| Background:Digestive system is the largest immune organ system in human body,which includes most of the human body's immune organs and tissues,such as liver,spleen and aggregate lymphoid tissues of gastrointestinal mucosa,therefore plays an important role in the body defense and the removal of foreign antigen.Lots of clinical data have confirmed that the autoimmune diseases(AID) of digestive system have high incidence and involves more organs.In additional,the immune diseases have more complex pathogenesis and clinical features.In recent years,the basic and clinical research on the immune diseases of digestive system progress very rapidly. And the research on mucosal immune system(MIS)—the major component of digestive system immune structure,has developed to be an independent discipline based on the specific structure and function of the MIS.Gastrointestinal mucosa is the place for food digestion and absorption.Meantime,the immune system of gastrointestinal mucosa also plays an important role in the body defense,the removal of foreign antigen and the maintenance of body's steady state.Once the mucosal immune system dysfunction occurs,which may lead to certain immune-related diseases.However epidermal growth factor(EGF),the major cell growth factor of gastrointestinal mucosal immune system,plays an important role in the maintenance of mucosa and the stability of gastrointestinal mucosal immune system.Therefore, the study of gastrointestinal diseases based on immune cell factors is practical and significant.In recent years,the studies of many topics have been focused on the cell growth factor indicators.We can see a broad prospect of the immunodiagnosis and immunotherapy on the digestive system diseases through these studies.Gastric ulcer(GU),i.e.peptic ulcer(PU),is a multi-causal disease.This disease has various incidence causes,such as gastric acid,pepsin,infection,inheritance, physique,environment,diet,life habits,neuropsychiatric factors and etc.These causes for disease ineidence enhance the invasion of gastric acid and the pepsin in gastric juice and/or weaken the defense mechanism through different ways or mechanisms,and cause to ulcer.The people suffering from gastric ulcer account for about 10 percent of the total global population.This disease was included in the areas of epigastralgia in the Chinese Traditional Medicine.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.In this study,we took the Chinese medicine theory as the guide,set the Epidermal Growth Factor(EGF) as the breakthrough point,referred to and improved some typical modeling methods,such as WATANABET,and then established the model for acetic acid induced gastric ulcer in rats.It's a chronic ulcer model,which short duration is eight to ten days,while the long duration is six months.In general,it can heal normally in about 60 days.This chronic ulcer is very similar to peptic ulcer through the naked eye observation and the histomorphological observation,which normal recurrence rate is about 30%.By detecting the changes of PH value of gastric juice in rats,observation of the gastric body samples,further microscopic pathological observation of the mucosal pathological sections at the model position,detecting the blood serum EGF through radioimmunoassay(RIA),we observed and discussed the treatment mechanism of WWF on the experimental ulcer in rats.Experimental Methods:1.SPF Wistar Male Rats:180～220g/rat,divided into nine groups randomly, eight rats for each group;①Normal Group②Model Group③Control Experiment Group④WWF High Dose Group⑤WWF Medium Dose Group⑥WWF Low Dose Group⑦Ranitidine Group⑧Baiji Sanqi Medium Dose Group⑨Baiji Sanqi High Dose Group.The rats in the Normal Group were given normal daily diet.The rats in the Model Group were killed after establishing model.The rats in the Control Experiment Group were observed for the healing of gastric ulcer,given normal diet after three days of establishing model,and killed at the same time with those in the drug groups.The rats in the remaining drug groups were given gastric perfusion with the corresponding drugs for 2ml/day/rat after three days of establishing model and killed after gastric perfusion for seven 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;vertically opened the abdominal walls for about 2cm layer by layer for the exposure of gastric body;pressured for 60 seconds with the filter paper with 5ram diameter and immerged by 100%acetic acid at the side serosal surface of gastric antrum nearby the juncture of anterior gastric wall and gastric antrum;placed the stomach in the body situ,sutured layer by layer and then disinfected the wounds with 75%alcohol.The rats were fasted for 24 hours but water was given 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.②Stripping stomach,ligating pylorus,snipping cardiac and taking out the stomach from the cardiac nearby duodenum and esophagus,crowing gastric juice onto the PH indicator paper for measured value.③Injecting 10ml 10% formaldehyde solution from cardiac into gastric cavity,ligating pylorus,placing into 10%formaldehyde solution for 30 minutes.④Opening stomach along the greater curvature,flattening,washing the inner gastric wall and searching ulcer surface at the model position;Selecting gastric tissue at the longest track of ulcer scar in longitudinal direction,obtaining and placing full-thickness gastric tissue into 10% formaldehyde solution and then further carrying through paraffin embedding and producing pathological sections.2.The selection,grouping,modeling,administration of experimental objects were the same as the above.Before collecting samples,the rats were fasted for 24 hours but water was given.4ml blood were sampled from each abdominal aorta after the rats were killed,the blood were transferred into vacuum tube and slant placed for 30 minutes.The tube was placed into low speed centrifuge,centrifuged for 30 minutes with the speed rate of 3000 r/min.2ml supernatant were transferred into EP tube,kept at the cold storage of-20℃and further for radioimmunoassay(RIA) analysis.Statistically:Lots of experimental data were showed with(?)±s.SPSS11.5 software was applied into One-way ANOVA for mean comparison based on multiple samples. When the variance was homogeneous,Least-significant difference(LSD) was applied for test and the value of P＜0.05 would mean significant difference.Major findings:1.Based on the observation of gross sample,healing of ulcer surface in the rats of drug groups showed a clear distinction with that of the Control Experiment Group and the Model Group.In particular,there was no perforation,less inflammatory exudation on ulcer surface which caused difficulty to identify ulcer surface,and no serious serous adhesion in the rats of the Ranitidine Group,the WWF Medium Dose Group and the WWF High Dose Group.Therefore thc healing effects of these three groups are better than that of the other groups.Based on the pathological observation by microscope,the WWF Medium Dose Group is closer to the Normal Group,which had basically repaired glands morphology,less inflammatory cells and no obvious pathological sub-mucous circulation.Therefore these two groups were in better recovery condition.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, and moderate atypical glands hyperplasia.In this group,the arterioles of serous layer showed an inflammatory pathological changes on the thrombus closed arterial intima. Based on the statistical results of PH value,these groups had F value of 4.202 and P value of 0.000 and showed significant difference.Of which,the Model Group had the highest value(3.288±0.300),which showed a significant difference with the three WWF groups and the Ranitidine Group(P＜0.05),and with the Normal Group(P＜0.01).2.The EGF in the rats of the Model Group were significantly higher than that of the Normal Group(the difference is significant,P＜0.01).The serum EGF concentrations of the drug groups were significantly lower than that of the Model Group(the difference between the drug groups and the Model Group is significant,P＜0.05,except of the BaiJi Sanqi High Dose Group).Of which,the Ranitidine Group, the WWF Medium Dose Group and the WWF High Dose Group dropped more obviously(P＜0.01),especially the WWF Medium Dose Group dropped to the lowest concentrations(0.253±0.055 ng/ml).Compared with the Model Group,the EGF of the Control Experiment Group also dropped,but the difference was not significant. Except of the BaiJi SanQi High Dose Group,the difference between the drug groups and the Control Experiment Group was significant(P＜0.05).Conclusion:1.The typical experimental model with strong stimulation established through treating acetic acid on the gastric serosa surface of rats has higher success rate, stronger feasibility and is easier to operate.2.Based on the observation of gross samples,healing of ulcer surface in the rats of drug groups had a clear distinction with that of the Control Experiment Group.In particular,the healing effects of the Ranitidine Group,the WWF Medium Dose Group and the WWF High Dose Group were better than that of the other groups.3.Based on the pathological observation by 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 sub-mucous circulation;therefore these two groups were in better recovery condition.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.The statistical results of gastric juice PH value showed that more serious ulcer, worse gastric acid secretion and higher gastric PH value(compared the Model Group with the Normal Group,P＜0.01).However the PH values of the three WWF groups were close to that of the Normal Group(compared the three WWF groups with the Model Group,P＜0.05) 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 the Western mcdicine. This is also in line with the results of observation on the gross samples.5.The measured results of serum EGF in rats showed that WWF has obvious effect for the healing of gastric ulcer.The data of the Ranitidine Group,the WWF Medium Group and the WWF High Group were closer to that of the Normal Group (0.239±0.432ng/ml)(compared the three groups with the Model Group,P＜0.01; compared the three groups with the Control Experiment Group,P＜0.05).Of which, the data of the WWF Medium Dose Group(0.253±0.055 ng/ml) was closest to that of the Normal Group.The efficacy of WWF was similar to that of Ranitidine,the routine drug for gastric ulcer.There was no statistical difference between them(the WWF Medium Dose Group has less difference).The BaiJi SanQi Group affected the serum EGF after modeling,but it was less effective than that of the three WWF groups and the Ranitidine Group.Of which,the effect between its high dose group and the Control Experiment Group had no significant difference,which showed that the efficacy of the combined detections of components in WWF is better than the efficacy of combination of BaiJi and SanQi.6.Combination of BaiJi and SanQi is the decomposed recipes of WWF.Based on the general observation,the pathological observation by microscope,the PH measurement and the serum EGF measurement,the mean PH value of the two decomposed recipes groups were higher than that of the three WWF groups and the Ranitidine Group.Of which,the difference between the BaiJi SanQi High Dose Group and the WWF Low Dose Group is significant(P＜0.05).In additional,the mean serum EGF value of the two decomposed recipes groups are 0.3050 and 0.3288 respectively,which were higher than the mean serum EGF of less than 0.2800 of the WWF Medium Dose Group,the WWF High Dose Group and the Ranitidine Group. Therefore,we can see that the combination of BaiJi and SanQi is less effective than WWF original formula and Ranitidine.In conclusion,the experimental model of this study is successful.WWF is effective for the treatment of gastric ulcer and the restoration of the normal physiological structure of gastric mucosa,so as to restore normal gastric acid secretion and maintain normal serum EGF concentrations.It showed that WWF has indirect immunity regulation function.The clinical usual dose of WWF is medium and the medium dose group has best efficacy which is equivalent to the efficacy of Ranitidine,the clinical usual acid inhibitor for gastric ulcer.However the decomposed recipes of WWF,i.e.combination of BaiJi and SanQi,are less effective than the original formula of WWF.
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