Irritable Bowel Syndrome And Abnormal Expression Proteins From Colonic Mucosa

[Background and Aims]Irritable bowel syndrome(IBS) is one of the most common functional gastrointestinal disorders(FGIDs).Although IBS has been intensively investigated in recent years,its pathogenesis remains unexplained.The previous study from our group applied proteomics methods to screen and identify abnormal expression of proteins in IBS with diarrhea(IBS-D) by the two dimensional gel(2-DE) and the mass spectrometry.we first identified the nine abnoamal proteins,which include alpha-enolase(α-enolase),aldolase A (ALDOA),ATP synthase subunit d(ATP5H),cytosolic acetyl coA acetyltransferase(CT),thioredoxin(Trx) and aldehyde dehydrogenase 2 (ALDH2),isobutyryl-CoA dehydro-genase(ACAD8),WD repeat-containing protein 1(WDR1) and T-complex protein 1 subunit alpha(tcp-1α).In this study, Western blot,immunohistochemistry staining(IHC) and fluorescent quantitation polymerase chain reaction(QPCR) were performed to verify the proteins. ATP content was detected in colonic mucosa from the IBS-D group,IBS with constipation(IBS-C) group and normal control group.All these researchs may provide new clues to further research on the molecular mechisms of IBS.[Materials and Methods]Eighteen IBS-D patients,18 IBS-C patients fulfilling on the RomeⅡand RomeⅢcriteria and 18 healthy volunteers as normal control group were enrolled in our current study.Biopsies were taken from the mucosa of cecum and sigmoid colon through standard colonoscopy.Five pieces were obtained separately at each site.Four out of five pieces were processed by cold saline water,and frozen in liquid nitrogen immediately while the other one was fixed in formalin and then embedded in paraffin three days later. Western blot and IHC were also performed for further verification of the proteins. Fluorescent QPCR were carried out for observing the level ofα-enolase mRNA, ACAD8 mRNA,Trx mRNA.Due to the limited sample numbers,samples were divided into three groups(a,b,c).Each group included 6 IBS-D patients,6 IBS-C patients and 6 healthy volunteers.The "a" group was applied to identify the expression of ALDOA andα-enolase and TCP-1α.The " b " group was applied to identify the expression of ACAT2 and Trx and ACAD8.The "c" group was applied to identify the expression of ATP5H and WDR1 and ALDH2.We enrolled 7 IBS-D patients,6 IBS-C patients both fulfilling on the RomeⅡandⅢcriteria and 5 normal controls.ATP content in colonic mucosa was observed by high performance liquid chromatograph(HPLC).[Results]Western blot and IHC showed that the expression of ALDOA,α-enolase and Trx were markedly decreased in mucosa of cecum and sigmoid colon from IBS-D and IBS-C groups than that of normal controls.α-enolase mRNA was markedly downregulated in mucosa of cecum from IBS-D.Trx mRNA was downregulated in mucosa of cecum from IBS-D and IBS-C groups.Trx mRNA were significantly upregnlated in mucosa of sigmoid colon from IBS-D and IBS-C groups comparing with control subjects.The expression of CT was significantly upregulated in mucosa of cecum from IBS-D and IBS-C groups.ACAD8 and ATP5H were significantly downregulated in mucosa of sigmoid colon from IBS-D groups and IBS-C groups.ACAD8 mRNA was downregulated in sigmoid colon from IBS-D and IBS-C groups.ALDH2 was markedly lower in cecum from IBS-D and IBS-C groups.ALDH2 was markedly higher in sigmoid colon from IBS-D and IBS-C groups than control.TCP-1αwas markedly lower in cecum and sigmoid colon from IBS-D and IBS-C groups. WDR1 were statistically significantly higer in sigmoid colon from IBS-C group. There were statistical significance on abnormal expression of above-mentioned proteins from colonic mucosa(P＜0.05 or P＜0.01).ATP content was markedly lower in sigmoid colon from IBS-C group and IBS-D groups(p＜0.05). [Conclusion]ATP5H,ACAD8,ALDH2 belong to "mitochondrial enzymes" that are abnormal in colonic mucosa from IBS group.The results indicated that mitochondrial molecule or function defect may involve in the pathogenesis of IBS;α-enolase,ALDOA,ACAD8,CT,ALDH2,ACAD8 were metabolism enzymes.The abnormal expression of the enzymes and reduction of ATP content showed that colonic mucosa from IBS patients may involve in dysregulation of energy metabolism.The abnormal expression of TCP-1α, WDR1 may influence colonic cystoskeleton,cellularity,cellular morphology and cellular movement.The different proteins had different expression in different area from colon which suggests complicated pathogenesis of IBS.