| Background:Gastric ulcer(GU) is a multi-causal disease, which is also one large kind of digestive system diseases and spreads cosmopolitanly. It has been estimated that 5%～10% people have suffered this disease in their lives. Its pathogenesy is complicated, many factors, such as gastric acid, pepsin, nfection, inheritance, physique, environment, diet, life habits, neuropsychiatric and etc., through different ways or mechanisms break the balance between aggressive factors of gastric mucosa and mucosa self-defend-reparative factors. It has been characterised by long course of desease, easily palindromia and some severe complication.At present, the drug to treat GU are mainly include proton pump inhibitor(PPI) and histamine-2-receptor antagonists(H2R), which are widely used in clinic make GU accrete rate predominance raisel, however, the problem of cost, side-effect, drug resistance, patient dependence makes the recurrence rate of GU maintained highly. Therefore, search therapeutics for GU is still a urge and important task. Recently, the exploitation of GU drugs turns to naturally occurring ones. The Chinese medicine practitioners have experienced a long history of theoretical research on this disease, accumulated a great deal of clinical treating experience for it and established the embryonic form of spleen-stomach theory in Jin and Yuan Dynasties, which landmark literature is Li Dongyuan's Spleen-Stomach Theory. My supervisor, Liu Xiaowei, has been making a great effort to study the classic of traditional Chinese medicine and referring to large numbers of traditional medical recipes, finally, she worked out a empirical prescription—Weiweifang (WWF), which made up of Fuling, Pugongying, Baiji and Sanqi, its significant effort of relieving GU symtoms and prevent palindromia had been proved by clinical practices again and again. In this study, we took the Chinese medicine theory as the guidance, set the MPO and H+-K+-ATPase as the breakthrough point, referred to and improved some classic modeling methods, such as Watanabet's, and then established the model for acetic acid induced gastric ulcer in rats. Compared with other methods, our technique have obvious advantage because it is a chronic ulcer model and its appearance of ulcer is very similar to human not only through the observation of naked eye but also of microscope. Its short duration is eight to ten days, while the longest duration is six months, in general, it can self-healing in some 60 days with some 30% natural recurrence rate. By observation of the mucosal pathological sections at the model position, measuring the activity of MPO and H+-K+-ATPase in gastric mucosa, we observed and discussed the treatment mechanism of WWF on the experimental ulcer in rats.Experimental Methods:SPF Wistar male rats:56 rats, weight between 180～220g, divided into 7 groups randomly,8 rats for each group:Normal Group, Model Group, Control Experiment Group, WWF high Dose Group, WWF Medium Dose Group, WWF Low Dose Group, Ranitidine Group. The rats in the Nomal Group were given normal daily diet. The rats in the Model Group were killed after establishing model 3 days. The rats in the Control Experiment Group were observed for the healing of gastric ulcer, and were killed at the same time along with those in the drug groups. The rats in the remaining groups were given gastric perfusion with corresponding drugs 2ml/day after 3 day since model established and killed after gastric perfusion for 7 days. Except of the Normal Group, the rats in the remaining groups were fasted for 24 hours but water was given. They were given intraperitoneal injection of 10% chloral hydrate for anesthesia; then pulled the body of stomach out through the incision which had been vertically cutted off in the abdominal wall layer by layer and was 2cm in length; pressured the filter paper, which diameter is 5mm and was immerged in 100% acetic acid, on the anterior gastric wall's serosal surface of juncture of gastric body and gastric antrum nearby gastric antrum for 60 seconds; place the stomach back in the body situ, sutured layer by layer and then disinfected the operative incision with 75% alcohol. The rats were fasted for 24 hours except water before collecting the samples. The concrete steps are as follows:①Weighting, giving abdominal anesthesia with 10% chloral hydrate, carrying through hair preparation and then fixing the rats;②Taking the stomach out of abdominal cavity, placing upon ice cubes, opening stomach along the greater gastric curvature, flattening, washing the inner gastric wall and observing sample of gastric mucosa in general;③Obtaining and placing stomachus glandularis tissue around the ulcer into 10% formaldehyde solution for 12～16h(4℃), then further carrying through paraffin embedding and producing pathological sections;④Using a slide to scrape the tissue and mucosal fluid off the stomachus glandularis on ice cubes gently, weighting the samples by using a balance with 0.0001 accuracy after freezing them at -80℃, then mixing these samples with cold normal saline at the rate of 1:9 and using usual method to make them into 10% homogenate, centrifuging for 10 minutes at the speed rate of 10000r/min, store the clear supernatant liquid of the mixture in refrigerator for mensurating the activity of MPO and H+-K+-ATPase.Statistical analysis:All experimental data is expressed as mean±standard deviation(x±s). SPSS 13.0 software was applied to One-Way ANOVA for mean comparison based on multiple samples. When the variance was homogeneous, Least-significance difference (LSD) was applied for test, otherwise Dunnett's T3 method was used. a=0.05 was the level of test.Results:1. Observations of gastric mucosa:Gastric mucosa of Normal Group rats were normal and smooth, without congestion, edema and erosion; Gastric mucosa of Model Group rats were congested and dropsical with many bleeding dots and ulcers, some substance of light coffee color also could be observed on its surface; In Control Experiment Group, coffee color also could be observed on its surface; In Control Experiment Group, rats'ectoptygma were congestion, edema and accretion, gastric mucosa, with pero-healing, were congestion and erosion. In WWF Medium Dose Group, the gastric mucosa of rats were smooth and erosion mildly, whereas it were edema and erosion mildly with little gore and bleeding dots in WWF High Dose Group and WWF Low Dose Group; Gastric mucosa of Ranitidine Group rats were smooth with erosion lightly, with few erosion dots, but without ulcer dots.2. Observations under microscope:Gastric mucosa of Normal Group rats were intact and smooth with gland in order, inflammatory cells and bleeding inclination could not be discover; Gastric mucosa of Model Group rats became thiner, ulcers could be found on it and some of which even gone into deep muscular layer. The capillary vessel in submucous layer were blocked, a great lot of inflammatory corpuscle could be observed in this layer as well as the disorder and rupture of smooth muscle cell. In Control Experiment Group, we found that gastric mucosa were broken through, the gland cells lined in disorder, inflammatory cells and bleeding inclination could be observed either. In WWF Medium Dose Group, we detected that epidermic cell of gastric mucosa were tumid and the clearance between glands were enlarged, at the same time, other tissue and the micro-circulation under mucosa have obvious no pathological changes, yet in WWF High Dose Group and WWF Low Dose Group the case was different, submucous layer were destroyed with light pathological changes. Rats in Ranitidine Group seemed had no special discrepancy compared with those in WWF Medium Dose Group, their gastric mucosa had no ulcer and the structure of glands seemed normal, but it had coloboma deep into glandular cell layer occasionally and muscular layer cells lined in disorder.3. Variety in activity of MPO:The activity of MPO in Model Group increased significantly compared with Normal Group (P=0.000). There was no significant difference between Control Experiment Group and Model Group (P=0.409). Those group, treated by WWF and ranitidine, whose activity of MPO is lower than that in Control Experiment Group (vs WWF High Dose Group,Medium Dose Group,Low Dose Group and Ranitidine Group P=0.000,0.000,0.048,0.001) and Medium Dose Group in particular (x±s=1.739±0.268). The therapeutic effect of WWF Medium Dose Group and WWF High Dose Group is not dose-dependented and the former was better. Compared with WWF High Dose Group and WWF Low Dose Group, Ranitidine Group hasn't significant difference(P=0.111,0.125) but significant lower than WWF Medium Dose Group(P=0.000).4. Variety in activity of H+-K+-ATP:As regards the activity of H+-K+-ATP, Normal Group and Control Experiment Group took superiority to Model Group(P=0.000,0.000). After drug treatment, the activity of H+-K+-ATP of each therapy group is approximate to Normal Group (Normal Group vs WWF High Dose Group,WWF Medium Dose Group,WWF Low Dose Group and Ranitidine Group P=0.081,0.053,0.136) and higher than Control Experiment Group (Control Experiment Group vs WWF High Dose Group,WWF Medium Dose Group,WWF Low Dose Group and Ranitidine Group P=0.025,0.040,0.000,0.013).Conclusion:1. The method of replicating gastric ulcer model by glacial acetic acid was in line with performance of human gastric ulcer, which has a high stability and good feasibility as well.2. According to the observation of gastric mucosa, the model achievement ratio is reach 100%. Control Experiment Group has bad autotherapy effort, while all the drug groups have more or less therapeutical effect, WWF Medium Dose Group in particular. The healing condition of Rats' Gastric Ulcer in the drug groups is enormously better than that in Control Experiment Group, especially in WWF Medium Dose Group and Ranitidine Group.3. Based on the pathological observation using microscope, the WWF Medium Dose Group was closer to the Normal Group, which had basically repaired glands morphology, less inflammatory cells and no obvious pathological submucous circulation, therefore, these facts could prove that WWF Medium Dose Group was in better recovery condition and the therapeutical mechanism of WWF maybe related to improving microcirculation, antiinflammatory and cell regeneration. However, the Model Group had more serious mucosa damage, mucosa defect involving muscular layer and completely destroyed glands at the model position, serious infiltration of inflammatory cells at the non-model position, glands deformation and obvious pathological sub-mucous circulation.4. In the treatment of drug groups, WWF expressed complicated dose-effect relationship, which manifests the dose of Traditional Chinese drags are important to therapeutical effect although in same conditions of medicinal substances selecting and decocting.5. The measured results of activity of MPO showed that WWF has obvious effect for the healing of gastric ulcer. The activity of each drug group is lower than both Model Group and Control Experiment Group (compared the WWF High,Mediumn,Low Dose Group and Ranitidine Group with the Model Group P=0.000,0.000,0.006,0.000, WWF High,Medium,Low Dose Group and Ranitidine Group vs Control Experiment Group, P=0.000,0.000,0.048,0.001). The value detected in WWF high Dose Group and WWF low Dose Group had little statistical significance compared with Ranitidine Group(P=0.111,0.125), but WWF Medium Dose Group had lower activity than it (P=0.000), which proved WWF Medium Dose Group had a superior antiinflammatory effect.6. The Statistical results of activity of H+-K+-ATPase showed that more serious ulcer, worse gastric acid secretion and lower H+-K+-ATPase's activity(compared the Model Group with Normal Group, P=0.000). However, the activity of the three WWF groups were close to that of the Normal Group(WWF High,Medium,Low Dose Group vs Normal Group, P=0.081,0.053,0.316) and also very close to that of the Ranitidine Group. It showed that WWF is effective in repairing the damaged gastric mucosa tissues and recovering the normal acid secretion of gastric mucosa. The efficacy of WWF is similar to that of Ranitidine, the clinical drug commonly used for gastric ulcer by modern medicine. This is also in line with the results of observation on the gross samples.Accordingly, the experimental model of this study is successful. By detecting gastric mucosa and gastric ulcer circa histopathologic slide, we found WWF is effective for the treatment of gastric ulcer and can exert best efficacy with clinical usual dose, which equal to WWF Medium Dose Group's. WWF can repair the normal physiological structure of gastric mucosa, so as to restore normal gastric acid secretion and resist inflammatory, moreover, it may be playing a hol-regulation role by improving microcirculation and accelerating cell regeneration. |
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