| Research background:Peptic ulcer refers to damage on gastrointestinal mucosa even to the depth of sub mucosa which are digested by stomach digestion, and it may occur in the esophagus, stomach, duodenum, as well as the gastrojejunostomy anastomosis, and Meckel's diverticulum that with gastric mucosa. Gastric ulcer and duodenal ulcer are more than any others in clinical.PU is one of the familiar and frequently encountered diseases in clinic, and epidemiological survey shows that about 10% of the population had suffered from the disease during their lifetime. With the H2 blockers and proton pump inhibitors having been published one after another, the curative ratio of ulcers has greatly increased, however ulcer recurrence has not been fundamentally resolved yet. To find effective drug therapy to improve the quality of ulcer healing becomes one of the research hot spots in medicine circles.Chinese medicine treatment on gastric ulcer with unique advantages attracts widespread attention now, which dues to the long history of significant curative effect.The pathogenesis of peptic ulcer is so complicated that it has not yet been fully clarified.In summary, this disease is caused by loss of balance between the stomach, duodenum mucosal damage (induced ulcer) factors and mucosal protection (mucosal barrier).Peptic ulcer is considered as a multi-cause disease now. Various disease-related factors such as gastric acid, pepsin, infections, genetic, physical, environment, diet, living habits, neuropsychological factors etc. through different channels or mechanisms, lead to the invasion enhanced or protection mechanisms weaken, and then ulcers occur.Recent researches show that many cellular factors are closely related to the occurrence, development and healing of peptic ulcer. IL-2 is a lymphoid factor mainly secreted by T lymphocytes, which can not only promote T cell proliferation and differentiation, promote B cell proliferation and secretion of antibody, but also can promote the synthesis and release of a variety of lymphocytes and antibody generation, making them play immune functions together. So it has an important role in the immune response, immune regulation and anti-injury. Its abnormal generation and expression can make a prompt to affect on regulating immune function, disease, injury and other indicative effects.The level of IL-2 can be an important indicator used to evaluate the cellular immune function.Weiweifang (WWF) is clinical experience on gastric ulcers, and researches on it were studied from nitric oxide, gastrin, epidermal growth factor, neurotensin, hexosamine, prostaglandin and other indicators.This topic is based on the observation on experimental gastric ulcer treatment effect, and then measures the serum levels of IL-2 by radioimmunoassay, and compare whether there were statistical difference between different doses of WWF and ranitidine on immunological index of gastric ulcer in rats.The purpose is not only to analyze causes of ulcer formation from the immunological view, but also to probe the possible mechanisms of ulcer healing, and then to provide experimental evidence for clinical application and development of Chinese medicine.Experimental Methods: 1.Experimental group and administration:Fifty-six male Wistar rats with SPF level weighing 220-260g were raised at a room of 22-26℃and quiet, and they were divided into seven groups according to a completely random method in strict. They are normal group that with normal feeding,astric ulcer model group,self-cure group,WWF group of high, middle and low dose and ranitidine group.Rats in model group were killed and drawn materials at the forth day after acid-induced gastric ulcer model made, and others in self-cure group were raised on normally after being modeled.Drug groups were drenched different doses of WWF and ranitidine once a day for seven times from the forth day after being modeled. Except model group, the others were executed at the eleventh day.2.Preparation of acid-induced gastric ulcer model:Models were prepared according to the classic and transformed glacial acetic acid method.The rats must fast diet more than 24 hours before being operated on but with normal drinking. Inject intraperitoneally 10% of Chloral Hydrate to anaesthetize rats, prepare for abdominal skin,fix limbs, disinfect with alcohol and pave surgical gauze, then cut one 1-2cm longitudinal incision from xiphoid along ventral on the left, remove the stomach from the stomach abdominal gently and expose it, and paste acid-soaked filter in diameter of 5mm for 30 seconds twice in the tunica serosa,wash the incision with saline cotton softly, put the stomach back and then suture layer by layer, disinfect the suture department again with 75% alcohol cotton balls.3.Specimen collection and main experimental methods:3.1 Serum IL-2:Collect about 3ml abdominal aortic blood into ordinary vacuum mining vessels and put them to the refrigerator at 4℃until natural solidification, then centrifugate them for 10min at the speed of 2000r/min, separate the serum and save them at-20℃refrigerator. Serum IL-2 was determined by radioimmunoassay in accordance with instructions.3.2 Gastric PH:Cut the left abdominal under the xiphoid, and expose the stomach. Ligate the cardia, cut out the stomach at the duodenum and esophagus, squeeze gastric juice on the PH test paper from the pylorus, then read and record the PH values.3.3 Ulcer size:Inject 10ml 10% neutral formalin solution to the stomach from the pylorus, cut the stomach along the greater curvature after 10 minutes and flat it. Measure the maximum transverse diameter and longitudinal diameter of ulcers with a caliper, and denote them by d1,d2. Ulcer areas are calculated according to the formula S=π×d1×d2×1/4(π=3.14).3.4 Preparation of gastric mucosal histopathology slide:Make use of the central mucosa that is parallel to the long axis of maximum diameter of the gastric ulcer scar, and fix placed in 10% neutral formalin solution, dehydrate them conventionally, packet wax, slice, HE staining and observe them with the optical microscope.4.Statistical analysisAll data were analyzed using statistical software SPSS13.0, and the value of P<0.05 would mean significant difference.The experimental results were expressed by X±S. Mean comparison of multiple samples was compared with One-way ANOVA.When the variance was homogeneous, least-significant difference(LSD) was applied for test.Major findings:1.Visual observation of gastric mucosa:Gastric mucosa of normal group rats were normal and smooth,without congestion,edema and erosion. There were crater-like ulcers at the sinuses ventriculi in rats of the model group and the bottom of ulcers were covered with yellow and white muscus.In self-cure group, rats' ectoptygma was congestion,edema, and was surrounded by adhesions, then gastric mucosa were insufficient healing. In ranitidine group, serosa had little adhesion with surroundings, and gastric mucosa ulcer in the gastric antrum almost basic healed, the surface was rough and white. In WWF of low-dose and high-dose groups, some serosa had adhesion with surroundings, and there were few ulcer points or scar tissue in the gastric antrum. In WWF of middle-dose group, there were less adhesion, and the majority of gastric mucosa has healed, only little ulcers points or small-scale scar exited.2.Observation under microscope:Gastric mucosa of normal group rats was normal and smooth,with gland in order. Gastric mucosa of gastric ulcer model rats were not intact,and the gland was completely destroyed and capillary vessel under ulcers were blocked and bleeding. Large amount of inflammatory cells infiltrated the sub mucosa and serosa. In self-healing group, some muscular mucosa were even defected and regenerative glandular cells arranged disorderly, there were inflammatory cell infiltration and apparent thrombosis in submucosa. In ranitidine group, there were smaller coloboma and new epithelial cells and glandular cells arranged in dense irregularly on the surface. Submucosa was infiltrated with inflammatory cells, while there was scar formation in muscular layer. In WWF of low-dose and high-dose groups, we can see defects up to glandular cell layer, and glandular cells regenerated with irregular density, then submucosa was infiltrated by inflammatory cell. There were on significant pathological changes in sub mucosal blood vessels and larger number of proliferated gland cell in high-dose group, however there was still infiltration of inflammatory cells. In WWF of middle-dose group, mucous membrane was almost integral, and larger number of glandular cell proliferated. There was fewer inflammatory cells infiltration in submucosa.3.Gastric PH:PH of model group was significantly higher compared with that of normal group, and the difference was statistically significant (P<0.001),difference between all drug groups and model group was statistically significant (P<0.05). There was no significant difference between WWF of middle-dose and low-dose groups and ranitidine group (P>0.05), and rats in WWF of middle-dose and low-dose groups are closer to the normal group.4.Ulcer size:The difference was statistically significant between ulcer areas of drug groups and self-healing group.There was significant difference (P<0.01) between WWF of middle-dose group and ranitidine group, which proved that the treatment effect of WWF of middle-dose was superior to ranitidine.5.Serum IL-2 in comparison:The difference was statistically significant between IL-2 contents of drug groups and self-healing group as well as between WWF middle-dose group and ranitidine group (P<0.05), while it was not middle and high-dose groups between normal group (P> 0.05)Conclusion:1.Animal model:The method of acetic acid cauterization serosa to ulcer was used in this experiment. Ulcers'performance was very similar to human ulcers through naked eye and microscopic observation. This modeling method with high success ratio is simple and easy to operate; it's now generally accepted and widely used to observe the efficacy.2.Through visual observation of gastric mucosa specimens, we found that ulcer of rats in drug groups heals well compared with that of model group and self-healing group, and ulcer area of each WWF group was less than that of self-healing group, and of which middle-dose group was the best.3.Through optical microscope observation of gastric mucosal histopathologic slide, we found that damage to mucosal layers of rat in model group was more serious and more obvious pathological changes in submucosal circulation, then drug groups healed obviously, in which WWF of middle-dose group was the best. 4. Gastric PH:PH values of the model group were significantly higher than the normal group, probably because that after modeling gastric mucosa was injured and gland was damaged, then acid secretion decreased. After treatment, ulcers healed in basic and the damaged gastric mucosa had been restored, glandular hyperplasia was promoted and acid secretion gradually became restored.5.Serum IL-2:There was significant difference between serum IL-2 of each WWF group and self-healing group, which suggested that WWF could heighten levels of IL-2, it could not only promote T lymphocyte proliferation and differentiation, but also improve the body cellular immune function and effectively kill the causative agents, in order to prevent ulcer development and promote ulcer healing. Compared with ranitidine, WWF of middle-dose group had more ability to improve cellular immune function.Experiments showed that WWF groups had better therapeutic effect,and WWF of middle-dose group was the best in all groups. We speculate that possible mechanisms for ulcer healing are related to increasing body's cellular immune function and levels of serum IL-2;however its relevant mechanisms have yet to be confirmed by further studies. |
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