矢车菊素-3-葡萄糖苷通过调控内质网应激增强RPE细胞屏障功能的机制研究

Cyanidin-3-glucoside (C3G) has many beneficial effects including antioxidant capacity and increasing the activity of antioxidant enzymes in cells. The blood-retinal barrier (BRB) is formed by the retinal pigment epithelial (RPE) tight junctions. Excessive light exposure causes endoplasmic reticulum (ER) stress and eventually disruptes RPE barrier function. In our previous studies, C3G protected the RPE cells by scavenging reactive oxygen species and reducing release of methylglyoxal. Therefore, C3G may possess potential efficacy for protecting RPE cells from photooxidation-induced ER stress and barrier dysfunction. To clarify the molecular mechanisms of C3G enhancing RPE barrier function, we will employ in vitro model that photooxidation-induced barrier dysfunction in RPE cells. Immunofluorescence staining, western blot, real time quantitative polymerase chain reaction and small-interfering RNAs will been carried out in this study. In addition, transepithelial electrical resistance and cell apoptosis measurement will been performed. We will demonstrate the related mechanisms that C3G enhances RPE barrier integrity via relieving ER stress. Furthermore, we will investigate the effects of C3G on Nrf2, MAPK and NF-κB pathways, and tight junction protein expression to elucidate primary target of C3G against RPE barrier dysfunction. The obtained results may provide theoretical basis for reasonable processing and consumption of black cereal products.

矢车菊素-3-葡萄糖苷（C3G）具有抗氧化和提高细胞内抗氧化酶活性等作用。视网膜色素上皮（RPE）细胞间紧密连接（TJ）构成血-视网膜屏障基础，过量光照可引起RPE细胞内质网应激，进而导致细胞屏障功能紊乱。本课题组前期研究发现C3G可抑制RPE细胞ROS蓄积和丙酮醛生成，具有缓解光氧化诱导的内质网应激和屏障功能紊乱的潜在功效。为解析C3G增强RPE细胞屏障功能的作用及分子机制，本项目将基于光氧化诱导的RPE细胞屏障功能紊乱模型，通过细胞免疫荧光、Western blot、实时荧光定量PCR、siRNA基因沉默等分子生物学手段，结合细胞跨膜电阻及细胞凋亡检测，重点从内质网应激角度揭示C3G增强RPE细胞屏障功能的作用机理，并结合C3G对Nrf2、MAPK、NF-κB通路和TJ蛋白表达的调控，阐明C3G增强RPE屏障功能的关键作用靶点，以期为合理加工和消费黑色谷物类食品提供理论基础。

视网膜色素上皮（RPE）细胞间紧密连接（TJ）构成血-视网膜屏障基础，过量光照可引起RPE细胞内质网应激，进而导致细胞屏障功能紊乱。矢车菊素-3-葡萄糖苷（C3G）具有缓解光氧化诱导的视网膜屏障功能紊乱的潜在功效，但具体作用机制尚不清楚。本项目重点从内质网应激角度揭示C3G增强RPE细胞屏障功能的作用机理，并结合C3G对Nrf2、MAPK、NF-κB通路和TJ蛋白表达的调控，阐明C3G增强RPE屏障功能的关键作用靶点。在此基础上，将研究内容进行了有效的拓展，研究C3G对RPE细胞溶酶体和线粒体结构及功能的保护效果，并初步探究C3G对细胞内鞘脂代谢的调控作用。基于以上研究内容，取得的主要研究结果包括：（1）C3G对RPE细胞内质网应激具有调控作用，通过抑制PERK-eIF2α-ATF4-CHOP通路，提高TJ蛋白（ZO-1和Claudin-5）表达，抑制细胞凋亡，增强RPE细胞屏障功能。（2）C3G激活Nrf2核转移，提高Nrf2下游抗氧化蛋白HO-1、NQO1和GCLC的表达；并且内质网应激相关蛋白IRE1α和PERK参与调控Nrf2通路。（3）C3G抑制MAPK通路中p38、JNK1/2及JNK1/2下游c-Jun磷酸化，从而抑制细胞凋亡。（4）C3G抑制NF-κB p65磷酸化，降低NF-κB通路下游炎症因子iNOS和COX-2表达。（5）C3G通过调节鞘脂代谢通路，降低神经酰胺、提高半乳糖基神经酰胺含量，保护RPE细胞。. 在研期间本项目组发表SCI期刊论文5篇，英文综述论文1篇，EI论文2篇，北大核心期刊论文1篇。以上研究结果为花色苷类功能因子保护视觉健康提供理论依据。