Department of Ophthalmology, The First Affiliated Hospital of China Medical University, Shenyang, Liaoning 110001, P.R. China.
Department of Ophthalmology, The Fourth People's Hospital of Shenyang, Shenyang, Liaoning 110031, P.R. China.
Mol Med Rep. 2019 Mar;19(3):2307-2316. doi: 10.3892/mmr.2019.9863. Epub 2019 Jan 15.
The present study aimed to investigate the significant role of β-elemene in mouse models of oxygen-induced retinopathy (OIR). C57BL/6J neonatal mice were used to establish OIR models. They were divided into four groups: Normoxia, OIR, OIR control and OIR‑treated. Mice in the OIR group were exposed to 75±5% oxygen for 5 days and returned to a normal oxygen environment on postnatal day 12 (P12). The OIR treated group was intravitreally injected with 1 µl β‑elemene on P12 and subsequently returned to a normal oxygen environment for 5 days (P12‑P17). Retinas were obtained on P17. Retinal neovascularization (RNV) was detected using adenosine diphosphatase staining and analyzed by counting the nuclei of neovascular endothelial cells. Vascular endothelial growth factor (VEGF) expression was determined by reverse transcription‑quantitative polymerase chain reaction, immunohistochemistry and western blot analysis. MicroRNA (miRNA/miR) microarrays were used to screen out differentially expressed miRNAs between the OIR and β‑elemene‑treated groups. Binding the 3'‑untranslated region (UTR) of VEGF and miR‑27a was confirmed using luciferase assays. It was found that high oxygen concentrations accelerated RNV and increased the number of preretinal neovascular cells; β‑elemene treatment reduced these effects. VEGF mRNA and protein expression was higher in the OIR and OIR control groups, compared with the normoxia and OIR‑treated groups. Further, it was shown that miR‑22, miR‑181a‑1, miR‑335‑5p, miR‑669n, miR‑190b, miR‑27a and miR‑93 were upregulated in the OIR‑treated group, and downregulated in the OIR group. The prediction websites TargetScan and miRanda revealed that VEGF contained a potential miR‑27a binding site in its 3'‑untranslated region (UTR). Luciferase assays demonstrated that miR‑27a directly bound to the 3'‑UTR of VEGF. In vitro experiments demonstrated that miR‑27a inhibited VEGF expression. In addition, β‑elemene treatment upregulate miR‑27a expression in vivo and in vitro. When miR‑27a expression was depleted by miR‑27a inhibitor, the protective effect of β‑elemene on RNV was eliminated. The present study demonstrated that β‑elemene reduced RNV in mouse OIR models via miR‑27a upregulation, leading to reduced VEGF expression. This finding may contribute to the development of novel therapeutic strategies for human retinopathy.
本研究旨在探讨β-榄香烯在氧诱导视网膜病变（OIR）小鼠模型中的重要作用。使用 C57BL/6J 新生小鼠建立 OIR 模型。将它们分为四组：常氧组、OIR 组、OIR 对照组和 OIR 治疗组。OIR 组的小鼠在出生后第 12 天（P12）前 5 天暴露于 75±5%氧气中，然后返回正常氧环境。OIR 治疗组在 P12 时玻璃体腔内注射 1µlβ-榄香烯，随后返回正常氧环境 5 天（P12-P17）。P17 时获取视网膜。用腺苷二磷酸酶染色检测视网膜新生血管（RNV），并通过计数新生血管内皮细胞核来分析。采用逆转录-定量聚合酶链反应、免疫组织化学和 Western blot 分析检测血管内皮生长因子（VEGF）的表达。使用 miRNA 微阵列筛选出 OIR 组和β-榄香烯治疗组之间差异表达的 miRNA。通过荧光素酶测定证实 VEGF 和 miR-27a 的 3'UTR 结合。结果发现，高氧浓度加速了 RNV 并增加了视网膜前新生血管细胞的数量；β-榄香烯治疗降低了这些作用。OIR 组和 OIR 对照组的 VEGF mRNA 和蛋白表达均高于常氧组和 OIR 治疗组。此外，结果表明 miR-22、miR-181a-1、miR-335-5p、miR-669n、miR-190b、miR-27a 和 miR-93 在 OIR 治疗组中上调，而在 OIR 组中下调。靶标扫描和 miRanda 预测网站显示，VEGF 在其 3'UTR 中含有一个潜在的 miR-27a 结合位点。荧光素酶测定表明 miR-27a 可直接结合 VEGF 的 3'UTR。体外实验表明 miR-27a 抑制 VEGF 表达。此外，β-榄香烯治疗可上调体内和体外的 miR-27a 表达。当 miR-27a 表达被 miR-27a 抑制剂耗尽时，β-榄香烯对 RNV 的保护作用被消除。本研究表明，β-榄香烯通过上调 miR-27a 减少小鼠 OIR 模型中的 RNV，从而降低 VEGF 表达。这一发现可能有助于开发人类视网膜病变的新治疗策略。
