Chlorogenic acid relieved oxidative stress injury in retinal ganglion cells through IncRNA-TUG1/Nrf2

<b>Objective</b>: To discover the possible underlying mechanism of Chlorogenic acid (CGA) in protecting against oxidative stress injury in glaucoma. <b>Methods</b>: LncRNA TUG1 and Nrf2 expressions were detected by qRT-PCR and Western blot. Retinal ganglion cell (RGC) viability and apoptosis were measured by MTT and flow cytometry, respectively. Reactive oxygen species (ROS) level was determined by reactive oxygen species assay kit. The interaction between lncRNA TUG1 and Nrf2 was confirmed by RNA pull-down and RIP assay. <b>Results</b>: IPL thickness and lncRNA TUG1 expression were significantly decreased in glaucoma mice model, and CGA treatment increased IPL thickness and lncRNA TUG1 expression. <i>In vitro</i> H2O2-induced RGCs, RGC viability was significantly decreased, and ROS level and cell apoptosis were significantly increased. CGA up-regulated lncRNA TUG1 and Nrf2 expressions, decreased cell apoptosis and ROS production in RGCs, and increased RGCs viability. We further verified the interaction between lncRNA TUG1 and Nrf2, and proved Nrf2 was positively regulated by lncRNA TUG1. We found CGA promoted Nrf2 expression through lncRNA-TUG1, and further verified CGA protected RGCs from oxidative stress through regulating lncRNA TUG1/Nrf2. <i>In vivo</i> experiments showed TUG1 knockdown abrogated therapeutic effect of CGA on glaucoma. <b>Conclusion</b>: CGA increased RGC viability and decreased ROS level and RGC apoptosis after oxidative stress injury through lncRNA TUG1/Nrf2 pathway, which protected against glaucoma.

**研究目的**：探讨绿原酸（Chlorogenic acid, CGA）对抗青光眼氧化应激损伤的潜在作用机制。
**研究方法**：采用实时荧光定量逆转录聚合酶链反应（qRT-PCR）与蛋白质印迹法（Western blot）检测长链非编码RNA TUG1（lncRNA TUG1）及核因子E2相关因子2（Nrf2）的表达水平；通过MTT法与流式细胞术分别测定视网膜神经节细胞（Retinal ganglion cell, RGC）的存活率与凋亡情况；利用活性氧检测试剂盒检测活性氧（Reactive oxygen species, ROS）水平；通过RNA pull-down实验与RNA免疫沉淀实验（RIP assay）验证lncRNA TUG1与Nrf2的相互作用。
**研究结果**：青光眼小鼠模型的内丛状层（Inner plexiform layer, IPL）厚度与lncRNA TUG1表达量均显著降低，而CGA干预可上调内丛状层厚度与lncRNA TUG1表达水平。在体外H₂O₂诱导的RGC损伤模型中，RGC存活率显著下降，ROS水平与细胞凋亡率显著升高；CGA可上调lncRNA TUG1与Nrf2的表达，降低RGC凋亡率与ROS生成量，并提升RGC存活率。本研究进一步验证了lncRNA TUG1与Nrf2的相互作用，证实lncRNA TUG1对Nrf2具有正向调控作用。研究发现，CGA可通过lncRNA TUG1促进Nrf2的表达，且进一步证实CGA可通过调控lncRNA TUG1/Nrf2通路保护RGC免受氧化应激损伤。体内实验结果显示，敲低TUG1可抵消CGA对青光眼的治疗效果。
**研究结论**：CGA可通过lncRNA TUG1/Nrf2通路提升氧化应激损伤后RGC的存活率，降低ROS水平与RGC凋亡率，从而发挥抗青光眼的神经保护作用。