Muller细胞对氧磷酶2表达改变在糖尿病视网膜病变中作用及其调控机制

Diabetic retinopathy (DR) is a leading cause of blindness; however its pathological mechanism remains largely unclear. Paraoxonase2 (Pon2), a pivotal anti-oxidative enzyme, is closely associated with diabetic complications, but the exact mechanism has not been elucidated. We, for the first time, demonstrated that expression of PON2 was significantly increased in retinas of diabetic mice, which was mainly colocalized with muller cell marker-glutamine synthetase. Systemic administration of Niacin markedly ugregulated Pon2 expression, attenuated 3-nitrotyrosine formation and reduced leukostasis in retinas of diabetic mice, which suggested us that Pon2 play an important role in development of DR. Thus, in this study, we will investigate association between diabetic retinopathy with PON2 gene polymorphisms, Pon2 expression or activity in peripheral blood mononuclear cells in Chinese population in Jiangxi. Furthermore, mouse model of diabetes will be setup by intraperitoneal injection of streptozotocin. Adeno-associated virus will be intravitreously delivered to diabetic mice. Generation of retinal reactive oxygen species, expression of adhesion molecules, vascular leakage, leukostasis, formation of accelular capillaries and pericyte gosts, muller cell gliosis and ganglion cell apoptosis will be evaluated in STZ mice. In addition, quantitative multiplex methylation-specific PCR assay (QMSP)and chromatin immunoprecipitation (ChIP) will be utilized to elucidate epigenetic regulation of Pon2 during diabetes. Taken together, the overall goal of this project is to further clarify pathological mechanism of DR and seek for novel therapeutic target of DR.

糖尿病视网膜病变(DR)是不可逆致盲眼病，发病机制未完全阐明。对氧磷酶2(PON2)为体内重要抗氧化酶，研究表明其基因多态性与糖尿病并发症密切相关，但具体机制尚不清楚。我们首次发现Pon2在糖尿病小鼠视网膜表达增加并与Muller细胞共定位，降脂药烟酸可上调Pon2表达并明显抑制视网膜氧化应激及血管处白细胞粘附。本课题拟在前期工作基础上，探讨江西地区人群中PON2基因多态性及外周血单个核细胞Pon2表达或活性与DR的相关性；建立DR动物模型，玻璃体腔注射过表达PON2的腺相关病毒载体，探讨其对DR活性氧自由基产生、炎症因子表达、血管渗漏及白细胞粘附、无细胞毛细血管和鬼影周细胞生成、胶质细胞活化及神经节细胞凋亡的作用；采用多重甲基化特异性定量PCR及ChIP解析DR中Pon2表达调控的表观遗传学机制。为丰富DR的发病机制及寻找治疗新靶点奠定基础。

糖尿病视网膜病变（DR）是工作年龄人群致盲的首要原因，微血管渗漏引起的黄斑水肿以及病理性视网膜新生血管生成是造成DR病人视力下降的主要病理生理学基础。既往研究提出过量活性氧自由基（ROS）产生介导的氧化应激是糖尿病并发症发生发展的始动因素。对氧磷酶2（Paraoxonase2， Pon2）是细胞内重要的抗氧化酶。我们前期研究发现Pon2在糖尿病小鼠视网膜表达增加并与Muller细胞共定位，但其表达改变在DR发生发展中的作用尚未阐明。本项目建立链脲佐菌素诱导的1型糖尿病动物模型以及氧诱导的新生小鼠视网膜病变模拟DR相关改变，给予含有烟酸的饮食喂养小鼠或者注射野生型Pon2腺相关病毒表达载体（AAV-Pon2）上调视网膜Pon2表达，我们发现可明显降低糖尿病小鼠视网膜ROS产生减轻氧化应激，减少炎症因子及VEGF产生，缓解视网膜血管处白细胞粘附，保护视网膜血管屏障功能，抑制视网膜血管渗漏、无细胞毛细血管及新生血管生成；此外，过表达AAV-PON2还可以抑制OIR小鼠视网膜胶质细胞活化及神经凋亡，表明无论从药物或者基因水平上调视网膜Pon2表达均可缓解糖尿病造成的视网膜损害。表观遗传学调控的组蛋白修饰在调控基因表达及DR代谢记忆中发挥重要作用。在此基础上，我们通过体外培养视网膜Muller细胞系rMC-1，给予组蛋白去乙酰化酶抑制剂SAHA，观察对PON2表达影响，发现高糖处理明显上调细胞中PON2表达，给予SAHA处理后PON2表达进一步增加并可抑制高糖诱导的炎症因子产生；动物实验也证实SAHA可以上调糖尿病小鼠视网膜PON2表达减轻DR血管渗漏，提示表观遗传机制可能参与DR中PON2的表达调控。受本项目资助已发表通讯作者SCI收录论文4篇，核心期刊论文4篇；6次应邀在国际学术会议作大会发言及壁报交流，4次应邀在国内会议作大会发言；培养并毕业博士研究生1名、硕士研究生3名。