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.
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。