The Effects Of Rebamipide On Tumor Necrosis Factor And Prostaglandin E₂,Nitric Oxide In Rats With Acute Alcoholic Gastric Injury

PrefaceRebamipide is the only kind of drug which has the function of both anti — free - oxygen - radical and stimulate synthesis of endogenic prostaglandin E₂ (PGE₂) , which may increase glycoprotein complex of gastric mucus and promote epithelial cells of injuried site proliferation. But its protective effect in a-cute alcohol gastric mucous membrane injury and its relationship with PGE₂, NO and TNF - a in repairing mechanism was seldom studied.In this study, acute gastric mucous membrane injury model was induced by injecting certain amount of alcohol in rats stomach. Observe rats' gastric mucous membrane pathological change and TNF - a localization and dynamic variation of PGE₂, NO at 0 min, 15 min, 30 min, 60 min. At the same time, compare it with mucous membrane which use Rebamipide intervene to further discuss the mechanism of acute alcohol gastric mucus and offer treatment.Experiment AnimalsPrepare 36 8 - week - aged male Wistar rats, whose weight are 170g ± 10g, offered by CMU experiment animal department, 8 in each cage to feed. Feed them with balance feedstuff for one week in room temperature and stable moisture with day - night period. Then, divide them into contrast group, alcohol group and prevention group, each of which contains 12 rats.Method1. Animal model and gastric juice amount, gastric mucous membrane injury index calculationEach group with the method of injecting alcohol through pipe in mouth. Alcohol group should be poured absolute alcohol lOg/kg;before pouring alcohol to prevention group , give Rebamipide lOmg/kg;pour equal volume of 0. 9% NaCl to contrast group.Kill 3 rats of each group at 0 min, 15 min, 30 min, 60 min, cardiac ex-sanguination to measure serum NO and plasma PGE2;open abdominal cavity, ligate cardia and pylorus, measure gastric volume first, then calculate gastric mucous membrane injury index.2. Pathological examinationTake obvious erosive gastric mucous membrane 0. 5 x 0. 5cm, fix it with neutral formallin, embed it with wax, and prepare 4|xm continuous slices, stain it with HE, observe pathological change of gastric under electrical microscope. Select at least 5 visual fields on each slice including center and peripheral.3. To measure TNF - a with Immunohistochemistry ABC methodAfter routinely Immune Immunohistochemistry staining as guider says, dehydrate, transparentize, envelop with neutral balata, observe under microscopy. Use PBS instead of antibody as negative contrast, cells with brown yellow granules in plasma are positive cells, calculate gastric mucous membrane TNF - a light density of alcohol group % prevention group and contrast group separately.4. Measurement of plasma PGE2 and serum NOMeasurement of plasma PGE2 and serum NO with ELISA and nitric acid — reductive enzyme method, strictly operate as guider of reagent box. Normal values are 20. 6 - 34. 2 pg/ml and 65.7 ± 15. 8|xmol/L.5. Statistical analysisStatistical analysis use SPSS11.5 software, measure data use average ± SD to express, use t - test to compare between groups, use ANOVA test inter-groups.Result1. General situationIn the course of making model, alcohol group rats appeared drunk phenomenon such as conjunctiva congestion, restless, vomitted etc. Prevention group rats reacted lightly.2. Pathologic change of gastric mucous membraneEpithelial cells degenerated on alcohol group rats'gastric mucus, some des-quamation, erosion, exudation could be seen. There was congestion and edema in mucus. Glands were injured. But only congestion and edema could be seen in prevention.3. TNF — a expression in gastric mucusCompare alcohol group and prevention group with contrast group, TNF - a expression increased in mucous, with obviously difference (P <0. 05). There was also obvious difference between alcohol group and prevention group (P <0. 05). As the prolong time of alcohol stimulating, alcohol group still increased more obviously than prevention ( P <0. 05 ) .4. Serum NO and plasma PGE2 concentration of each group30 mins after drinking, serum NO level of alcohol group was obviously under contrast group and Rebamipide prevention group (P <0. 01) , but there was no difference between prevention and contrast group 15 mins. After drinking PGE2 level of alcohol group and prevention group obviously reduced ( P < 0. 01) , but there was no difference between them;30 mins after drinking, PGE2 level of prevention group increased obviously, obvious difference with alcohol group (P<0.05);60 mins after drinking, PGE2 of prevention group even exceeded that of contrast group ( P < 0. 01) .5. Gastric fluid volume and gastric mucous membrane injury indexNo matter alcohol irritation or drug interruption, gastric fluid volume increased quickly and kept at a certain level, which was obviously different with contrast group, but there was no difference between alcohol group and prevention group;mucous membrane seriously injuried after alcohol irritation, whichwas obviously different with control group ( P < 0. 01 ) , but injury degree reduced after drug interfering, which was obviously different with alcohol group (P <0.05).DiscussionGastric mucous barrier (consist of mucous membrane, mucous - bicarbonate barrier, microcirculation, etc. ) can be destroyed by exogenous or endogenous factors, producing many kinds of inflammatory media and cytokines, such as TNF — a, which lead to or worsen the injury of the gastric mucous membrane. Alcohol is one of the exogenous factors. Many kinds of anti - injury mechanisms are motivated as the injury of tissue, including the producing and releasing some of biologic active substances, such as PG, NO, EGF and so on, which play an important role in the protection and repair of mucous membrane of digestive tract.In this study, as the increasing of the alcohol action time, the epithelium of the gastric mucous membrane degenerated, some of which took off and erosion, exudation, congestion and swelling in the mucous membrane and the injury of glands could be seen;the expression of TNF - a of the gastric mucous membrane was sharply increased (p <0.05). And as the increasing of the alcoholic stimulating time, the expression of TNF - a gradually increased, leading to the acute injury of the gastric mucous membrane.The level of PG and NO decreased apparently as the increasing of the alcoholic acting time. After taking alcohol for 15 mins, the plasma level of PGE2 of alcoholic group decreased apparently compared with the control group (p < 0. 01) , and decreased rapidly than NO. But after taking alcohol for 60 mins, the level of PGE2 increased apparently even to the level before taking alcohol. This is due to harmful object do harm to the gastric mucosa and assumed the tissue PG rapidly ( PG is not stored in cells, but is produced as soon as it has been secreted ). Without harmful object loading, the PG has been secreted rapidly to repair the injury tissue and act a " cell protection" role.Rebamipide, a kind of protector of gastric mucous membrane, (commodityname;Mucosta) , is a kind of remarkable gastric mucous membrane repairing medicine, which can rise the level of PGE2 in gastric mucous membrane of animals and human , make the mucus greater in volume in the superficial layer of the gastric mucous membrane, and increase the soluble mucus and blood flow of gastric mucous membrane, promoting the healing of ulcer and so on. In the consequence of the research, Rebamipide can not only inhibit the expression of TNF — a in tissue, but also increase the level of NO and PG in blood, which may protect cells and promote the repair of injured tissue.Conclusion1. The research demonstrated and reappeared that alcohol could cause the acute injury of the gastric mucous membrane.2. The stimulation of alcohol to the gastric mucous membrane could increase the expression of TNF - a, while decrease the level of NO and PGE2.3. Rebamipide can obviously prevent the tissue from alcohol, reduce the expression of TNF - a in tissue and increase the level of NO, PGE2.