Pim-1抑制剂对TNBS诱导的小鼠炎症性肠病的疗效

Effects of Pim-1 inhibitor on mouse model of inflammatory bowel disease induced by TNBS

目的:观察莫洛尼小鼠白血病病毒的前病毒插入点激酶1(provirus integration site for Moloney murine leukemia virus kinase 1,Pim-1)抑制剂对2,4,6-三硝基苯磺酸(2,4,6-trinitrobenzenestdfonic acid,TNBS)诱导的小鼠炎症性肠病的治疗作用,同时研究Pim-1抑制剂对肠黏膜中T细胞分化的影响。方法:将45只BALB/c小鼠随机分成5组(n=9):溶媒对照组、TNBS模型组、小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组。用TNBS和乙醇混合液灌肠诱导小鼠炎症性肠病模型,小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组造模后分别采用5,10 mg/kg Pim-1抑制剂每天1次腹腔注射,激素治疗组给予0.1 mg泼尼松每天1次灌胃,共5 d。观测各组小鼠的一般情况、疾病活动指数(disease activity index,DAI)评分、小鼠结肠长度、结肠大体标本评分及病理学评分,评估Pim-1抑制剂对小鼠炎症性肠病的治疗作用,并与激素治疗组比较。运用RT-PCR、Western印迹分别检测Th 1,Th 2,Th 17,免疫调节性T细胞(T regulatory cell,Treg)的特异性转录因子T细胞表达的T盒(T-box expressed in T cells,T-bet)、GATA连接蛋白3(GATA binding protein3,GATA-3)、维甲酸相关孤儿受体γ(RA orphan receptorγ,RORγt)、叉头蛋白P3(forkhead box P3,Foxp3)基因及蛋白的表达。结果:溶媒对照组小鼠无腹泻、便血,体重平稳增加,TNBS模型组小鼠逐渐出现便血、腹泻、体重减轻;与溶媒对照组比较,TNBS模型组结肠长度明显缩短,DAI评分、结肠大体标本评分、病理学评分明显升高(P<0.05);小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组小鼠便血、腹泻、体重减轻较TNBS模型组明显好转,DAI评分、结肠长度的缩短、结肠大体标本评分、病理学评分较TNBS模型组明显降低(P<0.05);小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组比较,差别无统计学意义(P>0.05)。RT-PCR和Western印迹结果显示:与溶媒对照组比较,TNBS模型组GATA3和RORγt表达升高,Foxp3表达明显下降(P<0.05),而T-bet无明显差异;与TNBS模型组比较,小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组结肠组织中GATA3,RORγt的表达明显下降,Foxp3的表达明显升高(P<0.05),小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组之间比较,差别无统计学意义(P>0.05)。RT-PCR结果显示:与TNBS模型组比较,小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组结肠组织中T-bet的表达明显下降(P<0.05);小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组之间比较,差别无统计学意义(P>0.05)。Western印迹结果显示:与TNBS模型组比较,小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组结肠组织中T-bet的表达稍下降,差别无统计学意义(P>0.05),但激素治疗组表达明显下降(P<0.05);小剂量Pim-1抑制剂组、大剂量Pim-1抑制剂组、激素治疗组之间比较,差别无统计学意义(P>0.05)。结论:Pim-1抑制剂能抑制BALB/c小鼠炎症性肠病模型Th2特异性表达因子GATA3,Th17特异性表达因子RORγt的表达,促进Treg细胞的特异性表达因子Foxp3的表达,从而发挥抑制以Th 2和Th 17为主的免疫炎症反应,促进向Treg细胞分化的作用,对小鼠炎症性肠病具有一定的治疗作用。

Objective： To explore the role of Pim-1 in the pathology of inflammatory bowel disease and the potential effect of Pim-1 inhibitor on treating such disease.Methods： Forty-five BALB/c mice were randomly divided into 5 groups （n=9）： A normal control group, a inflammatory bowel disease group, two different dose of Pim-1 inhibitor treatment groups,and steroidhormone treatment group. The model of inflammatory bowel disease was induced by intracolonic administration of 2, 4, 6-trinitrobenzenestdfonic acid （TNBS） and ethanol mixture.Mice were treated with Pim-1 inhibitor [intraperitoneal inject, 5 or 10 mg/（kg.d）] for 5 days and prednisone （intragastric administration, 0.1 mg/d） for 5 days. The DAI, colon length, gross score and pathological grade were evaluated. The expressions of T cell master transcription factors T-box expressed in T cells （T-bet）, GATA binding protein 3 （GATA-3）, RA orphan receptorγ （RORγt） and forkhead box P3 （Foxp3） were measured by Real-time PCR and Western blot, respectively.Results： Pim-1 inhibitor and prednisone showed therapeutic effect on acute TNBS colitis in vivo.GATA3 and RORγt were significantly up-regulated in acute TNBS colitis （P〈0.05）. In contrast,the expression of Foxp3 was suppressed in the inflammatory bowel disease group, whereas it did not cause any significant change in T-bet expression （P〉0.05）. Administration of Pim-1 inhibitor and prednisone resulted in suppression of GATA3, RORγt expression, and the increase of Foxp3 expression （P〈0.05）. Administration of Pim-1 inhibitor and prednisone resulted in inhibition of T-bet mRNA expression （P〈0.05）, but only prednisone could inhibit T-bet protein expression （P〉0.05）.Conclusion： Pim-1 inhibitor significantly suppresses Th2- and Th17-type immune responses. Furthermore, Pim-1 inhibitor could induce T-cell differentiation towards a Treg phenotype. Pim-1 inhibitor has therapeutic effect on acute TNBS colitis.