence Of Acetic Acid Gastric Ulcer In Rats

1. BackgroundPeptic ulcer is characterized by recurrence and difficulty in prevention. Clinical observation indicated that non-steroidal anti-inflammatory drug (NSAIDs) is an important factor causing recurrence. Aspirin is a representative drug of NSAIDs. The aspirin is one of the most consumed drugs all over the world because of its extensive pharmacological action. However, aspirin produces severe side effects that cause gastritis, gastric ulcer and inducing recurrence and so on while it cures some disease.The mechanism of peptic ulcer recurrence is still not clear. Therefore, the acetic acid ulcer model was adopted in this study. After the ulcer healed, the recurrence was induced with aspirin. The purpose of this paper was to study the effect and mechanism of aspirin on peptic ulcer recurrence. It has an important significance to prevent recurrence and cure ulcer thoroughly.2. Materials and methods2.1. Methods: Male Wistar rats were used in this study. The ulcer model was established according to the previous method.2.2. Grouping: Experiments were divided into two steps: the first step: at subsequent regular intervals (day 3, 15 and day 25) after establishment of ulcer model, some of samples were killed with diethyl ether and the appearance of ulcer was observed; The second step: at day 25, other samples were randomly assigned to the following three groups: Fasting control group; Saline control group; Aspirin group (aspirin was given). These samples were collected and analyzed at day 55.2.3. Observation of ulcerative appearance: The ulcerative appearance was observed and the ulcer area was measured under anatomical lens.2.4. Evaluation of QOUH: The dilated glands, the thickness of regenerate mucosa, the number of capillary and the number of inflammatory cell below mucosa tissue were evaluated under light microscope.2.5. Determination of gastric mucosa PGE₂ and gastric acid secretion: The volume of gastric juice and the pH were measured. The PGE₂ was determined with enzyme-linked immunoassay (ELISA).2.6. Cycloxygenase expression: The expression of gastric mucosa COX-1 and COX-2 mRNA was detected by RT-PCR, and the gastric mucosa COX-1 and COX-2 protein was analyzed by western blot.2.7. Determination of gastric mucus, gastric mucosal blood flow (GMBF) and endothelin (ET): The gastric mucus was determined according to the previous method, GMBF was determined by H₂ gas clearance technique, and ET in plasma was determined with ELISA.2.8. Determination of growth factor: Epidermal growth factor (EGF) and transforming growth factor-α(TGF-α) in gastric mucosa and vascular endothelial growth factor (VEGF) in serum were determined with ELISA.2.9. Determination of inflammatory factor: Tumor necrosis factor-α(TNF-α) in serum and interleukin-1β(IL-1β) in plasma were determined with ELISA.2.10. Statistical analysis: Date was expressed as mean±SD. SPSS (Version 11.0) software was used. One-way ANOVA with LSD or rank sum test with Mann-Whitney U was performed for analysis among groups. The significance level was set at P<0.05.3. Results3.1. Effect of aspirin on ulcerative appearance: At day 25, the ulcers healed completely. 2 and 5 of 8 rats in Aspirin group showed the recurrence respectively at day 45 and day 55.3.2. Effect of aspirin on QOUH: The number of dilated glands and inflammatory cells in aspirin group increased compared with fasting or saline control group (P<0.01) at day 55. Thickness of regenerate mucosa and the number of microvessel in aspirin group decreased compared with fasting or saline control group (P<0.01) at day 55.3.3. Effect of aspirin on PGE₂ in gastric mucosa and gastric acid secretion: The PGE₂ in aspirin group decreased compared with fasting or saline control group (P<0.01) at day 55. The volume gastric juice in aspirin group increased compared with fasting or saline control group (P<0.01) at day 55. The pH of gastric juice decreased compared with fasting or saline control group (P<0.01).3.4. Effect of aspirin on COX expression: At day 55, the expression of COX-1 mRNA and protein showed no difference among three groups and was identical essentially with that in the ulcer area at day 15. However, the expression of COX-2 mRNA and protein in aspirin group was more than those in fasting or saline control group (P<0.01) and was not different from that in the ulcer area at day 15.3.5. Effect of aspirin on gastric mucus, GMBF and ET: The gastric mucus and the GMBF in aspirin group decreased compared with fasting or saline control group (P<0.01) at day 55. However, the ET in plasma increased compared with fasting or saline control group (P<0.01) on 55 day.3.6. Effect of aspirin on growth factor: The EGF and TGF-αin gastric mucosa in aspirin group decreased compared with fasting or saline control group (P<0.01) at day 55. The VEGF in serum in aspirin group decreased compared with fasting or saline control group (P<0.01) at day 55.3.7. Effect of aspirin on inflammatory factor: The TNF-αin serum and IL-1βin plasma in aspirin group increased compared with fasting or saline control group (P<0.01) at day 55.4. DiscussionThe ulcers healed completely in 25 days. After 25 day, aspirin was given to rats by gavage. At day 55,5 of 8 rats showed the ulcerative recurrence, and there is statistical significance. It indicated that aspirin could induce recurrence successfully.4.1. Effect of aspirin on morphology of acetic acid ulcer: At day 3, a large and round ulcer was observed. After that, the ulcer heals gradually. At day 25, the ulcers also healed completely under naked eye and microscope. At day 55, it showed the recurrence in aspirin group under naked eye and microscope. These results indicated that aspirin induced recurrence at healed ulcer area.4.2. Effect of aspirin on QOUH: At day 55, the number of dilated glands and number of inflammatory cell in aspirin group increased compared with fasting or saline control group. Thickness of regenerate mucosa and number of microvessel decreased compared with fasting or saline control group (P<0.01). The results indicated mat aspirin might reduce QOUH. However, the appearance of mucosa without the ulcerative recurrence in aspirin was similar to the normal mucosa. It indicates that satisfactory QOUH might counteract the effect of aspirin damaging gastric mucosa effectively. This may explain why the recurrence emerged at different time as well as at the healed area.4.3. Effect of aspirin on PGE₂ in gastric mucosa and gastric acid secretion: The PGE2 might inhibit gastric acid secretion, regulate bicarbonate secretion, expand blood vessel, increase GMBF, and promote gastric epithelium mucosae renewing and generation. The present study showed that the PGE₂ in aspirin group decreased compared with fasting or saline control group (P<0.01). It suggested that aspirin might inhibit production of PGE2 in mucosa. Gastric hypersecretion might damage the mucus-bicarbonate barrier and the mucosal barrier. The present study showed that the gastric juice volume in aspirin group was more than those of fasting and saline control groups at day 55 (P<0.01), while the pH value of gastric juice in aspirin group was lower than that of other two groups. It suggested that aspirin might increase gastric acid secretion. Its mechanism might be presumed mat aspirin inhibited production of mucosal PGE₂ and resulted in increasing of gastric acid secretion because PGE₂ could inhibit gastric acid secretion.4.4. Effect of aspirin on COX expression: The COX is a rate-limiting enzyme synthesizing prostaglandin. The present study showed that COX-2 mRNA and protein were expressed in large quantities at ulcer and ulcerative recurrence. Thus PGE2 should increase. However, the present study showed that PGE2 in aspirin group decreased. Therefore, it was presumed that aspirin inhibit the activity of COX-1 and COX-2 enzyme activities rather than inhibit their expressions.4.5. Effect of aspirin on the gastric mucus, the GMBF and the ET: Gastric mucus and HCO₃^- form a layer of mucus-bicarbonate barrier. The barrier constitutes the first line of defence that protects gastric mucosa from harmful factor. The GMBF can provide oxygen and nutrient substances for gastric epithelial cells. It maintains the function of mucus-bicarbonate barrier and the function of mucosal barrier. The mucosal barrier constitutes the second line of defence that protects gastric mucosa from harmful factor. The ET possesses an intense effect on contraction of blood vessel. The present study showed that the gastric mucus and the GMBF in aspirin group decreased compared with fasting or saline control group (P<0.01), while the ET increased compared with fasting or saline control group (P<0.01). It indicated that aspirin could decrease the gastric mucus and the GMBF, and increase the ET. It suggested that the decrease of GMBF might result from decrease of PGE2 and increase of the ET. The decrease of GMBF can result in gastric mucosa ischemia. It causes mucus and bicarbonate to decrease so as to weaken the function of gastric mucosa barrier and cause ulcerative recurrence.4.6. Effect of aspirin on growth factor: The EGF can inhibit gastric acid secretion, and also promote gastric mucosa epithelium to differentiate, proliferate, and increase GMBF. The present study showed that the EGF in aspirin group decreased compared with fasting or saline control group (P<0.01). Thus it was presumed that aspirin caused decrease of the EGF in gastric mucosa, which resulted in increase of gastric acid secretion. The decrease of GMBF reduced the QOUH, which resulted in the recurrence. The TGF-αhas an important effect in the maintaining renewal and integrity of gastrointestinal tract mucosa. The present study showed that the TGF-αin aspirin group was decreased compared with fasting or saline control group (P<0.01). Thus it was presumed that aspirin caused decrease of TGF-αin gastric mucosa, which reduced renewing capability of the mucosa. The reduction of renewing capability of mucosa promoted the recurrence. The VEGF can promote vasculogenesis. The present study showed that the VEGF in aspirin group decreased compared with fasting or saline control group (P<0.01). Thus, decrease of vasculogenesis in gastric mucosa resulted in decrease of GMBF, which promoted the recurrence.4.7. Effect of aspirin on inflammatory factor: The TNF-αand the IL-1βare important inflammatory factors. The present study showed that the TNF-αand the IL-1βin aspirin group decreased compared with fasting or saline control group (P<0.01). It indicated that aspirin could induce releasing of the TNF-αand the IL-1β. The TNF-αand the IL-1βpromoted inflammatory reaction and resulted in recurrence.5. ConclusionAspirin can induce ulcerative recurrence. The mechanism that aspirin causes ulcer recurrence may be: 1. Aspirin can inhibit COX activities and decrease PGE₂ synthesis. Deficiency of PGE2 results in increase of gastric acid secretion and decrease of GMBF. 2. The decrease of GMBF results in the decrease of mucus production and mucosal renewal. 3. The increase of ET causes gastric vasoconstriction and gastric mucosa ischemic. 4. The decrease of the EGF and the TGF-αcause the increase of gastric acid secretion and the decrease of mucosa renewing ability. 5. The decrease of VEGF and vasculogenesis cause decrease of GMBF. 6. The increase of the TNF-αand the IL-1βinduce inflammatory reaction in gastric mucosa, which damage gastric mucosa. Thus aspirin induce ulcerative recurrence under multiple factor and complex action.