DSCR1调控CaN-CREB-Bcl-2信号轴抗氧化应激诱导视网膜神经节细胞凋亡的机制研究

Retinal ganglion cells (RGCs) apoptosis induced by oxidative stress is the major reason of irreversible blindness caused by glaucoma and other degenerative diseases of optic nerve. However, the exact pathogenesis and protective mechanism is still unclear. It was reported that calcineurin (CaN) inhibit the phosphorylation of CREB, subsequently reduced the transcription of Bcl-2, thus apoptosis of neuron occurred. DSCR1 is the endogenous regulator of CaN, which could inhibit the activity of CaN, then protects the neurons from apoptosis induced by oxidative stress. Based on our previous study, we proposed hypothesis that DSCR1 increases in RGCs under oxidative stress, CaN-CREB-Bcl-2 signaling axis was activated subsequently, RGCs apoptosis was blocked therefore. In the current study, optic nerve crush mice and RGCs treated with hydrogen peroxide were used to study. DSCR1 was up or down regulated in vivo and in vitro. The regulation of DSCR1 on CaN-CREB-Bcl-2 signaling axis was explored. The effects of DSCR1 on RGCs’ mitochondrial function, apoptosis and visual function were assessed on molecular level, structural and functional level. The purpose of the current study is to illustrate the mechanism of DSCR1 protection of RGCs from apoptosis induced by oxidative stress, which might provide a new target on the treatment of glaucoma and other degenerative diseases of optic nerve.

氧化应激引起的视网膜神经节细胞（RGCs）凋亡是青光眼等视神经退行性疾病不可逆性致盲的重要原因，但其具体致病及保护机制仍未明确。研究表明，CaN能抑制CREB磷酸化，从而减少Bcl-2转录，诱导神经元凋亡。DSCR1是CaN的内源调节因子，能抑制CaN活性，保护氧化应激诱导的神经元凋亡。我们在前期研究基础上提出假说：氧化应激状态下RGCs内DSCR1增加，CaN-CREB-Bcl-2信号轴激活，RGCs凋亡受抑制。本研究以RGCs内DSCR1为靶点，以视神经碾压伤动物模型和过氧化氢诱导的RGCs为研究对象，通过体内外干预DSCR1表达，探究DSCR1对CaN-CREB-Bcl-2信号轴的调控，从蛋白、形态及功能层面分析其对RGCs凋亡的影响，从分子、细胞及动物整体水平分层次阐明DSCR1保护氧化应激诱导RGCs凋亡的机制。该研究有望为青光眼等视神经退行性疾病的治疗提供新靶点，意义深远。

不可逆的视网膜神经节细胞（retina ganglion cell, RGC）丢失是青光眼进展过程中的关键过程。既往研究表明唐氏综合症关键区域1（Down syndrome critical region 1, DSCR1）具有抑制神经元凋亡的作用。在本项研究中，我们主要探讨DSCR1对RGC的神经保护机制。为此，我们首先利用DBA / 2J小鼠模型、建立视神经碾压伤（optic nerve crush，ONC）大鼠模型和H2O2诱导的原代RGC氧化应激模型等多个模型明确DSCR1蛋白的定位和表达。通过蛋白质印迹实验（western blot，WB）和实时PCR技术分别从蛋白质水平和基因水平检测DSCR1表达。我们发现：DSCR1表达于正常SD（Sprague-Dawley）大鼠的RGC上，在DBA / 2J小鼠，ONC大鼠模型和H2O2处理的RGC中，可观察到DSCR1表达显著上调且具有时间依赖性，呈现“先升高后降低”的趋势，其中DSCR1蛋白表达量和mRNA水平在6个月龄的DBA / 2J小鼠，大鼠ONC后5天和在经H2O2处理后24小时的RGC中达到峰值。在体外干预DSCR1基因表达并通过WB实验发现在H2O2处理组中，过表达DSCR1基因，RGC中cAMP应答元件结合蛋白（CREB）磷酸化水平，B细胞淋巴瘤-2（Bcl-2）转录表达和RGC存活率显著增加，caspase 3的表达显著降低；敲除DSCR1基因，RGC中CREB磷酸化水平，Bcl-2转录表达和RGC存活率显著降低，caspase 3的表达显著增加。此外，敲除CREB基因表达可抑制DSCR1的表达。以上结果表明，RGC中存在DSCR1-CREB反馈环路，且DSCR1可能通过CREB–Bcl-2途径抑制氧化应激诱导的RGC凋亡，这可能为将来治疗青光眼提供理论依据。