| 刘 旭,万鹏飞,杨维佳,贾 俊,何 媛,王 霞.PDE2A通过影响脉络膜血管生成和NADPH氧化酶/ROS/NF-κB通路参与年龄相关性黄斑变性[J].现代生物医学进展英文版,2023,(6):1033-1040. |
| PDE2A通过影响脉络膜血管生成和NADPH氧化酶/ROS/NF-κB通路参与年龄相关性黄斑变性 |
| PDE2A Participates in Age-Related Macular Degeneration by Affecting Choroidal Angiogenesis and NADPH Oxidase/ROS/NF-κB Pathway |
| Received:September 21, 2022 Revised:October 18, 2022 |
| DOI：10.13241/j.cnki.pmb.2023.06.007 |
| 中文关键词: 年龄相关性黄斑变性 脉络膜新生血管 磷酸二酯酶2A NADPH氧化酶 活性氧 NF-κB |
| 英文关键词: Age-related macular degeneration Choroidal neovascularization Phosphodiesterase 2A NADPH oxidase Reactive oxygen species NF-κB |
| 基金项目:陕西省教育厅专项科学研究计划项目（No.19JK0758）；西安医学院第二附属医院一般项目（23KY0108） |
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| 中文摘要: |
| 摘要 目的：探究磷酸二酯酶2A（Phosphodiesterase 2A，PDE2A）在年龄相关性黄斑变性（Age-related macular degeneration，AMD）中的可能作用及其对脉络膜新生血管（Choroidal neovascularization，CNV）形成的影响。方法：通过qRT-PCR法检测36例湿性AMD患者（AMD组）和36例健康体检者（Healthy组）的血清PDE2A水平。将恒河猴脉络膜血管内皮细胞系（RF/6A）分为Normoxia组、Hypoxia组、Hypoxia+NC组、Hypoxia+PDE2A组。使用Lipofectamine2000分别将NC-shRNA和PDE2A-shRNA慢病毒转染入Hypoxia+NC组和Hypoxia+PDE2A组，转染后，根据分组情况，在RPMI 1640培养基中添加200 mM CoCl2来对RF/6A细胞进行低氧处理。建立了激光诱导的CNV小鼠模型后，将36只建模成功的小鼠随机分为Model组、NC-shRNA组和PDE2A-shRNA组，每组12只。选取12只未建模的小鼠作为Control组。分别对NC-shRNA组和PDE2A-shRNA组小鼠玻璃体腔注射相应慢病毒，共治疗7 d。然后对小鼠进行眼底荧光血管造影（fundus fluorescein angiography，FFA）检查和眼球HE染色。使用相应试剂盒检测小鼠脉络膜组织中ROS、SOD、MDA的含量。通过qRT-PCR或Western blot检测RF/6A细胞或脉络膜组织中PDE2A、VEGFA、VEGFR2、HIF-1α、NOX2、NOX4和NF-κB p65的表达。结果：与Healthy组相比，AMD组患者的血清PDE2A水平显著升高（1.00±0.23 vs 3.09±1.46， P<0.001）。与Hypoxia组相比，Hypoxia+PDE2A组RF/6A细胞的闭合管腔数量减少（P<0.05），PDE2A、VEGFA、VEGFR2和HIF-1?琢的mRNA和蛋白表达水平均降低（P<0.05）。与Model组相比，PDE2A-shRNA组CNV小鼠脉络膜病变明显减轻，血管生成和脉络膜增明显减少，CNV相对荧光强度降低（P<0.05），脉络膜组织中的PDE2A、VEGFA、VEGFR2和HIF-1α的mRNA和蛋白表达水平均降低（P<0.05）。与Model组相比，PDE2A-shRNA组小鼠脉络膜组织中的ROS和MDA含量均降低，SOD含量升高（P<0.05）。与Model组相比，PDE2A-shRNA组小鼠脉络膜组织中的NOX2、NOX4和细胞核NF-κB p65蛋白相对表达量均降低（P<0.05）。结论：PDE2A通过影响脉络膜血管生成和NADPH氧化酶/ROS/NF-κB通路参与年龄相关性黄斑变性。 |
| 英文摘要: |
| ABSTRACT Objective: To reveal the possible role of phosphodiesterase 2A (PDE2A) in age-related macular degeneration (AMD) and its effect on choroidal neovascularization (CNV). Methods: The serum PDE2A levels in 36 wet AMD patients (AMD group) and 36 healthy subjects (Healthy group) were detected by qRT-PCR. Rhesus monkey choroidal vascular endothelial cell line (RF/6A) was divided into Normoxia group, Hypoxia group, Hypoxia+NC group and Hypoxia+PDE2A group. NC-shRNA and PDE2A-shRNA lentivirus were transfected into Hypoxia+NC group and Hypoxia+PDE2A group by Lipofectamine2000, respectively. After transfection, according to the grouping, 200 mM CoCl2 was added to RPMI1640 medium to treat RF/6A cells under hypoxia. After the laser-induced CNV mouse model was established, 36 successfully modeled mice were randomly divided into model group, NC-shRNA group and PDE2A-shRNA group, with 12 mice in each group. Twelve unmodeled mice were selected as the Control group. The corresponding lentiviruses were injected into the vitreous cavity of mice in the NC-shRNA group and the PDE2A-shRNA group, respectively, for a total of 7 days of treatment. The mice were then subjected to fundus fluorescein angiography (FFA) and eye HE staining. The contents of ROS, SOD and MDA in mouse choroidal tissue were detected using corresponding kits. The expressions of PDE2A, VEGFA, VEGFR2, HIF-1α, NOX2, NOX4 and NF-κB p65 in RF/6A cells or choroidal tissues were detected by qRT-PCR or Western blot. Results: Compared with Healthy group, the serum PDE2A level in AMD group was significantly increased (1.00±0.23 vs 3.09±1.46, t=8.507, P<0.001). Compared with Hypoxia group, the number of closed lumens of RF/6A cells in Hypoxia+PDE2A group decreased (P<0.05), and the mRNA and protein expressions of PDE2A, VEGFA, VEGFR2 and HIF-1α were decreased (P<0.05). Compared with Model group, the choroidal lesions of the CNV mice in PDE2A-shRNA group were significantly reduced, angiogenesis and choroidal hyperplasia were significantly reduced, and the relative fluorescence intensity of CNV was decreased (P<0.05), the mRNA and protein expression levels of PDE2A, VEGFA, VEGFR2 and HIF-1α in choroidal tissue were decreased (P<0.05). Compared with Model group, the contents of ROS and MDA in the choroidal tissue of PDE2A-shRNA group were decreased, and the content of SOD was increased (P<0.05). Compared with Model group, the relative expressions of NOX2, NOX4 and nuclear NF-κB p65 proteins in the choroidal tissue of PDE2A-shRNA group were all decreased (P<0.05). Conclusion: PDE2A participates in age-related macular degeneration by affecting choroidal angiogenesis and NADPH Oxidase/ROS/NF-κB pathway. |
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