高糖环境诱导ROS及内质网应激与糖尿病相关干眼的关系

Dry Eye often occurred in diabetes mellitus (DM) patients, even induced the loss of vision, while its pathogenesis was unclear. High concentration of sugar could induce the accumulation of reactive oxygen species (ROS), then oxidative stress and endoplasmic reticulum stress (ERS) happened, which led cells to be damaged, even apoptosis. Here also showed our study results as followed: ① the analysis of tear proteomics hinted that oxidative stress and apoptosis related proteins were up-expressed; ② in the condition of high osmotic pressure, ROS was accumulated in corneal epithelial cells (ECs) and induced the expression of active p-JNK; ③ the corneal ECs has been damaged in the patients with DM when dry eye did not take place. Then we suggest that because of DM patients’ tears with high concentration of glucose, much ROS are accumulated in conjunctival ECs and corneal ECs; and the ROS induce the damage, even apoptosis of conjunctival and corneal ECs through oxidative stress/ERS; which finally cause dry eye. In this project, we will observe that how high concentration of sugar-induced ROS regulate the damage of the conjunctival and corneal ECs and how ROS contribute to ERS, leading to the apoptosis of ECs; then an animal model of DM related dry eye will be set up; in which we will investigate the relationship of conjunctival and corneal ECs when damages occur, in addition, if ROS-blocked agent can protect the ECs or not. The results of this project can further clarify the pathogenesis of DM related dry eye and support the important academic evidence for its individualized treatment.

糖尿病人群的干眼发病率高，严重者甚至失明。其发病机制不清楚。高糖环境诱导活性氧(ROS)的累积，引起氧化应激和内质网应激(ERS)，可导致细胞损伤，甚至凋亡。基于我们前期实验，①糖尿病患者泪液中氧化应激反应和凋亡路径相关蛋白表达上调②角膜上皮细胞在高渗透压干预下，产生大量ROS，诱导磷酸化JNK表达(活化)③糖尿病患者未发生干眼时，已出现角膜上皮细胞的损伤，我们推测糖尿病患者泪液的高糖微环境诱导结膜/角膜上皮细胞产生大量ROS，引起氧化应激和ERS，介导结膜/角膜上皮细胞的双重损伤，从而发生干眼。本项目拟在体外观察高糖环境诱导的ROS对结膜/角膜上皮细胞损伤的调控作用及引起细胞凋亡的作用机制；然后建立糖尿病相关干眼的动物模型，探索结膜/角膜上皮细胞损伤之间的关系，并观察ROS抑制剂对结膜/角膜上皮细胞的保护作用。通过上述研究，进一步阐明糖尿病相关干眼的发病机制，为其个体化治疗提供重要依据。

本课题项目尝试探索高糖环境下ROS内质网应激与糖尿病性相关干眼的关系，按照实验计划完成体外实验和体内实验部分。 .体外实验部分（1）完成兔角膜上皮原代细胞的获取、培养及细胞鉴定，（2）采用流式细胞检测完成高糖状态下角膜上皮细胞活性氧的生成及活性氧抑制剂对高糖状态下细胞活性氧生成的干预作用研究，（3）采用细胞增殖能力检测方法CCK-8和Westernblot方法完成角膜上皮细胞中ROS对ERS的调控机制的研究，（4）采用Westernblot方法和PCR方法基本完成角膜上皮细胞中ROS介导凋亡相关路径的调控机制部分的研究。.体内实验部分（1）采用大体实验和组织化学方法顺利完成糖尿病小鼠干眼模型的构建及鉴定，（2）采用Westernblot方法和PCR方法完成糖尿病干眼小鼠模型角膜氧化应激反应部分的研究，证实抗氧化应激因子对糖尿病干眼小鼠模型角膜上皮细胞具有保护作用。.综上所述，本课题通过体外实验和体内实验部分，发现高泪糖诱导兔角膜上皮细胞活性氧生成增加，细胞活性降低，活性氧抑制剂NAC可以逆转这种改变。高泪糖诱导生成的活性氧通过激活内质网应激信号通路诱导细胞凋亡。细胞凋亡通路研究中发现高糖环境下，p-PERK和CHOP表达水平上升，保护性p-Akt表达水平降低，从而诱导细胞凋亡。目前已知高泪糖作为糖尿病患者的伴随症状，其对角膜上皮细胞有负面作用，可以作为一个独立的干眼发病的影响因素，从而符合糖尿病相关性干眼患者其干眼症状较正常人群更明显、角膜上皮细胞损伤更严重的临床现象。因此，我们认为泪液的高糖环境与干眼发生有密切关系，高泪糖的微环境可以通过ROS-内质网应激-凋亡路径引起糖尿病性相关干眼的发生，采用抗氧化应激因子对糖尿病干眼小鼠模型角膜上皮细胞的凋亡进程具有抑制作用，该理论的证实为后续糖尿病性相关干眼的诊断和治疗方法的精准化提供理论依据，为靶向药物研发提供思路。