| Background and ObjectiveChronic atrophic gastritis(CAG)is one of the subtypes of chronic gastritis.Atrophic gastritis with intestinal metaplasia(IM)and/or dysplasia(Dys),are closely associated with gastric cancer.Atrophic gastritis,as a most common pre-cancerous condition of gastric cancer,is developed from non-atrophic gastritis.The incidence and detection rate of CAG increase with age.CAG has been thought as a serious threat to the health of our population.At present,for the etiology and pathogenesis is unclear,there is no effective drug treatment for CAG.CAG advances slowly,diagnosis and curative effect evaluation demands gastroscope inspection,and gastric mucosa pathological changes often uneven distribute,which makes difficult to conduct clinical trials on patients.Copying the animal disease model to shorten the duration of the disease and remove unnecessary factor for disease,especially to avoid ethical issues,can effectively promote the etiological study of the disease,also for the research methods of prevention and cure.Due to the complexity of the etiology,there is no ideal medicine for CAG.Syndrome differentiation of Chinese medicine treatment shows curative effect on CAG.Chinese medicine not only can alleviate the symptoms,but also reverse the pathological progress to slow down disease.At present the CAG rats model building method is numerous,but objective evaluation lacks.This project is to evaluate the superiority of two different composite methods with time to establish the model,success rate,serum pepsinogen,mortality.Use quantitative proteomics technology screening different proteins of chronic atrophic gastritis model rats and normal rats.Bioinformatics analysis for the differentially expressed proteins including Molecular Function,Cellular Component and Biological Process,then the Gene Ontology(GO)annotation,and Cluster of GO,in order to detect the biological function of differential proteins.Protein-protein interaction analysis and enrichment analysis of pathway were conducted to determine the metabolic pathway and the signal transduction pathways which differential proteins involved.And screening of target proteins,using enzyme-linked immunosorbent assay and Quantitative real-time PCR analysis for mRNA eacpression reaction to verified results.To observe the effects of prescription of Xinkaikujiang Yiqihuoxue principle on general situations,mortality and pathological changes of chronic atrophic gastritis rats.And to study the functions of this prescription for target proteins.The experiment is divided into the following three parts.1.The research for animal model of chronic atrophic gastritisMaterials and methodsExperimental animals are 60 SPF healthy male SD rats,randomly divided into three groups.Normal group 8 rats.MNNG with high salt mold heat starch paste group(M1)26 SD rats.MNNG with alcohol group(M2)26 SD rats.The normal group of rats were given an SPF normal water,and a 5ml saline solution every day.M1 group were ranitidine 0.03%fodder fed,free drinking 2%salicylic acid sodium solution,lavaged daily 120 mu g/mLMNNG once,fast 18 hours every week.Since the 14th week,lavaged high-salt hot starch paste after fast.M2 group were ranitidine 0.03%fodder fed,daily drinking freely 2%salicylic acid sodium solution,lavaged daily 120 mug/mL MNNG once,fast 18 hours every week.Since the 14th week,lavaged 35%alcohol.Evaluate the superiority of two different composite methods with time to establish the model,success rate,serum pepsinogen,mortality.Results1.MNNG with alcohol lavaged and MNNG with high salt hot starch paste are both success in replicating the CAG lesions.Gastric mucosa pathological integral,serum pepsinogen level showed no statistical difference between two groups(P>0.05).Animal mortality and complications of MNNG with alcohol model rats was lower.It can be used for subsequent application research of atrophic gastritis.2.Identification of Chronic atrophic gastritis model rats differentially expressed proteins in gastric mucosa3.Materials and methodsUsing quantitative proteomics TMT technology screened the difference of gastric mucosa protein beteen MNNG with alcohol chronic atrophic gastritis rats and normal rats.Bioinformatics analysis for the differentially expressed proteins includingMolecular Function,Cellular Component and Biological Process,then the Gene Ontology(GO)annotation,and Cluster of GO,in order to detect the biological function of differential proteins.Protein-protein interaction analysis and enrichment analysis of pathway were conducted to determine the metabolic pathway and the signal transduction pathways which differential proteins involved.And screening of target proteins.Results(1)through high-throughput proteomics identified the CAG rats model of gastric mucosa and normal gastric mucosa of rats,a total of 6937 differentially expressed proteins,and 363 proteins significant.Differential proteins mainly are the binding proteins,distribute in the cytoplasm and extracellular foreign body,the extracellular space,mitochondria and cytoplasm,sticky spot around the nucleus and cytoplasm regions,etc.They are involving in biological process of responsing to drugs,dealing with ethanol,dealing with hunger,protein hydrolysis,anoxia,complement other classical reaction,cell adhesion.Signaling transduction involved are sticky spot,MAPK signal pathways,amoebiasis,viruses cause cancer,closely connected,tumor of proteoglycans,complement system,protein digestion and absorption,etc.(2)According to the node connection degree analysis of protein,select FLNa,TGFR-betal,Bad protein,using ELISA and RT-PCR methods to verify the proteomics experiments.(3)By analyzing the difference proteins,the Casp3 decreased with Bcl-2 associated death promoter(Bad)increased.The Caspase family is downstream from the bcl-2 family.Bad’s effects on apoptosis depends on the combination of the bcl-2,and the proteomics results do not show the bcl-2 expression anomaly.In order to explore how the rise of Bad expression influences the regulation of apoptosis by real time PCR method to identify Bcl-2 mRNA expression in gastric antrum mucosa of the CAG model rats and normal rats3.The effect of prescription of acrid to diffuse and bitter to descend,benefiting qi for activating blood circulation in chronic atrophic gastritis rats and study on the functions of this prescription for target proteins.Materials and methodsThe 100 SPF SD rats were randomly divided into two groups based on their weight.There were 10 rats in normal group(N group)fed on an SPF standard diet,and the daily irrigation of saline water was done 1 times,continuing to the end of the experiment.The rest 90 rats was made into CAG,by ranitidine 0.03%fodder fed,daily drinking freely 2%salicylic acid sodium solution,lavaged daily 120 mug/mL MNNG once,fast 18 hours every week.Since the 14th week,lavaged 35%alcohol.Rats wre executed in the 18th,20,22,24 and 26 weekends,the mucosa was used to observe pathological changes.After the models were successful,the remaining rats were randomly divided into four groups by weight.Model groups(M group),model controlled way(group C),high dose group of TCM(group G),low dose group of TCM(group D).N,M group rats were put to death immediately after models success.Rats in group C drank the physiological saline 3 ml/kg to fill the stomach,once a day;Group G,group D lavaged respectively agreement preparation 5 ml of granules of TCM,respectively,the equivalent of granules 3 g/kg/D,1.5 g/kg/D(the equivalent of raw herbs 21.9 g/kg/D,11 g/kg/D)to fill the stomach,once a day.Treatment stage last 8 weeks,after that rats were put to death.Observed rats’ gastric mucosa pathological changes to evaluate the effect of Chinese medicine.Using enzyme linked immunosorbent assay(ELISA)determination of N,M group gastric mucosa TGF-beta 1,FLNa,Bad protein expression levels,Real-time polymerase chain reaction(RT-PCR)assay N,M group,the group G,gastric mucosa TGF-beta 1 D group,FLNa,Bad,the Bcl-2 mRNA levels.The results(1)traditional Chinese medicine(TCM)can significantly improve the CAG model rats’gastric mucosa atrophy(P<0.05),hyperplasia(P<0.05),intestinal integral compared with model group rats decreased,but no significantly statistical differences.(2)ELISA method for determination of TGF-beta 1,FLNa,Bad protein in the M and C group rat gastric mucosa express the increase in the N group(P<0.05);Rt-pcr assay group D,C group of rat gastric mucosa TGF-beta 1 mRNA,FLNa mRNA,Bad mRNA expression increased compared with rats in the N group(P<0.05),consistent with the results of proteomics.M group,the rats of group C and N group rats the Bcl-2 mRNA levels showed no obvious difference.(3)The RT-PCR determination of Chinese traditional medicine group of TGF-beta 1,FLNa,Bad mRNA levels can be fell back compared with rats in the M group and the C group(P<0.05).Conclusions(1)This research reference related literature and improved,compared with MNNG solution with alcohol and MNNG with heat starch paste.Results showed MNNG with alcohol is easier to operate,and has good stability,but time-consuming is longer,needs to be improved in the future.(2)High-throughput protein analysis showed that the CAG lesions involved multiple,complex biological pathways,affect the gelling spot,MAPK,closely connected,changes in proteoglycan,phosphatidyl inositol signal system.According to node and connection degree analysis of protein we selected FLNa,Bad and TGF-betal as the key proteins.ELISA and RT-PCR validation results were consistent with proteomics.(3)Chinese tradional medicine recipe can effectively improve the general situations of chronic atrophic gastritis rats,reverse the pathological changes of atrophy and gastric precancerous lesions and block the development of gastric cancer.Its effective mechanism may be reversing expression of FLNa、Bad、TGF-betal. |
