| Objective:To explore different feature of the BALB / C mice colitis model induced by different does of TNBS ethanol enemas,and study the best animal model suited for this experimental requirements which determined by the DAI,survival rate,pathological changes and so on.Methord:BALB / C mice which were fasted but drunk water for 24h before the experiment,were randomly divided into 5 groups and each group had 16 of them. The mice in first group were administered intracolonically with 1.0mg TNBS in 50% ethanol enemas once in the 1st day , the other groups were administered intracolonically with 1.5,2.0,2.5mg TNBS in 50% ethanol enemas respectively,and TNBS was raplaced with saline in the control grpup,All mice were examined for signs of colitis including DAI,weight loss,stool consistency,and feces occult blood,we calculated survival rate per day,and calculated gross survival rate on day 7. Mice were killed under anaesthesia on day 7,colon tissue were collected for the following experiments , which included inflammatory scores and mucosa morphological changes under light microscopy and the level of NF-κB p65 in intestinal mucosal examained by immune histochmistry method.Result:DAI in all groups(in order of TNBS l.0mg,1.5mg,2.0mg,2.5mg,control group)respectively is 5.12±0.68,6.36±0.71,11.54±1.28,12.75±1.62,0;Inflammatory scores is 4.26±0.78,5.26±0.98,10.32±1.15,10.72±1.35,1.95±0.50;Survival rate is 100%,93.7%,62.5%,37.5%,100%;The positive cell of NF-κB p65 is 23.06±2.88,25.26±2.37,50.26±4.26,55.26±4.68,1.26±0.86. We can found that from the results: With the increase in TNBS dose , the DAI score , intestinal histopathological score increased gradually,these in 1.0mg,1.5mg TNBS group,compared with TNBS 2.0mg,2.5mg group had statistical difference (P<0.05),compared with these in saline control group had statistical difference too(P<0.05), but there are no significant difference between 1.0mg and 1.5mg TNBS group(P>0.05),and no significant difference between 2.0mg and 2.5mg TNBS group(.P>0.05)With the increase in TNBS dose,the survival rate decreased rapidly,there are statistical differencein 2.0mg TNBS group compared with 1.0,1.5,2.5mg TNBS group(P<0.05). The positive cells of NF-κB p65 are statistical difference(P<0.05)between 2.0mg and 2.5mg TNBS group,and no statistical difference in 2.0mg TNBS group compared with 1.0mg,1.5mg TNBS group(P>0.05).Conclusion:We can find that 2.0 mg TNBS in 50% ethanol enemas can finally induce the best animal model for research from comprehensive comparison of the performance of mice,the survival rate and intestinal pathological changes.and so on,So 2 mg TNBS is the best dose of enemas in the experiment. Objective:To evaluate the effects of NF-κB p65 antisense oligonucleotides on inflammation,expression of cytokines such as tumor necrosis factor alpha (TNF-α),interleukin(IL-1β,IL-10)in colonic intestinal mucosa of experimental colitis in BALB/C mice,and to study the effects and mechanisms of NF-κB p65 antisense oligonucleotides treatment to experimental colitisMethods: The 36 BALB/C mice established IBD model induced by 2,4,6- trinitrobenzene sulfonic acid(TNBS). were randomly divided into models control group(UC),antisense oligonucleotides treatment group (ASOND)and missense oligonucleotides treatment group (MSOND). Moreover, a normal control group was used for comparison. In ASOND group and MSODN group Antisense oligonucleotides or missense oligonucleotides were instilled into colon by 25 nmol ever mouse at next 24 hour after the model established. all mice were killed on day 7, colon tissue were collected for the study of Inflammatory scores ,the expression of TNF-αmRNA,IL-1βmRNA measured by RT-PCR,positive cells of TNF-α,IL-1β,IL-10,NF-κB p65 in colonic mucosa examined by immune histochmistry methodand the level of NF-κB p65 in nuclear extraction of colon tissue by Western blot .Result:DAI in all group(sin order of NC,ASODN,MSODN,UC group)respectively is 1.02±0.26,4.36±0.54,11.76±1.21,12.02±1.18;inflammatory scores is 1.95±0.50,5.26±0.88,10.40±1.28,10.32±1.25;the expression of TNF-αmRNA measured by RT-PCR is 0.36±0.04,0.64±0.05,1.29±0.05,1.25±0.07,IL-1βmRNA is 0.21±0.04,0.46±0.03,1.00±0.07,1.00±0.03;positive cells of TNF-αexamined by immune histochmistry s-p method is 64.17±11.29,82.68±14.30,166.49±11.63,168.48±11.89,IL-1βis 11.68±3.52,42.42±5.77,120.43±28.2,115.74±27.63 , IL-10 is 168.32±10.66,146.68±6.02,58.44±10.92,62.50±11.57,NF-κB p65 is 2.42±1.37,22.66±5.32,61.09±3.73,58.38±4.49;the level of NF-κB p65 in nuclear extraction of colon tissue examined by Western blot is 0.372±0.02,1.05±0.09,2.34±0.09,2.36±0.08. From the results We can find that: DAI,inflammatory scores,the expression of TNF-αmRNA,IL-1βmRNA,positive cells of TNF-α,IL-1β,NF-κB p65,the level of NF-κB p65 in nuclear extraction of colon tissue in ASODN group decreased compared with these in UC group,MSODN group(P<0.05),increased compared with NC group(P<0.05),but positive cells of IL-10 increased in ASODN group compared with these in UC group,MSODN group(P<0.05). decreased compared with NC group(P<0.05),there are no statistical difference in all indexes between UC group and MSODN group(P>0.05).Conclusion:1,The expression of NF-κB p65 in the colonic mucosa of animals with experimental colitis decreased indicates NF-κBp65 ASODN can attenuate NF-κB2 NF-κB p65 ASOND can effectually abrogates some proinflommatory such as TNF-α,IL-1βcytokine or promote other proinflommatory as IL-10 cytokine expression, all of those indicate that ASODN could be useful for the treatment of inflammatory bowel disease.,it would become a new kind of medicine in treating IBD. |
