The Observation Of The Effect And Treatment Of The NF-κBp65Antisense Oligonucleotide On Intestinal Ivact Fibrosis Of Chronic Colitis Of Mice Induced By Trinitrobenzene Sulfonic Acid

Background:The inflammatory bowel disease (IBD) is a chronic, non-specific inflammatorydisease of gastrointestinal tract, including Crohn disease (CD) and Ulcerative colitis(UC). It is reported in the global that it is a continuous increasing trend of theincidence and prevalence of this disease and the related colorectal cancer, that causesgreat harm to people's health. The efficacy and safety of traditional and emergingdrug on the treatment of inflammatory bowel disease are not satisfied. Long-termchronic intestinal inflammation induces fibrosis and thus leads to intestinalobstruction. It is will be happened again that the intestinal fibrosis and strictureinduced by the enteritis or the change of extracellular matrix after the surgicalresection. Studies have shown that NF-κB plays an important role when colitis wasinduced by such as Dextran Sodium Sulfate (DSS), Trinitrobenzene Sulfonic acid(TNBS) and etc. So the NF-κB has become the target of the research anddevelopment on new drugs of inflammatory bowel disease treatment.Purpose:To investigate the effect of the NF-κBp65antisense phophorothioate oligode-oxynucleotide(ASOND)enema on the BALB/C mice with chronic intestinal fibrosisinduced by TNBS, and study the best treatment time of NF-κBp65ASOND.Observing the health conditions of the mice in each group, analyzing histologicalpathological changes, hyperplasia degree of collagen fibers, the contents ofIL-1Β,TNF-α, Col-Ⅲ α1mRNA and the protein contents of NF-κBp65, TGF-β1, toinvestigate the importance of NF-κBp65ASOND in the intestinal fibrosis treatment,explore the mechanism of NF-κBp65ASOND. Then provide experimental evidencefor a new potential development drug for IBD treatment and find a new way for IBDtreatment.Methods:BALB/C female mice weighing about19to23g were randomly divided to eight groups,12per group. The eight groups are: blank control group (blank group),TNBS model group (TNBS group), NF-κBp65missense oligonucleotide Ⅰ,Ⅱ,Ⅲgroup (MSONDⅠ,Ⅱ,Ⅲ group),NF-κBp65Antisense oligonucleotide Ⅰ, Ⅱ, Ⅲgroup (ASOND Ⅰ, Ⅱ, Ⅲ group). Blank group, twice of100ul physiological salineenema per week, for six weeks; TNBS group,100ul2mg TNBS/50%alcohol enemasix weeks continuous, the day before TNBS gets100ul physiological salineenema;group MSOND I and ASOND I,100ul2mg TNBS/50%alcohol enema sixweeks continuous, the day before TNBS of first two weeks, each group gets100ulMSOND or ASOND enema, and the last four weeks each group gets100ul physiol-ogical saline enema; group MSONDⅡand ASONDⅡ,100ul2mg TNBS/50%alcohol enema for six weeks, the day before the TNBS, each group gets the100ulMSOND or ASOND enema in the third and fourth weeks, and100ul physiologicalsaline enema in the rest four weeks; group MSONDⅢand ASONDⅢ,100ul2mgTNBS/50%alcohol enema for six weeks, each group gets100ul MSOND orASOND enema in the last two weeks, and100ul physiological saline enema in therest four weeks. All groups of mice get executed one week after the last enema forcolon tissue acquisition, evaluating the degree of inflammation of the colon tissue byHE staining, assessing the degree of intestinal fibrosis by VG staining, RT-PCRdetection of IL-1β, TNF-α and Col-Ⅲ α1mRNA contents, immunohistochemicaltests for the protein contents degree of NF-κBp65and TGF-β1of colon tissue.Result:1. The mice in group TNBS, MSONDⅠ, Ⅱ and Ⅲ, developed various degreesof symptoms and gradually worsened after the TNBS enema every time, andgradually reduced from the third to fourth days. The symptoms in the first threeweeks were worse than the last three weeks. The symptoms of group ASONDⅠafterthe TNBS enema in the first two weeks were lighter than other groups excepted theblank group,It was worse than the first two weeks in the third week while lighterthan others excepted the blank group, The fourth week was worse than the thirdweek,And it was begins to lessen from the fifth week;The symptoms of groupASONDⅡ after the TNBS enema were severe in the first two weeks and lessenedfrom the third week; The symptoms of group ASONDⅢ were more severe with the TNBS enema in the first three weeks, and lessened from the third week, especially inthe fourth and fifth week; The blank group had no obvious above symptoms.2. Group TNBS and MSOND groups could be found that congestion, edema,stiffness, twisted, distorted at the lesions colon, causing part of bowel stenosis bymacroscopic observation of the mice colon specimens. Enlarged PP lymph nodes andintestinal adhesions were seen in small part of the mice colon specimens. Thesymptoms of congestion, edema in the ASOND groups were lighter than the groupTNBS, MSOND groups. There were no obvious stricture and deformation in theintestine of ASOND groups, especially the group ASONDⅡ. Through the pathologyobservation, all the groups except the blank group could find various degrees ofintestinal inflammation and intestinal fibrosis symptoms, included inflammatory cellinfiltration, reduced number of goblet cells, glandular organ destruction, epithelialcell disruption in the lamina propria of colon tissues; the colon tissue microscopeobserved that a large number of collagen deposited under the mucosa and on theserosa, and the muscularis propria was significantly thicker. The inflammation scoresand fibrosis scores in the group TNBS and MSOND groups were higher than theblank group and ASOND groups (P<0.05). And the inflammation scores and fibrosisscores in the group ASONDⅠand Ⅲ were higher than the blank group and thegroup ASONDⅡ (P<0.05). The group ASONDⅡ's inflammation score was onlyhigher than the blank group (P<0.05). And it was no significant to compare thefibrosis scores between the group ASONDⅡ and the blank group (P>0.05).3. The contents of IL-1β, TNF-α and Col-Ⅲα1mRNA of the mice colon tissuein the group TNBS and MSOND groups were more than the blank group andASOND groups (P<0.05), while the contents of the group ASONDⅠand Ⅲ werehigher than the blank group and the group ASONDⅡ. The contents of IL-1βandTNF-α mRNA in the group ASONDⅡ was only higher than the blank group. It wasno significant to compare the Col-Ⅲ α1between the group ASOND and blank group(P>0.05)4. The protein contents of NF-κB p65,TGF-β1of the mice colon tissue in thegroup TNBS and MSOND groups were higher than the blank group and ASONDgroups (P<0.05). The protein content in the ASONDⅡ was less than the group ASONDⅠand Ⅲ, and only higher than the blank group (P<0.05)Conclusions:1.It confirms that TNBS induces the animal model of chronic intestinal fibrosisby observing the health conditions, colon gross specimens, colonic pathology andfibrosis of the mice of the TNBS group.2. The NF-κBp65ASOND reduces the inflammation and fibrosis of the colon inmice by decrease the proinflammatory cytokines like TNF-a, IL-1β and Col-Ⅲ α1'smRNA contents and NF-κBp65and TGF-β1's protein contents. The NF-κBp65ASOND could be a new effective drug for IBD therapy. On the other hand, the NF-κBp65MSOND has no above function.3. It is effective to inhibit the mice of colonic inflammation and fibrosis withNF-κBp65ASOND for two weeks. The effects are different for the interventiontimes. The medication effect is the best in the third and fourth weeks.