XBP1对视网膜色素上皮细胞Nrf2信号通路的调控及其对细胞凋亡的保护作用

Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly. Retinal pigment epithelium (RPE) apoptosis is the early sign for AMD, however, the mechanism for RPE protection is unclear. We for the first time demonstrated that X-box binding protein 1 (XBP1) protected the RPE from hydroquinone induced apoptosis. Moreover, we found that XBP1 was necessary for anti-oxidative gene expression in RPE cells, which implied that XBP1 might regulate anti-oxidative response in the RPE. NF-E2-related factor 2 (Nrf2) is a central regulator for oxidative stress. In order to approach the influence of XBP1 on Nrf2 signaling pathway in the RPE, we will infect RPE cells with adenovirus overexpressing spliced XBP1 or shRNA-XBP1, and detect the influence on Nrf2 signaling and RPE apoptosis using luciferase assay, Western Blot, real-time RT PCR, immunocytochemistry and TUNEL staining. We will treat cells with pAkt/p38/Erk/PKCδ inhibitor and detect Nrf2 level to explore the mechanism by which XBP1 regulates Nrf2 expression. We will infect mice RPE with adenovirus overexpressing spliced XBP1 or shRNA-XBP1 by subretinal injection of the virus, and approach the influence on Nrf2 signaling in vivo. This proposal aims at finding molecular mechanisms for RPE protection, thus providing new therapeutic target for AMD.

年龄相关性黄斑变性(AMD)是老年人群致盲的首要原因。视网膜色素上皮细胞(RPE)凋亡是AMD早期的病理特征，而保护RPE免于凋亡的分子机制尚不明确。我们首次发现内质网应激调节蛋白XBP1能保护促氧化剂诱导的RPE凋亡，并发现其正常表达是RPE中SOD1、SOD2等抗氧化基因表达的必要条件，提示XBP1对RPE氧化应激有调节作用。Nrf2是氧化应激的关键调节基因。为阐明XBP1对RPE中Nrf2信号通路的调节，本课题用腺病毒在RPE中过表达或沉默XBP1基因，免疫印迹法、TUNEL染色法等探讨其对Nrf2通路及RPE凋亡的影响。用pAkt、p38、Erk、PKCδ抑制剂分别处理细胞后检测Nrf2水平，探讨XBP1调控Nrf2表达的机制。通过视网膜下腔注射腺病毒在小鼠RPE内干预XBP1表达，检测其在体内对Nrf2通路的影响。本研究旨在揭示保护RPE凋亡的分子机制，为AMD防治提供新靶点。

年龄相关性黄斑变性(AMD)是老年人群致盲的首要原因。视网膜色素上皮细胞(RPE)凋亡是AMD早期的病理特征，而保护RPE免于凋亡的分子机制尚不明确。我们首次发现内质网应激调节蛋白XBP1能保护促氧化剂诱导的RPE凋亡，提示XBP1对RPE氧化应激有调节作用。Nrf2是氧化应激的关键调节基因。本课题旨在阐明XBP1对RPE中Nrf2信号通路的调节。XBP1的活性形式是剪切型XBP1。本课题用腺病毒在RPE中过表达剪切型XBP1后，发现Nrf2在细胞核和细胞浆中的水平均升高。用药物QT和4μ8C抑制XBP1剪切后，Nrf2的蛋白水平显著降低。在RPE特异性XBP1基因敲除的小鼠，RPE中Nrf2及其下游基因的表达均下降。从RPE特异性XBP1基因敲除小鼠中分离的原代RPE中，Nrf2的正常表达及应激表达均下降。这些结果说明在RPE中，XBP1对Nrf2通路有调节作用。机制研究方面，我们发现过表达剪切型XBP1并不影响Nrf2的mRNA水平，抑制XBP1剪切并不加速Nrf2蛋白降解，我们推测XBP1可能影响Nrf2的蛋白翻译，这尚需更多研究证实。同时我们发现Nrf2对XBP1并无明显反向调节。我们的研究揭示了保护RPE凋亡的新的分子机制，为AMD防治提供了新靶点。