DNA甲基化抑制剂Zebularine治疗葡萄膜炎的分子机制研究

In China, uveitis leads to many legally blind cases every year. Although recent genetic studies discovered many gene loci that contribute to the pathogenesis of uveitis, such as Behcet’s disease and Vogt–Koyanagi–Harada syndrome, the advance of therapies for uveitis falls significantly behind. Steroid therapy remains the first line approach treating uveitis. However, about 1/3 patients with uveitis are resistant or intolerant to current therapies. A new solution for controlling uveitis effectively is needed. Our recent unpublished study found that Zebularine, an epigenetic drug (DNA methylation inhibitor) for cancer therapy, could control the onsite and ocular pathology of uveitis in a murine experimental autoimmune uveitis (EAU) model in vivo. To further investigate the molecular mechanism by which Zebularine controls the intraocular inflammation, we propose to profile the genome-wide changes of DNA methylation (5 methylcytosine) and gene expression in inflammatory CD4+ T cells as well as retinal pigment epithelium (RPE) in response to Zebularine treatment in vitro. Furthermore, the genes of interest in response to Zebularine treatment will be tested in EAU model to define their in vivo roles mediating the DNA methylation inhibitor’s drug effects. Identification of target genes regulated by Zebularine will significantly improve our understanding of how to use this drug efficiently by enhancing the therapeutic and minimizing the side effects.

在我国，葡萄膜炎仍是致盲率极高的一种眼病。虽然近年来的研究发现了多个可能诱发葡萄膜炎的相关基因突变，但是针对非感染性葡萄膜炎的治疗手段并未有所进步。以糖皮质激素为主的免疫抑制剂仍是控制眼内炎症反应的主要治疗方法。然而，至少1/3的葡萄膜炎病人对糖皮质激素以及其它免疫抑制剂的治疗作用不敏感。因而开发有效的治疗葡萄膜炎的新方法已经成为当前眼免疫学研究的热点问题。我们应用小鼠葡萄膜炎动物模型的前期研究表明，一种临床上被批准用于肿瘤表观遗传治疗的DNA甲基化抑制剂Zebularine可以有效的减轻眼内炎症反应并降低组织损伤。为了进一步明确Zebularine的抗炎作用，研究其在眼内可能的抗炎分子机理，本课题拟应用全基因组分析Zebularine引起的DNA甲基化变化和基因表达调控的方法，找到该药物的分子靶点，分析其药物作用分子机理，从而提高其治疗的有效性和安全性。

过去的研究证明，CD4+T细胞的异常激活是葡萄膜炎的主要发病机制之一。目前，糖皮质激素，免疫抑制剂，以及生物制剂如抗细胞因子抗体等是主要的葡萄膜炎治疗药物。然而，各类免疫抑制药物仍存在副作用多、少数病人无法耐受或无反应等疗效缺陷。因而，我们观察了一种DNA甲基化抑制剂-Zebularine-是否能通过抑制CD4+T细胞进而抑制眼内的炎症反应。首先，我们通过CD4+T细胞的体外分化实验入手，发现了Zebularine对小鼠与人的CD4+T细胞分化成IFN-gamma+的Th1细胞或者是IL-17+的Th17细胞都有抑制作用。而且，Zebularine还能在实验性葡萄膜炎小鼠模型中抑制眼内的炎症。最后，我们发现Zebularine可能是通过抑制了Foxp基因启动子的甲基化，进而增强其表达来增加抑制性T细胞Treg的功能，从而抑制了IFN-gamma和IL-17的表达。因而，我们的研究明确了Zebularine对实验性葡萄膜炎的抗炎作用，并且找到了这一抗炎作用的可能分子机制。为Zebularine成为可能的全新葡萄膜炎治疗药物提供了初步的理论证据。