| Background:Peptic Ulcer(PU)is a common disease with a high clinical incidence.With the development of anti-ulcer drugs,it is no problem to heal it in the near future.However,the frequent recurrence of PU is a major obstacle and hotspot in clinical treatment.Gastric Ulcer(GU) of PU is a complex process involving many factors.Though some molecular pathogenesis studies on GU recurrence have been undertaken successfully on the gene(DNA)and transcription(mRNA)levels,the exact pathomechanism is still unclear.Currently,there still no effective and specific molecular marker for the evaluation of GU prognosis.The proteome analysis of gastric tissues may directly and comprehensively explore the pathomechanism of GU recurrence,which is expected to provide some useful clues for futher study of GU recurrence pathogenesis, precaution,diagnosis,treatment and novel drug discovery.Thus, Proteomics and its development and consummation offer new tools for GU recurrence research and help to probe the molecule mechanisms of GU recurrence process and ulcer-related diseases in the whole protein level.Objective:1.Investigate the gastric tissue proteins expression of GU recurrence rats induced by interleukin-1β(IL-1β)and the pathogenesy related to GU recurrence.2.Analysis the differential expression of gastric tissue proteins and effective mechanisms of GU recurrence rats induced by IL-1βintervention with JianWeiYuYang granule(JWYY).Method:1.The preparation of GU models and intervention with JWYY:①80 rats were randomly divided into four groups:normal control group (n=10),sham operated control group(n=10),GU model group(n=40)and JWYY group(n=20).All rats were induced ulcer by the method of Okabe acetic squash method with some modification except for the GU control group and sham operated group.JWYY physic liquor(0.081g/1ml/100g, once a day,totally 90 days)was given orally by gastric gavage to the rats of JWYY group and distilled water(1ml/100g,once a day)was given orally by gastric gavage to other groups.At 91stday,rats of GU model group were randomly divided into the GU model group and the GU model recurrence group.GU recurrence model rats were duplicated by IL-1β(1μg/kg)injected intraperitoneally except for the rats of GU model group which were injected intraperitoneally with saline.②At 8thand 92th day(92thday is a day which ulcer recurrence was induced 48 hours later), rats of each group were put to death twice planly and the stomachs were taken out.After stomachs were observed by macroscopic and the gastric ulcer index(GUI)was measured,the gastric tissues which contain ulcer (draw the materials from corresponding sites of stomach of rats without ulcer)were obtained for histological investigation by hematoxylin and eosin stain(HE stain),Two-dimensional electrophoresis(2-DE),Immunohistochemistry(IH), Reverse transcriptoin-polymerase chain reaction(RT-PCR) and Western blot.2.Proteome analysis of gastric tissues in GU recurrence rats and intervention with JWYY:In the first place,the total proteins of rat stomach tissues at different time spot including normal,GU model,GU model recurrence and JWYY group were separated by means of immobilized pH gradient(IPG)based 2-DE and Coomassie brilliant blue staining.The expression of differential protein spots was analyzed by PDquest and then identified by matrix-assisted laser desorption/Ionization time of flight mass spectrometry(MALDI-TOF-MS).The acquired peptide mass fingerprints(PMFs)were used to search for matches in SWISS-PROT and NCBI databases with Mascot software.The functions of identified proteins were further analysed according to bioinformatics resources.3.Validating of differential expression protein spots of Heat Shock Protein 27(HSP27)and Glucose-Regulated Protein 78(GRP78):In order to validate the reliability of the identified proteome analysis results,the expression of 2 differential protein spots HSP27 and GRP78 in protein and mRNA levels,which maybe correlated with GU recurrence and JWYY intervention,was detected by immunohistochemical,Western blot and RT-PCR analysis.RESULTS.1.The preparation of GU models and intervention with JWYY:At the 8thday after models were made,the ulcer foci in rats was observed macroscopically and histologically at different degree.GUI of GU model group was the highest,while the GUI of JWYY group was significantly lower than that of GU model group(P＜0.05).At the 92thday,the ulcer relapse rate were 90%and 20%in GU model recurrence group and JWYY group respectively,there was significant difference between the GU model recurrence group and the JWYY group(P＜0.01).2.Proteome analysis of gastric tissues in GU recurrence rats and intervention with JWYY:①The results showed that the well-resolved, reproducible 2-DE patterns of rat stomach tissues of GU recurrence, JWYY and normal group were obtained.At 8thday,for gastric tissues of normal,GU model and JWYY group,average spots of 3 gels were 935±29,1012±23 and 1002±43 and average matching rate were 92.7%, 94.6%and 92.6%.At 92thday,for gastric tissues of GU model,GU model recurrence and JWYY group,average spots of 3 gels were 1068±32,1109±24 and 1023±36 and average matching rate were 90.8%, 88.4%,91.5%.The average position deviation of matched spots were 0.901±0.125mm in IEF direction and 1.108±0.102mm in SDS-PAGE direction.②Analysis different protein spots of 2-DE each group,there were 282 different spots between the GU model group and normal group, 65 spots increased in GU model group while 217 spots decreased.At 8th, there were 216 different spots between JWYY and GU model group,157 spots increased in JWYY group while 59 spots decreased,there were 189 different spots between GU model recurrence and normal group,73 spots increased in GU model recurrence group while 116 spots decreased.At 92th,there were 168 different spots between JWYY and GU model recurrence group,115 spots increased in JWYY group while 53 spots decreased.③Through comparative proteome analysis of gastric tissues of normal,GU model recurrence and JWYY group,15 differential expression protein spots which maybe related to GU recurrence and JWYY intervention were identified by MS.The identified protein spots were categorized into several protein groups according to their functions,including molecular chaperones,cystoskeleton,substance metabolism and conveying,energy generate,anti oxidative stress,signal transduction and blood proteins.3.Validating of differential expression protein spots of HSP27 and GRP78:Used the methods of immunohistochemical,Western blot and RT-PCR,the expression of HSP27 and GRP78 was up-regulated in GU model group at 8thday(P＜0.01),while that was increased significantly in JWYY group compared with other groups at 8thday and 92thday(P＜0.01), which was consistent with our proteome analysis results.Conclusions: 1.JWYY can help to enhance the GU heal induced by acetic acid and cut down the GUI,improve QOUH and degrade the GU recurrence rate of GU recurrence rats induced by IL-1β.2.The 2-DE reference maps of gastric tissues of rats including normal group,GU recurrence group and JWYY group were established successfully.15 differential expression protein spots were identified.It indicated that the GU recurrence is a complicated process refer to diverse proteins participate and the comparison proteome analysis was a reliable method to sieve related protein spots for GU recurrence pathological changes.For several protein spots expression of gastric tissues in GU recurrence rats was regulated by JWYY,it provides contribution to the effect characteristic of multitarget.3.The expression of liSP27 and GRP78 in protein and mRNA levels with the methods of immunohistochemical,Western blot and RT-PCR analysis was consistented with our proteome analysis results.Thus identificated the stability and reliable of proteome analysis.On the other hand,the results suggested that the expression of HSP27 and GRP78 was coincidence in protein and gene transcription levels,JWYY could up-regulate the expression of HSP27 and GRP78 in protein and mRNA levels,which maybe one of the mechanisms of JWYY's anti-ulcer relapse effect. |
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