siRNA 介导的 RhoA 表达下调可减少氧化诱导的视网膜神经节细胞凋亡。
siRNA Mediated Downregulation of RhoA Expression Reduces Oxidative Induced Apoptosis in Retinal Ganglion Cells.
机构信息
Henan Provincial People's Hospital, Henan Eye Institute, Henan Eye Hospital and Zhengzhou University People's Hospital, Zhengzhou, 450003, China.
出版信息
Curr Mol Med. 2024;24(5):630-636. doi: 10.2174/1566524023666230511095628.
BACKGROUNDS
Glaucoma is the second leading cause of blindness. Apoptosis of retinal ganglion cells (RGCs) is an important mechanism of glaucomatous optic injury. Rho kinase expression is significantly increased in apoptotic RGCs. This study aimed to investigate the role of RhoA, a Rho GTPase, on the survival of RGCs and further to explore its potential therapeutic applications.
METHODS
RGCs were treated with siRhoA for 24 hours . Knockdown of RhoA was confirmed with quantitative RT-PCR. Oxidative stress was induced by treating the RGCs with 200 μM of HO for 1 hour, and apoptosis of RGCs was quantified with TUNEL assay , and with flow cytometry. The mRNA expression levels of RhoA, Nogo receptor, caspase 3 and Bcl-2 were evaluated by quantitative RT-PCR, and the protein levels of RhoA, ROCK1, ROCK2, Nogo receptor, caspase 3 and Bcl-2 were evaluated by Western blot. We found siRhoA treatment efficiently downregulated the expression of RhoA in RGCs and protected against HO-induced injury in RGCs . Apoptosis of RGC cells under oxidative stress was quantified in situ using TUNEL assay and confirmed with flow cytometry (FCM).
RESULTS
With the knockdown of RhoA, the expression of ROCK1, ROCK2, Nogo Receptor, Casepase-3 were decreased, while the expression of Bcl-2 was increased in both mRNA and protein level. Our data indicated that siRhoA prevented HO-induced apoptosis in RGC cells by modulating the RhoA/ROCK pathway.
CONCLUSION
The results suggested that siRhoA may exert potentially effective neuroprotection for RGCs by reducing injury.
背景
青光眼是导致失明的第二大原因。视网膜神经节细胞（RGCs）的凋亡是青光眼视神经损伤的重要机制。Rho 激酶在凋亡的 RGCs 中的表达显著增加。本研究旨在探讨 Rho GTPase RhoA 对 RGCs 存活的作用，并进一步探讨其潜在的治疗应用。
方法
用 siRhoA 处理 RGCs24 小时。用定量 RT-PCR 证实 RhoA 的敲低。用 200μM 的 HO 处理 RGCs1 小时诱导氧化应激，并通过 TUNEL 测定和流式细胞术定量 RGCs 的凋亡。用定量 RT-PCR 评估 RhoA、Nogo 受体、caspase3 和 Bcl-2 的 mRNA 表达水平，用 Western blot 评估 RhoA、ROCK1、ROCK2、Nogo 受体、caspase3 和 Bcl-2 的蛋白水平。我们发现 siRhoA 处理能有效下调 RGCs 中 RhoA 的表达，并能抵抗 HO 诱导的 RGCs 损伤。用 TUNEL 测定和流式细胞术（FCM）原位定量氧化应激下 RGC 细胞的凋亡。
结果
敲低 RhoA 后，ROCK1、ROCK2、Nogo 受体、Casepase-3 的表达降低，而 Bcl-2 的表达在 mRNA 和蛋白水平均增加。我们的数据表明，siRhoA 通过调节 RhoA/ROCK 通路，防止 HO 诱导的 RGC 细胞凋亡。
结论
结果表明，siRhoA 可能通过减少损伤对 RGCs 发挥潜在有效的神经保护作用。
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