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Abstract：
BACKGROUND: Retinopathy of Prematurity (ROP) is a proliferative retinal vascular disease occurring in the retina of premature infants and is the main cause of childhood blindness. Nowadays anti-VEGF and retinal photocoagulation are mainstream treatments for ROP, but they develop a variety of complications. Hydrogen (H2) is widely considered as a useful neuroprotective and antioxidative therapeutic method for hypoxic-ischemic disease without toxic effects. However, whether H2 provides physiological angiogenesis promotion, neovascularization suppression and glial protection in the progression of ROP is largely unknown.This study aims to investigate the effects of H2 on retinal angiogenesis, neovascularization and neuroglial dysfunction in the retinas of oxygen-induced retinopathy (OIR) mice.
METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining.
RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression.
CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.
METHODS: In this study, mice that were seven days old and either wild-type (WT) or Nrf2-deficient (Nrf2-/-) were exposed to 75% oxygen for 5 days and then returned to normal air conditions. Different stages of hydrogen gas (H2) inhalation were administered. Vascular obliteration, neovascularization, and blood vessel leakage were analyzed and compared. To count the number of neovascularization endothelial nuclei, routine HE staining of retinal sections was conducted. Immunohistochemistry was performed using DyLight 594 labeled GSL I-isolectin B4 (IB4), as well as primary antibodies against proliferating cell nuclear antigen (PCNA), glial fibrillary acidic protein (GFAP), and Iba-1. Western blots were used to measure the expression of NF-E2-related factor 2 (Nrf2), vascular endothelial growth factor (VEGF), Notch1, Dll4, and HIF-1α. Additionally, the expression of target genes such as NQO1, HO-1, Notch1, Hey1, Hey2, and Dll4 was measured. Human umbilical vein endothelial cells (HUVECs) treated with H2 under hypoxia were used as an in vitro model. RT-PCR was used to evaluate the mRNA expression of Nrf2, Notch/Dll4, and the target genes. The expression of reactive oxygen species (ROS) was observed using immunofluorescence staining.
RESULTS: Our results indicate that 3-4% H2 does not disturb retinal physiological angiogenesis, but ameliorates vaso-obliteration and neovascularization in OIR mice. Moreover, H2 prevents the decreased density and reverses the morphologic and functional changes in retinal astrocytes caused by oxygen-induced injury. In addition, H2 inhalation reduces microglial activation, especially in the area of neovascularization in OIR mice. H2 plays a protective role in vascular regeneration by promoting Nrf2 activation and suppressing the Dll4-induced Notch signaling pathway in vivo. Also, H2 promotes the proliferation of HUVECs under hypoxia by negatively regulating the Dll4/Notch pathway and reducing ROS levels through Nrf2 pathway aligning with our findings in vivo.Moreover, the retinal oxygen-sensing mechanisms (HIF-1α/VEGF) are also involved in hydrogen-mediated retinal revascularization and neovascularization suppression.
CONCLUSIONS: Collectively, our results indicate that H2 could be a promising therapeutic agent for POR treatment and that its beneficial effect in human ROP might involve the activation of the Nrf2-Notch axis as well as HIF-1α/VEGF pathways.
摘要：
背景：早产儿视网膜病变（ROP）是一种发生在早产儿视网膜上的增生性视网膜血管疾病，是导致儿童失明的主要原因。抗VEGF和视网膜光凝是目前ROP的主流治疗方法,但是它们会出现各种并发症。氢气（H2）被广泛认为是缺氧缺血性疾病的有用的神经保护和抗氧化治疗方法，而没有毒性作用。然而,H2是否提供生理血管生成促进，ROP进展中的新生血管抑制和神经胶质保护作用在很大程度上是未知的。本研究旨在探讨H2对视网膜血管生成的影响,氧诱导的视网膜病变（OIR）小鼠视网膜的新生血管形成和神经胶质功能障碍。
方法：在本研究中，将7日龄和野生型（WT）或Nrf2缺陷（Nrf2-/-）的小鼠暴露于75％的氧气中5天，然后恢复到正常的空气条件。给予不同阶段的氢气(H2)吸入。血管闭塞,新生血管形成,并对血管渗漏进行分析比较。计算新生血管内皮细胞核的数量，行视网膜切片常规HE染色。使用DyLight594标记的GSLI-isolectinB4（IB4）进行免疫组织化学，以及针对增殖细胞核抗原（PCNA）的一级抗体，胶质纤维酸性蛋白（GFAP），Iba-1蛋白质印迹法检测NF-E2相关因子2(Nrf2)的表达,血管内皮生长因子（VEGF），Notch1、Dll4和HIF-1α。此外,测定靶基因如NQO1、HO-1、Notch1、Hey1、Hey2和Dll4的表达。在低氧条件下用H2处理的人脐静脉内皮细胞(HUVECs)用作体外模型。RT-PCR检测Nrf2、Notch/Dll4和靶基因的mRNA表达。使用免疫荧光染色观察活性氧（ROS）的表达。
结果：我们的结果表明，3-4％H2不会干扰视网膜生理性血管生成，但改善OIR小鼠的血管闭塞和新生血管形成。此外，H2可防止密度降低，并逆转氧诱导损伤引起的视网膜星形胶质细胞的形态和功能变化。此外,H2吸入减少小胶质细胞活化，特别是在OIR小鼠的新生血管形成领域。H2通过在体内促进Nrf2活化和抑制Dll4诱导的Notch信号通路在血管再生中起保护作用。此外，H2通过负调节Dll4/Notch途径并通过Nrf2途径降低ROS水平来促进低氧下HUVECs的增殖，这与我们在体内的发现一致。此外，视网膜氧敏感机制(HIF-1α/VEGF)也参与氢介导的视网膜血管再生和新生血管抑制.
结论：总的来说，我们的结果表明,H2可能是一种有前景的POR治疗药物,其在人ROP中的有益作用可能涉及Nrf2-Notch轴以及HIF-1α/VEGF通路的激活.
方法：在本研究中，将7日龄和野生型（WT）或Nrf2缺陷（Nrf2-/-）的小鼠暴露于75％的氧气中5天，然后恢复到正常的空气条件。给予不同阶段的氢气(H2)吸入。血管闭塞,新生血管形成,并对血管渗漏进行分析比较。计算新生血管内皮细胞核的数量，行视网膜切片常规HE染色。使用DyLight594标记的GSLI-isolectinB4（IB4）进行免疫组织化学，以及针对增殖细胞核抗原（PCNA）的一级抗体，胶质纤维酸性蛋白（GFAP），Iba-1蛋白质印迹法检测NF-E2相关因子2(Nrf2)的表达,血管内皮生长因子（VEGF），Notch1、Dll4和HIF-1α。此外,测定靶基因如NQO1、HO-1、Notch1、Hey1、Hey2和Dll4的表达。在低氧条件下用H2处理的人脐静脉内皮细胞(HUVECs)用作体外模型。RT-PCR检测Nrf2、Notch/Dll4和靶基因的mRNA表达。使用免疫荧光染色观察活性氧（ROS）的表达。
结果：我们的结果表明，3-4％H2不会干扰视网膜生理性血管生成，但改善OIR小鼠的血管闭塞和新生血管形成。此外，H2可防止密度降低，并逆转氧诱导损伤引起的视网膜星形胶质细胞的形态和功能变化。此外,H2吸入减少小胶质细胞活化，特别是在OIR小鼠的新生血管形成领域。H2通过在体内促进Nrf2活化和抑制Dll4诱导的Notch信号通路在血管再生中起保护作用。此外，H2通过负调节Dll4/Notch途径并通过Nrf2途径降低ROS水平来促进低氧下HUVECs的增殖，这与我们在体内的发现一致。此外，视网膜氧敏感机制(HIF-1α/VEGF)也参与氢介导的视网膜血管再生和新生血管抑制.
结论：总的来说，我们的结果表明,H2可能是一种有前景的POR治疗药物,其在人ROP中的有益作用可能涉及Nrf2-Notch轴以及HIF-1α/VEGF通路的激活.
