The Influence Of NF-κBp65 Antisense Oligonucleotide On Intestinal Tract Fibrosis Of Chronic Colitis Of BALB/C Mice Induced By Trinitrobenzene Sulfonic Acid

Background:Inflammatory bowel disease including crohn's disease and ulcerative colitis, it is a group of disease of gastrointestinal tract that etiology and pathogenesis are still not clear and,its features is chronic and repeated inflammation.the inflammation of crohn's disease exists in each layer of intestinal wall,long-term chronic inflammation causes a large of abnormal extracellular matrix deposited, and initiates the luminal fibrosis narrowing, eventually leads to intestinal obstruction. More than 40 percents of the crohn's patients have different degrees of intestinal obstruction, and of which about 80 percents eventually need endoscopic or surgical treatment, but the effect is actually unsatisfactory. However,the mechanism of intestinal fibrosis is not clear and there is lack of effective drugs to treat fibrosis.Many studies confirm that NF-κBp65 antisense oligonucleotide can down regulate the expression of NF-κB,thereby down regulate the expression of inflammatory cytokines, restrain the development of inflammation,if it can effectively reduce intestinal tract fibrosis of chronic colitis of experimental animal model, it will undoubtedly become new biological agent to anti-inflammatory and anti-fibrosis for inflammatory bowel disease.Objective:To explore the influence of NF-KBp65 antisense oligonucleotide by enema on colon fibrosis of chronic colitis of BALB/C mice which induced by TNBS/alcohol. Through analysing the disease activity index(DAI),the change of colon pathology, collagen fiber hyperplasia degree,NF-theκBp65 and TNF-a expression level of mice of each group,to research whether preventive use of NF-KBp65 antisense oligonucleotide can reduce intestinal fibrosis and inflammation induced by TNBS/ alcohol,then discusses the action mechanism of NF-κBp65 antisense oligonucleotide, and provides the theory basis of gene therapy for intestinal tract fibrosis of 1BD.Methods:BALB/C female mice,were randomly divided into four groups,they were control group,TNBS group,NF-κBp65 antisense oligonucleotide group,NF-κB p65 missense oligonucleotide group,each group has 12,and the weigh was range from 20 to 24 grams.The mice in control group were only given 100ul physiological saline,1 time a week, for 6 weeks continuously. And TNBS group, ASOND group, MSOND group were respectively given 100ul physiological saline,100ul NF-κBp65 antisense oligonucleotide(25nmol),100ul NF-κBp65 missense oligonucleotide enema at first,the following day,they were given 2mgTNBS+50% alcohol 100ul enema for 6 weeks continuously.A week after the last enema,all of the mice are killed, and collect colon tissue.the inflammation and the fibrosis degree of colon tissue is assessed by HE dye and VG dye respectively,NF-κBp65 protein expression level is assessed by immunohistochemistry.Results:1.The mice appeared different symptoms in 24 hours after each given TNBS enema, For example less activities and fed less,lasted 2 to 3 days,it would alleviate, in general,it was most serious at former 3 months,and each group had mice death,but at the fourth week,the symptoms becomed relatively stable, and the number of deaths decrease.The weight of the mice declined at 2 to 4 days after enema,but it will increased.Compared with control group,DAI (Disease activity index) scores of TNBS group,MSOND group and ASOND group were higher (P< 0.05), but DAI scores of TNBS group and MSOND group were higher than ASOND group (P< 0.05).2.Macroscopic observation of colon specimens as follow:blank control had normal colonic organization,no visible hyperemia,edema,and was felt soft;however, TNBS group and MSOND group, the colon tissue was still slightly reddening and swelling,and the most important,lesions colon becomed stiff, distortion,narrow, even some appeared that intestinal tract was adhered each other,intestinal tube becomed shorted,PP lymph node becomed bigger.However, ASOND group had not such change, except the control group, three groups appeared intestinal inflammation in different degree,such as fossae damage, inflammatory cells infiltrate lamina propria of colon tissue, the number of goblet cells decrease.The inflammation scores of TNBS group and MSOND group were significantly higher than control group (P< 0.05), but between ASOND group and control group, the difference was not obvious (P>0.05).3.In TNBS group and MSOND group inherent muscularis thickens obvious, a lot of collagen fibers deposits on submucosal, however,it was not so serious in ASOND group.Compared with control group and ASOND group,fibrosis hyperplasia in TNBS group was remarkable (P< 0.05), but between control group and ASOND group, there was obvious difference (P<0.05).4.We can find four groups have NF-κBp65 protein expression,but the positive rate of control group and ASOND group was obviously lower than TNBS group and MSOND group(P< 0.05),but between the former two groups,the difference was not evident(P> 0.05).In addition, positive cells mainly existed in epithelial cells in the former two groups,and mainly cytoplasm dyeing, however,many positive cells mainly existed in lamina propria in the later groups,and mainly nuclei dyeing.5.Compared with control group and ASOND group,the expression of TNF-αprotein in mice colon of TNBS group and MSOND group were increased (P< 0.05),but between control group and ASOND group,there was not statistically significant (P>0.05)Conclusion:1. Activity index(DAI), colon pathology and colon VG suggested that chronic colitis and bowel wall fibrosis animal model induced by 2mgTNBS+50%ethanol is successful.2. NF-κBp65ASOND can reduce the expression of NF-κBp65 protein and TNF-αprotein,however NF-κBp65MSOND had not such effects.3. NF-κBp65ASOND can reduce intestinal inflammation and intestinal fibrosis of BALB/C mice,but NF-κBp65MSOND had not such biological effects.4. NF-κBp65ASOND is promising to become the new gene drugs for treating the chronic inflammation and fibrosis of intestinal tract of IBD.