| The incidence of gastric disease has increased in recent years,and many gastric diseases are related to gastric mucosal injury.Heavy or long-term drinking is one of the main causes of gastrointestinal diseases.Therefore,it is significant to establish an alcoholic gastric mucosal injury model.Plant polysaccharide not only has little toxic and side effects,also has the function of protecting gastric mucosa.Bletilla striata polysaccharide is one of the plant polysaccharides.It has been reported that the Bletilla striata polysaccharide has significant effect on the protection and treatment of acetic acid,stress and ethanol type gastric ulcer in mice.In the existing research reports,there are few studies on alcoholic gastric mucosal injury models,and the mechanism of action needs further research.Also there is no report on the role of Bletilla striata polysaccharides in protecting gastric mucosa in vitro.In the pre-laboratory study,the resources of Bletilla striata were analyzed,and the structure of Bletilla striata polysaccharide was identified.On this basis,this subject establishes an alcoholic gastric mucosal injury model in vivo and in vitro,and studies the protective effect of Bletilla striata polysaccharide on the gastric mucosa,and further studies the mechanism.The purpose of this study is to develop Bletilla striata polysaccharide as a health care drug and provide experimental evidence for its application in drinkers.The main contents and results are as follows:1.The preparation of Bletilla striata polysaccharideThe crude polysaccharides from Bletilla striata were extracted by water extraction and alcohol precipitation.The crude polysaccharides from Bletilla striata were obtained by debinding,repeated freezing and thawing,removing protein and dialysis.It was further purified by DEAE-52 cellulose column chromatography and dextran gel G-150 column chromatography,and ultra pure water was selected as eluant.The neutral polysaccharide with homogeneous molecular weight was obtained,and the content of BSP was 92.7%by phenol-concentrated sulfuric acid method.2.Protective effect of BSP on ethanol induced human gastric epithelial cell line(GES-1)(1)Using ethanol as a damage medium,we established an ethanol induced injury model of GES-1 cell line by measuring cell viability.When ethanol concentration was 100 M and the action time was 6 h,the cell state was stable,and the survival rate was about 50%.So the final concentration of ethanol is 100 M,and the time of action is 6 h,which is the condition of making model.It is concluded that ethanol induced gastric mucosal injury is concentration dependent.Because ethanol has volatiles,with the prolongation of its action time,ethanol concentration decreases,cell viability increases,and cell damage is reduced.(2)LDH method was used to detect the toxicity of BSP with different concentrations(0.25 mg/ml,0.5 mg/ml,1 mg/ml,2 mg/ml,4 mg/ml,8 mg/ml)on GES-1 cells.The results showed that BSP of 0.25?4 mg/ml was not toxic to GES-1 cell line,and it had obvious toxicity at 8 mg/ml.(3)The effects of Bletilla striata polysaccharides at different concentrations(0.25 mg/ml,0.5 mg/ml,1 mg/ml,2 mg/ml,4 mg/ml,8 mg/ml)on the activity of GES-1 cells at different time(12 h,24 h,48 h)were observed.The result of CCK-8 method showed that BSP promoted proliferation of GES-1 cell line in 0.5?4 mg/ml,and the proliferation was not obvious at 0.25 mg/ml(P>0.05).At 8 mg/ml,cell viability decreased and cell growth was inhibited,which was toxic,which was consistent with the result of LDH assay.(4)The protective effects of BSP at 0.25 mg/ml,1 mg/ml and 4 mg/ml were observed.After BSP cells were pretreated with 24 h,the cells were damaged by ethanol.Two methods of CCK-8 and Live/dead staining were used to observe the survival of the cells,and 4 mg/ml BSP had protective effect on GES-1(P<0.05).However,when the concentration of BSP is 4 mg/ml,the concentration of drug is high and the solution is thicker.There is no effect on protection of cell in vitro.3.Protective effect of BSP on acute gastric mucosal injury in mice.(1)Establishing an acute ethanol type gastric mucosal injury model:The mice were divided into normal group,model group,BSP high dose group(100 mg/kg),middle dose group(50 mg/kg),low dose group(25 mg/kg)and positive control group(OMe),each group was 10.Normal saline was given to the normal group and model group.The other groups were given different concentrations of BSP and OMe respectively.After 10 days of continuous feeding medicine,except normal group,the other groups were given anhydrous ethanol at 0.009 ml/g,and the fasting was 48 h before treatment,so that the models of each group were as consistent as possible.Through naked eye observation and Guth score,we could see that the model was successful.(2)Observing the protective effect of BSP on acute ethanol type gastric mucosal injury:Compared with the model group,result of Guth score shows that the gastric mucosal damage index of each group was reduced.However,there was no significant change in pepsin content in each group.The protective effect of 100 mg/kg BSP was similar to that of the positive control group.H&E staining showed that BSP could reduce bleeding and infiltration of inflammatory cells.It indicates that BSP has an obvious protective effect on acute alcoholic gastric mucosal injury.4.Protective mechanism of BSP on alcoholic gastric mucosal injury in miceThe activity of SOD,GSH-Px,CAT,MDA and LPO in the homogenate of gastric tissue was measured.Compared with the model group,BSP increased the vitality of SOD,GSH-Px and CAT,and reduced the content of MDA and LPO.Gastric paraffin sections in each group were examined by Tunel staining to detect gastric tissue apoptosis.The apoptotic index was observed and calculated.Compared with the normal group,the apoptosis rate in the model group was significantly higher than that in the normal group(P<0.001),and the apoptotic index in the drug delivery group was lower than that in the model group.The expression of PGC-1a,HO-1,Bcl-2,Bax and NF-kB mRNA in gastric tissue was detected by real time fluorescence quantitative PCR,and the expression of HO-1,PGC-1a and Bcl-2 were up-regulated,and the expression of Bax and NF-kB was decreased.It is concluded that BSP has protective effect on gastric mucosal injury induced by ethanol in vivo.Presumably,it may play a protective role by enhancing the antioxidant capacity of mice and inhibiting the apoptotic pathway.The protective effect of BSP in vitro is not significant. |
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