玻璃体内注射雷公藤内酯可减轻激光诱导的小鼠模型中视网膜下纤维化。

Intravitreal injection of triptolide attenuates subretinal fibrosis in laser-induced murine model.

机构信息

State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, 54 South Xianlie Road, Guangzhou, Guangdong 510060, China.

Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA 52242, USA.

出版信息

Phytomedicine. 2021 Dec;93:153747. doi: 10.1016/j.phymed.2021.153747. Epub 2021 Sep 12.

DOI:10.1016/j.phymed.2021.153747

PMID:34620548

Abstract

BACKGROUND

Choroidal neovascularization (CNV) is a common cause of irreversible blindness in elderly patients in developed countries, and subretinal fibrosis is an advanced stage of CNV. Currently, there is no effective clinical treatment for subretinal fibrosis.

PURPOSE

To investigate whether intravitreal injection of triptolide (TP) could attenuate subretinal fibrosis and determine its underlying mechanisms.

METHODS

CNV was induced by laser photocoagulation in C57BL/6J mice. Immediately after laser photocoagulation, 1 μl of free TP (10 μg), TP-nanolip-PEG (TP-loaded PEGylated nanoliposomes containing 10 μg TP), or the same volume of phosphate-buffered saline (PBS) was intravitreally administered to each respective group. Areas and ratios of subretinal fibrosis were calculated seven days after laser injury. Additionally, expression levels of M2 macrophage-related markers, molecules of the transforming growth factor (TGF)-β1/Smad signaling pathway, and markers for epithelial-mesenchymal transition (EMT) and endothelial-to-mesenchymal transition (EndoMT) were detected both in vitro and in vivo.

RESULTS

The areas of subretinal fibrosis were significantly reduced in both the free TP (10993.87 ± 2416.90 μm) and TP-nanolip-PEG (7695.32 ± 2121.91 μm) groups when compared with the PBS group (15971.97 ± 3203.10 μm) (p < 0.05, n = 6). The ratio of subretinal fibrosis in the free TP monomer (20.8 ± 4.2%) and TP-nanolip-PEG (12.5 ± 4.0%) groups was lower than that in the PBS control group (41.7 ± 5.3%) (p < 0.01, n = 6). Moreover, both TP and TP-nanolip-PEG suppressed the polarization of M2 macrophages and downregulated gene expressions of TGF-β1, Smad 2, Smad 3, α-SMA, and collagen I (p < 0.05), but upregulated the gene expression of E-cadherin (p < 0.05), thus reversing TGF-β1 induced EMT/EndoMT and attenuating subretinal fibrosis.

CONCLUSIONS

TP could attenuate subretinal fibrosis by suppressing the polarization of M2 macrophages and TGF-β1 induced EMT/EndoMT. TP-nanolip-PEG enhanced the inhibitory effects of TP on subretinal fibrosis, suggesting its therapeutic potential for CNV-related subretinal fibrosis.

摘要

背景

脉络膜新生血管（CNV）是发达国家老年患者致盲的常见原因，而视网膜下纤维化是 CNV 的晚期阶段。目前，尚无有效的临床方法治疗视网膜下纤维化。

目的

探讨玻璃体内注射雷公藤甲素（TP）能否减轻视网膜下纤维化，并确定其潜在机制。

方法

通过激光光凝在 C57BL/6J 小鼠中诱导 CNV。激光光凝后立即向各组分别玻璃体内注射 1 μl 游离 TP（10 μg）、TP-纳米脂质体-PEG（载有 10 μg TP 的聚乙二醇化纳米脂质体）或等量磷酸盐缓冲盐水（PBS）。激光损伤后 7 天计算视网膜下纤维化的面积和比例。此外，还在体外和体内检测了 M2 巨噬细胞相关标志物、转化生长因子（TGF）-β1/Smad 信号通路分子以及上皮-间充质转化（EMT）和内皮-间充质转化（EndoMT）的标志物。

结果

与 PBS 组（15971.97 ± 3203.10 μm）相比，游离 TP（10993.87 ± 2416.90 μm）和 TP-纳米脂质体-PEG（7695.32 ± 2121.91 μm）组的视网膜下纤维化面积明显减少（p < 0.05，n = 6）。游离 TP 单体（20.8 ± 4.2%）和 TP-纳米脂质体-PEG（12.5 ± 4.0%）组的视网膜下纤维化比例均低于 PBS 对照组（41.7 ± 5.3%）（p < 0.01，n = 6）。此外，TP 和 TP-纳米脂质体-PEG 均抑制了 M2 巨噬细胞的极化，并下调了 TGF-β1、Smad 2、Smad 3、α-SMA 和胶原 I 的基因表达（p < 0.05），但上调了 E-钙黏蛋白的基因表达（p < 0.05），从而逆转了 TGF-β1 诱导的 EMT/EndoMT，并减轻了视网膜下纤维化。