Screening And Identification Of Colon Mucosal Proteins In Patients With Irritable Bowel Syndrome With Diarrhea

[Background and Aims]Irritable bowel syndrome(IBS)is one of the most common functional gastrointestinal disorders.A variety of mechanisms are supposed in its pathogenesis,including abnormal gastrointestinal motility,altered gut sensitivity,brain-gut reactions,disturbed neuro-immuno-endocrine network and so on.However,the actual cells or molecules involved have not been identified.It is known that proteins provide the functions of cells and organs,and are closely associated with diseases.In our study,we focus on the screening of the related proteins to IBS with diarrhea(IBS-D)in order to reveal the possible clues or molecular mechisms for this disorder.[Materials and Methods]We enrolled five IBS-D patients on the RomeⅢcriteria and five healthy volunteers as normal control group.Biopsies were taken from the cecum and sigmoid colon under direct vision.Four pieces were taken separately at each site.Three pieces were processed by cold saline water,which contained 0.1%PMSF,and frozen in liquid nitrogen immediately while the other one was fixed in formalin and then embedded in paraffin three days later.For the proteomic procedures,we mixed equal amounts of each individual's cecum and sigmoid colon samples,extracted total proteins from the mixed samples and ran on the two-dimensional gel electrophoresis(2-DE).The proteins on 2-DE were visualized by the silver stain,and then gel maps were scanned and analyzed with the Image Master 2D Elite.Ten of the obvious abnormal protein spots were identified by the mass spectrometry.Western blot and immunohistochemistry were also performed for further verification of one of the proteins.[Results]Protein spots were 1504±69 in control group and 1356±104 in IBS-D group,with 41 spots significantly altered in abundance(Volume value=2.0 times).Among the 41 spots,11 protein spots indicated higher expression and 30 were lower in abundance.Five upregulated protein spots and five downregulated ones were identified by mass spectrometry.The upregulated proteins were ATP synthase subunit d,sulfotransferase 1A3/1A4,cytosolic acetyl-coA acetyltransferase, thioredoxin and aldehyde dehydrogenase 2.The downregulated proteins were immunoglobulin kappa chain(Igk),isobutyryl-CoA dehydrogenase, WD repeat-containing protein 1,T-complex protein 1 subunit alpha and alpha-enolase.Western blot showed Igk markedly reduced in IBS-D group compared with control(P＜0.05),and Igλwas not significant different between the two groups(P＞0.05).Igk and Igλwere expressed on plasma cells in the lamina propria,interstitial substance among colonic glands,small vessal wall and basal lamina of cecum and sigmoid colon by immunohistochemistry.In 24 IBS-D patients,Igk was lower in integrated optical density of immunostaining,compared with those in the 19 controls(P＜0.05).However,the difference of Igλbetween the two groups was not significant(P＞0.05).[Conclusion]The pathogenesis of IBS-D may be associated with the downregulation and upregulation of the following proteins:ATP synthase subunit d,sulfotransferase 1A3/1A4,cytosolic acetyl-coA acetyltransferase,thioredoxin and aldehyde dehydrogenase 2,Igk,isobutyryl-CoA dehydrogenase,WD repeat-containing protein 1,T-complex protein 1 subunit alpha and alpha-enolase. Igk was expressed on plasma cells,interstitial substance,small vessal wall and basal lamina of cecum and sigmoid colon.Both western blot analysis and immunohistochemistry showed Igk markedly reduced in IBS-D group compared with control group.