Development of the epigenetic treatment against inflammatory bowel disease with targeting chromatin remodeling

1.观察小鼠实验性结肠炎的治疗效果评价DNA甲基化抑制剂（Aza-dC）和组蛋白去乙酰化酶抑制剂（FK228）单独或联合给药对小鼠DSS结肠炎的治疗效果。用3% DSS诱导Balb/c小鼠结肠炎4天。我们将其分为DNA甲基化抑制剂（Aza-dC）单给药组、HDAC抑制剂（FK228）单给药组、联合给药组和对照组，探讨治疗的预防和治疗效果。结果表明，Aza-dC单给药组结肠炎有加重的趋势，而联合给药组结肠炎有所改善，与对照组比较差异无统计学意义，但证明了FK228组治疗的预防和治疗效果。我们接下来对小鼠TNBS结肠炎进行了研究，其中FK228组治疗仅在DSS结肠炎中显示出显著的预防和治疗效果。HDAC抑制剂抗炎作用机制的体内分析采用ELISA法对小鼠TNBS结肠炎结肠组织中多种细胞因子谱进行了研究。结果表明，TNFα、IFN-γ和IL-6的表达呈剂量依赖性降低，而IL-10的表达水平无显著差异。此外，我们从DSS结肠炎中分离出固有层淋巴细胞（LPMC），使用抗乙酰组蛋白（H3）抗体进行Western blotting，检测到H3的乙酰化以FK228剂量依赖性的方式进行。实验性结肠炎表观遗传异常的检测我们利用cDNA阵列分析基因随化学给药的表达变化，并对沉默被取消和表达的候选基因进行启动子区染色质重塑状态的检测。

1.Examination of therapeutic effect in the mouse experimental colitisA single or combination administration of DNA methylation inhibitor (Aza-dC) and histone deacetylase (HDAC) inhibitor (FK228) was evaluated on therapeutic efficacy for the mouse DSS colitis.Colitis was induced by 3% DSS for four days ad libitum in Balb/c mouse.We divided it into our groups : DNA methylation inhibitor (Aza-dC) single administration group, HDAC inhibitor (FK228) single administration group, combined administration group, and control group and we explored the preventive and therapeutic effect of treatment.As a result, colitis rather accepted a tendency to worsen in an Aza-dC single administration group, while colitis was ameliorated in the combination group, which did not reach a statistically significant difference comparing with the control group, but preventive and therapeutic effect of treatment in FK228 group was proven.We next examined mouse TNBS colitis, where significant preventive and therapeutic effect of treatment in FK228 group was only demonstrated in DSS colitis.2.The in vivo analysis of the anti-inflammatory action mechanism of HDAC inhibitorWe reviewed various cytokine profiles using colonic tissue in the mouse TNBS colitis by ELISA.As a result, TNFα, IFN-γ, and IL-6 expression was decreased in a dose-dependent manner, but a significant difference was not found in IL-10 expression level.Furthermore, we isolated lamina propria lymphocytes (LPMC) from DSS colitis, and acetylation of H3 was detected in a FK228 dose-dependent manner revealed by Western blotting using an anti acetyl-histone (H3) antibody.3.Examination of the epigenetic abnormality in the experimental colitisWe genome-widely analyze gene expression changes with the chemical administration by cDNA array, and candidate genes whose silencing is canceled and expressing are going to be checked the chromatin remodeling state of the promoter region of those in future.