The Observation Of The Effect And Treatment Of The PLOD2Antisense Oligonucleotide On The Trinitrobenzene Sulfonic Acid Induced Intestinal Tract Fibrosis Of Chronic Colitis In Mice

Background:Ulcerative colitis(UC) and Crohn disease(CD) are two parts of Inflammatorybowel disease(IBD),as chronic and non-specific intestinal tract inflammation for itscharacteristic,its etiology and pathogenesis is unclear up to now,and people give highattention to it as its morbility is continue increasing in recent years.Long term chronicinflammation can stimulate intestinal wall fibrosis,and the lesion of Crohn diseaseinvolve a wide range,it’s intestinal wall full-thickness sexual inflammation,lead tomass extracellular matrix(ECM) abnormal sediment induce every dissection ofintestinal wall occur fibrosis,which can easy generate cicatrical stricture that lead toileus.At present,with regard to chronic intestinal wall fibrosis,the treatment outcomeof traditional therapies and surgery are undesirability and have poor prognosis,sothere is lack of full effective therapies yet.According to research findings domesticand abroad,the depositon of ECM is given priority to the depositon of collagen.Whereas lysyl hydroxylase(LH) plays an important role in compounding collagen andmaintaining its stable, in which LH2especially plays the key role， and PLOD(procollage-lysine,2-oxiglutarate,5-dioxygerase) encods LH,PLOD2is expected tobe a new research direction for prevent or treat chronic intestinal wall fibrosis.Purpose:Priliminary study has shown that use trinitrobenzene Sulfonic acid (TNBS)enema on the BALB/C mice can succeed in inducing the modeling of chronicintestinal wall fibrosis.On this basis,We synthesis PLOD2antisense phophorothioateoligode-oxynucleotide（ASOND）and explore the effect and the mechanism of itenema on the BALB/C mice with chronic intestinal fibrosis.Via compare DAI ofevery group laboratory mice,observe pathologic changes and the hyperplasia degreeof collagen fibers of colon tissue,comparative analysis the expression level of thecontents of TNF-α,PLOD2,Col-ⅢmRNA and the protein contents of Col-Ⅲ,thenanalysis the effect of PLOD2ASODN on the TNBS induced chronic intestinal fibrosis in BALB/C mice and explore the mechanism of it,to provide the experimentaland theoretical basis for the treatment of PLOD2ASODN on chronic intestinal wallfibrosis and the exploitation of related gene therapy drugs.Methods:Divided forty weight about20to25g,yearling6-8weeks female BALB/C miceinto four groups randomly,10per group, respectively are normal saline blank controlgroup (blank control group), TNBS model group (TNBS group),PLOD2missenseoligonucleotide negative control group(MSODN group) and PLOD2antisensephosphorothioate oligodeoxynucleotide treatment group(ASODN group).Adoptenema on the BALB/C mice to establish animal model,give twice of continuous100ul physiolo-gical saline enema per week to Blank group;TNBS group give100ul2mg TNBS/50%alcohol enema first then give100ul physiological saline enema24hours later per week; PLOD2MSODN group and PLOD2ASODN group were allgiven100ul2mg TNBS/50%alcohol enema firstly and then respectively given100ulMSOND and ASOND enema24hours later per week;Every group continuouslyenema six weeks,every group of mice will be executed by cervical dislocation methoda week after the last enema.Then gather colon tissue from every mice and will beused as follows:assessing the degree of inflammation of the colon tissue by HEstaining, assessing the degree of fibrosis of the colon tissue by VG staining,detectionthe express of the contents of TNF-α,PLOD2,Col-Ⅲ mRNA by RT-PCR and theexpress of the protein contents of Col-Ⅲ by immunohistochemical.Results:1. TNBS group,MSODN group and ASODN group appeared varying degreesof symptoms after every enema,such as eat less,body curled up,loose stools, stoolsoccult blood even the naked eye bloody stools,fur dull and so on,but these symptomsgradually relieved after three or four days.The weight of most mice had varyingdegrees of decline at the second to the forth day,but part of them gained weight afterthe forth day,even exceed the weight before enema.During the whole experiment,theheaviest symptoms focus on the first three weeks,then the symptoms relieved after theforth week.Three groups of mice all had individual death in the first three weeks.The situation of feed and activity in blank group had no significant change and no micedied. The DAI score of blank group is lower than the other three groups and hasstatistical significance(P<0.05),but the other three groups compared between groupshave no statistical significance (P>0.05).2. Macroscopic observe the mice colon specimens,we found that TNBSgroup,MSODN group and ASODN group have varying degrees of congestion, edema,adhesion and so on,split the intestinal wall we can see varying degrees of erosion andulcer, part of bowel appeared incrassation, stenosis, stiffness and distortion,but blankgroup had no above changes.Microscopic observate the colon tissue of HE stainingwe can find there is no obvious inflammation manifestation in blank group,groupTNBS,MSODN and ASODN all can find inordinately broken of epithelial cells,glands and crypts,reduced number of goblet cells,infiltrated of inflammatory cellssuch as lymphocyte and monocyte, hyperplasia of lymphoid follicle,part of colontissue had ulcer in mucous layer even reached deep in muscularis mucosae.Theinflammation score of group TNBS,MSODN and ASODN were higher than blankgroup(P<0.05), and the three groups compared between groups have no statisticalsignificance (P>0.05). Microscopic observate the colon tissue of VG staining,therewas no obvious fibrosis change in blank group,group TNBS and MSODN had plentyof collagen deposit in colon tissue,incrassated of muscularis propria inordinately,evencould see septate fiber.But the degree of the deposit of collagen and incrassated ofmuscularis propria in group ASODN was lighten than group TNBS and MSODN.Thefibrosis score of group TNBS,MSODN and ASODN were higher than blankgroup(P<0.05),group TNBS compared with group MSODN there was no statisticalsignificance (P>0.05),but the score of ASODN was lower than group TNBS andMSODN(P<0.05).3. The expression of PLOD2mRNA in group TNBS and MSODN were higherthan group blank and ASODN(P<0.05),group ASODN was higher thanblank(P<0.05), but there was no statistical significance between group TNBS andMSODN(P>0.05).For the expression of TNF-αmRNA and Col-IIImRNA,groupblank was lower than the other groups(P<0.05),but there was no statisticalsignificance in the other three groups(P>0.05). 4. For the expression of the protein contents of Col-III in mice colontissue,group TNBS,MSODN and ASODN were higher than blank group(P<0.05),andgroup ASODN was lower than group TNBS and MSODN(P<0.05),but there was nostatistical significance between group TNBS andASODN(P>0.05).Conclusion:We adopted PLOD2ASODN to therapy the chronic intestinal fibrosis induced byTNBS/50%EtOH in mice,via inhibited the expression of PLOD2,lower the activity ofLH2,thus it could reduce the synthesis of Col-Ⅲ and lower the degree of intestinalfibrosis.