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Abstract：
BACKGROUND: NADPH oxidase (NOX), a primary source of endothelial reactive oxygen species (ROS), is considered a key event in disrupting the integrity of the blood-retinal barrier. Abnormalities in neurovascular-coupled immune signaling herald the loss of ganglion cells in glaucoma. Persistent microglia-driven inflammation and cellular innate immune system dysregulation often lead to deteriorating retinal degeneration. However, the crosstalk between NOX and the retinal immune environment remains unresolved. Here, we investigate the interaction between oxidative stress and neuroinflammation in glaucoma by genetic defects of NOX2 or its regulation via gp91ds-tat.
METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study.
RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst.
CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.
METHODS: Ex vivo cultures of retinal explants from wildtype C57BL/6J and Nox2 -/- mice were subjected to normal and high hydrostatic pressure (Pressure 60 mmHg) for 24 h. In vivo, high intraocular pressure (H-IOP) was induced in C57BL/6J mice for two weeks. Both Pressure 60 mmHg retinas and H-IOP mice were treated with either gp91ds-tat (a NOX2-specific inhibitor). Proteomic analysis was performed on control, H-IOP, and treatment with gp91ds-tat retinas to identify differentially expressed proteins (DEPs). The study also evaluated various glaucoma phenotypes, including IOP, retinal ganglion cell (RGC) functionality, and optic nerve (ON) degeneration. The superoxide (O2-) levels assay, blood-retinal barrier degradation, gliosis, neuroinflammation, enzyme-linked immunosorbent assay (ELISA), western blotting, and quantitative PCR were performed in this study.
RESULTS: We found that NOX2-specific deletion or activity inhibition effectively attenuated retinal oxidative stress, immune dysregulation, the internal blood-retinal barrier (iBRB) injury, neurovascular unit (NVU) dysfunction, RGC loss, and ON axonal degeneration following H-IOP. Mechanistically, we unveiled for the first time that NOX2-dependent ROS-driven pro-inflammatory signaling, where NOX2/ROS induces endothelium-derived endothelin-1 (ET-1) overexpression, which activates the ERK1/2 signaling pathway and mediates the shift of microglia activation to a pro-inflammatory M1 phenotype, thereby triggering a neuroinflammatory outburst.
CONCLUSIONS: Collectively, we demonstrate for the first time that NOX2 deletion or gp91ds-tat inhibition attenuates iBRB injury and NVU dysfunction to rescue glaucomatous RGC loss and ON axon degeneration, which is associated with inhibition of the ET-1/ERK1/2-transduced shift of microglial cell activation toward a pro-inflammatory M1 phenotype, highlighting NOX2 as a potential target for novel neuroprotective therapies in glaucoma management.
摘要：
背景：NADPH氧化酶（NOX），内皮活性氧（ROS）的主要来源，被认为是破坏血-视网膜屏障完整性的关键事件。神经血管偶联免疫信号异常预示着青光眼神经节细胞的丢失。持续的小胶质细胞驱动的炎症和细胞先天免疫系统失调通常导致恶化的视网膜变性。然而,NOX与视网膜免疫环境之间的串扰仍未解决。这里,我们通过NOX2的遗传缺陷或通过gp91ds-tat调节来研究青光眼氧化应激与神经炎症之间的相互作用。
方法:来自野生型C57BL/6J和Nox2-/-小鼠的视网膜外植体的离体培养物经受正常和高静水压力(压力60mmHg)24小时。在C57BL/6J小鼠中诱导高眼压（H-IOP）两周。压力60mmHg视网膜和H-IOP小鼠均用gp91ds-tat(NOX2特异性抑制剂)处理。对对照进行蛋白质组学分析，H-IOP,并用gp91ds-tat视网膜处理以鉴定差异表达蛋白（DEP）。该研究还评估了各种青光眼表型，包括IOP,视网膜神经节细胞（RGC）功能，和视神经（ON）变性。超氧化物(O2-)水平测定，血-视网膜屏障降解，胶质增生,神经炎症，酶联免疫吸附测定(ELISA),西方印迹,和定量PCR在这项研究中进行。
结果：我们发现NOX2特异性缺失或活性抑制可有效减弱视网膜氧化应激，免疫失调，内部血-视网膜屏障（IBRB）损伤，神经血管单元（NVU）功能障碍，RGC损失,和H-IOP后轴突变性。机械上,我们首次揭示了NOX2依赖性ROS驱动的促炎信号，其中NOX2/ROS诱导内皮源性内皮素-1（ET-1）过表达，激活ERK1/2信号通路，介导小胶质细胞活化向促炎M1表型转移，从而引发神经炎症爆发。
结论：总的来说，我们首次证明NOX2缺失或gp91ds-tat抑制减弱iBRB损伤和NVU功能障碍，以挽救青光眼RGC丢失和ON轴突变性，这与ET-1/ERK1/2转导的小胶质细胞活化向促炎M1表型转移的抑制有关，强调NOX2是青光眼治疗中新型神经保护疗法的潜在靶点。
方法:来自野生型C57BL/6J和Nox2-/-小鼠的视网膜外植体的离体培养物经受正常和高静水压力(压力60mmHg)24小时。在C57BL/6J小鼠中诱导高眼压（H-IOP）两周。压力60mmHg视网膜和H-IOP小鼠均用gp91ds-tat(NOX2特异性抑制剂)处理。对对照进行蛋白质组学分析，H-IOP,并用gp91ds-tat视网膜处理以鉴定差异表达蛋白（DEP）。该研究还评估了各种青光眼表型，包括IOP,视网膜神经节细胞（RGC）功能，和视神经（ON）变性。超氧化物(O2-)水平测定，血-视网膜屏障降解，胶质增生,神经炎症，酶联免疫吸附测定(ELISA),西方印迹,和定量PCR在这项研究中进行。
结果：我们发现NOX2特异性缺失或活性抑制可有效减弱视网膜氧化应激，免疫失调，内部血-视网膜屏障（IBRB）损伤，神经血管单元（NVU）功能障碍，RGC损失,和H-IOP后轴突变性。机械上,我们首次揭示了NOX2依赖性ROS驱动的促炎信号，其中NOX2/ROS诱导内皮源性内皮素-1（ET-1）过表达，激活ERK1/2信号通路，介导小胶质细胞活化向促炎M1表型转移，从而引发神经炎症爆发。
结论：总的来说，我们首次证明NOX2缺失或gp91ds-tat抑制减弱iBRB损伤和NVU功能障碍，以挽救青光眼RGC丢失和ON轴突变性，这与ET-1/ERK1/2转导的小胶质细胞活化向促炎M1表型转移的抑制有关，强调NOX2是青光眼治疗中新型神经保护疗法的潜在靶点。
