EGFL7 Inhibits the Sestrin2/Nrf2 Signaling Pathway，promotes Angiogenesis，and aggravates Diabetic Retinopathy in Rats
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摘要:
目的 基于Sestrin2/核因子红细胞2相关因子2（nuclear factor erythroid 2-related factor 2，Nrf2）信号通路，探究表皮生长因子样结构域7（epidermal growth factor-like domain 7，EGFL7）对糖尿病视网膜病变血管生成的影响。 方法 用大鼠构建糖尿病视网膜病变模型。苏木精-伊红（hematoxylin-eosin，HE）染色用于观察视网膜病理学形态。视网膜胰蛋白酶消化实验用于检测视网膜血管形态。实时定量聚合酶链式反应（real-time quantitative polymerase chain reaction，RT-qPCR）和蛋白免疫印迹（Western blot）分别用于分析视网膜组织EGFL7、Sestrin2、Nrf2、血红素氧合酶1（heme oxygenase-1，HO-1）的mRNA表达以及EGFL7、Sestrin2、分化簇31（cluster of differentiation 31，CD31）、血管内皮生长因子受体2（vascular endothelial growth factor receptor 2，VEGFR2）、Nrf2、HO-1的蛋白表达。相应试剂盒用于检测眼内房水中丙二醛（malondialdehyde，MDA）、超氧化物歧化酶（superoxide dismutase，SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase，GSH-Px）的水平。 结果 与对照组相比，模型组大鼠的视网膜组织呈现病理学损伤，视网膜组织中血管数量增加，EGFL7、CD31、VEGFR2、MDA升高（P < 0.05），SOD、GSH-Px、Sestrin2、Nrf2、HO-1降低（P < 0.05）；与模型组相比，IgG组大鼠的上述病理学指标无显著变化（P > 0.05）；与IgG组相比，anti-EGFL7组大鼠视网膜组织的病理学损伤减轻，视网膜组织中血管数量减少，EGFL7、CD31、VEGFR2、MDA降低（P < 0.05），SOD、GSH-Px、Sestrin2、Nrf2、HO-1升高（P < 0.05）。 结论 EGFL7可能通过促进氧化应激来加剧糖尿病视网膜病变大鼠视网膜的血管新生，其机制可能与抑制Sestrin2/Nrf2信号通路相关。 -
关键词:
- EGFL7 /
- 糖尿病视网膜病变 /
- Sestrin2/Nrf2 /
- 氧化应激
Abstract:Objective To investigate the impact of epidermal growth factor-like domain 7 （EGFL7） on angiogenesis in diabetic retinopathy, based on the Sestrin2/nuclear factor erythroid 2-related factor 2 （Nrf2） signaling pathway. Methods A diabetic retinopathy model was established in the rats. Retinal pathological morphology was examined using hematoxylin-eosin （HE） staining, while retinal vascular morphology was assessed via the retinal trypsin digestion assay. The mRNA expression levels of EGFL7, Sestrin2, Nrf2, and heme oxygenase-1 （HO-1） in retinal tissues were analyzed by RT-qPCR. Protein expression levels of EGFL7, Sestrin2, cluster of differentiation 31 （CD31）, vascular endothelial growth factor receptor 2 （VEGFR2）, Nrf2, and HO-1 were detected by Western blot. Corresponding assay kits were used to measure the levels of malondialdehyde （MDA）, superoxide dismutase （SOD）, and glutathione peroxidase （GSH-Px） in the intraocular aqueous humor. Results Compared with the control group, rats in the model group exhibited pathological damage in retinal tissues, increased retinal vascular density, and elevated levels of EGFL7, CD31, VEGFR2, and MDA （P < 0.05）, whereas levels of SOD, GSH-Px, Sestrin2, Nrf2, and HO-1 were decreased （P < 0.05）. No significant changes were observed in the above pathological indicators in the IgG group compared with the model group （P > 0.05）. In contrast, compared with the IgG group, rats in the anti-EGFL7 group showed alleviated retinal pathological injury, reduced retinal vascular density, decreased levels of EGFL7, CD31, VEGFR2, and MDA （P < 0.05）, and increased levels of SOD, GSH-Px, Sestrin2, Nrf2, and HO-1 （P < 0.05）. Conclusion EGFL7 may exacerbate retinal neovascularization in diabetic retinopathy rats by promoting oxidative stress, a mechanism potentially associated with the inhibition of the Sestrin2/Nrf2 signaling pathway. -
Key words:
- EGFL7 /
- Diabetic retinopathy /
- Sestrin2/Nrf2 /
- Oxidative stress
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图 1 大鼠视网膜中EGFL7蛋白的表达（$ \bar x \pm s $，n = 8）
A：Western blot条带图；B：EGFL7/GAPDH统计图；C：视网膜中EGFL7 mRNA表达。与对照组相比，*P < 0.05；与IgG组相比，#P < 0.05。
Figure 1. Western blot detection of EGFL7 protein expression in the retina of rats in each group （$ \bar x \pm s $，n = 8）
图 2 HE染色检测各组大鼠的视网膜病理学改变（400×，标尺：50 μm）
INL内核层、ONL外核层。
Figure 2. Retinal pathological changes detected by HE staining in rats in each group （400×，scale: 50 μm）
图 3 视网膜胰蛋白酶消化实验检测各组大鼠视网膜的血管形态（200×，标尺：100 μm）
Figure 3. Vascular morphology of the retina of rats in each group was detected by retinal trypsin digestion experiment （200×，scale: 100 μm）
图 4 大鼠视网膜中CD31、VEGFR2蛋白的表达（$ \bar x \pm s $，n = 8）
A：Western blot条带图；B：CD31/GAPDH；C：VEGFR2/GAPDH；与对照组相比，*P < 0.05；与IgG组相比，#P < 0.05。
Figure 4. The expression of CD31 and VEGFR2 proteins in the retina of rats （$ \bar x \pm s $，n = 8）
图 5 各组大鼠眼内房水中氧化应激相关指标的比较（$ \bar x \pm s $，n = 8）
A：眼内房水MDA水平；B：眼内房水SOD水平；C：眼内房水GSH-Px水平；与对照组相比，*P < 0.05；与IgG组相比，#P < 0.05。
Figure 5. Comparison of oxidative stress-related indexes in intraocular aqueous humor of rats in each group （$ \bar x \pm s $，n = 8）
图 6 各组大鼠视网膜中Sestrin2、Nrf2、HO-1 mRNA表达的比较（$ \bar x \pm s $，n = 8）
A：视网膜中Sestrin2 mRNA表达；B：视网膜中Nrf2 mRNA表达；C：视网膜中HO-1 mRNA表达；与对照组相比，*P < 0.05；与IgG组相比，#P < 0.05。
Figure 6. Comparison of mRNA expressions of Sestrin2，Nrf2 and HO-1 in the retina of rats in different groups （$ \bar x \pm s $，n = 8）
图 7 Western blot实验检测各组大鼠视网膜中EGFL7分子及Sestrin2/Nrf2信号通路相关分子的表达（$ \bar x \pm s $，n = 8）
A：Western blot条带图；B：Sestrin2/GAPDH；C：Nrf2（总）/GAPDH；D：HO-1/GAPDH；E：Nrf2（胞浆）/GAPDH；F：Nrf2（胞核）/Histone；与对照组相比，*P < 0.05；与IgG组相比，#P < 0.05。
Figure 7. Western blot experiment detected the expression of EGFL7 molecules and Sestrin2/Nrf2 signaling pathway-related molecules in the retina of rats in each group （$ \bar x \pm s $，n = 8）
表 1 引物信息表
Table 1. Primer information table
引物名称 引物序列 引物长度（bp） EGFL7-F 5'- TGCTGATGTGGCTTCTGGTGTTG -3' 21 EGFL7-R 5'- GGTGGTGAGGAAGGGCTGGTAC -3' 22 Sestrin2-F 5'- CTTCATCCCAGTGGAGGAGATCC -3' 23 Sestrin2-R 5'- CCAGAAGCTGCTAAGGTAGTCG -3' 21 Nrf2-F 5'- CCCATTGAGGGCTGTGATCT -3' 20 Nrf2-R 5'- GCCTTCAGTGTGCTTCTGGTT -3' 20 HO-1-F 5'- ATTTGTCCGAGGCCTTGAA -3' 19 HO-1-R 5'- CCAGGGCCGTATAGATATGGTA -3' 22 GAPDH-F 5'- ACAGCAACAGGGTGGTGGAC -3' 20 GAPDH-R 5'- TTTGAGGGTGCAGCGAACTT -3' 20 
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