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中华细胞与干细胞杂志(电子版) ›› 2025, Vol. 15 ›› Issue (02) : 75 -81. doi: 10.3877/cma.j.issn.2095-1221.2025.02.002

论著

微重力环境下中性粒细胞胞外诱捕网在大鼠视网膜炎症改变中的作用
刘猛1,2, 郑汉文1, 任飞1, 王宇宇1,2, 张荣1, 高彩云1, 宋飞龙1, 高惠1, 顾建文2,(), 聂闯1,()   
  1. 1. 100101 北京,中国人民解放军总医院第九医学中心
    2. 100049 北京,中国科学院大学医学院
  • 收稿日期:2024-11-11 出版日期:2025-04-01
  • 通信作者: 顾建文, 聂闯
  • 基金资助:
    2021年度中心博士毕业生科研启动基金项目(21ZX05)

The role of neutrophil extracellular traps in inflammatory changes in rat retinas under microgravity conditions

Meng Liu1,2, Hanwen Zheng1, Fei Ren1, Yuyu Wang1,2, Rong Zhang1, Caiyun Gao1, Feilong Song1, Hui Gao1, Jianwen Gu2,(), Chuang Nie1,()   

  1. 1. The Ninth Medical Center of PLA General Hospital,Beijing 100101,China
    2. Medical School,University of Chinese Academy of Sciences,Beijing 100049,China
  • Received:2024-11-11 Published:2025-04-01
  • Corresponding author: Jianwen Gu, Chuang Nie
引用本文:

刘猛, 郑汉文, 任飞, 王宇宇, 张荣, 高彩云, 宋飞龙, 高惠, 顾建文, 聂闯. 微重力环境下中性粒细胞胞外诱捕网在大鼠视网膜炎症改变中的作用[J/OL]. 中华细胞与干细胞杂志(电子版), 2025, 15(02): 75-81.

Meng Liu, Hanwen Zheng, Fei Ren, Yuyu Wang, Rong Zhang, Caiyun Gao, Feilong Song, Hui Gao, Jianwen Gu, Chuang Nie. The role of neutrophil extracellular traps in inflammatory changes in rat retinas under microgravity conditions[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2025, 15(02): 75-81.

目的

探讨构建模拟微重力环境大鼠模型视网膜结构及炎性因子的变化,并探究NETs 是否在其中发挥作用。

方法

40 只大鼠随机分为对照组和尾悬吊组,每组各20 只,采用尾悬吊法建立大鼠失重模型,悬吊时间为4 周,HE 染色观察视网膜结构变化,OCTA 检测视网膜灌注面积和厚度。采用免疫组化的方法对两组大鼠视网膜中NETosis 相关调节因子[髓过氧化物酶( MPO)、瓜氨酸组蛋白H3( CIT-H3)、中性粒细胞弹性酶( NE)]进行定性评估,并通过Western blot 定量分析模拟微重力前后NETosis 相关调节因子[MPO、蛋白精氨酸脱亚氨酶4( PAD4)、CIT-H3和NE]。此外,采用ELISA方法定量检测促炎因子[白细胞介素-1( IL- 1)、IL-6和肿瘤环死因子-α( TNF-α)]及抗炎因子( IL-10)的表达水平。两组间比较采用独立样本t 检验。

结果

与对照组比较,尾悬吊组视网膜灌注面积[0-6mm:(26.93 ± 0.18) 比( 24.96 ± 0.26) mm2]和厚度[0-6mm:(255.42 ± 3.37)比( 230.77 ± 4.13)μm]均升高( P 均 < 0.05),视网膜各细胞层厚度呈现不同程度增加,同时其排列变得稀疏紊乱;尾悬吊组促炎因子IL-1β [(10.59 ± 0.31)比( 8.25 ± 0.21)pg/(mg·pro)],IL-6[(32.97 ± 1.11)比( 22.89 ± 0.87) pg(/ mg·pro)],TNF-α[(102.47 ± 4.01) 比( 65.84 ± 3.48)pg/( mg·pro)]水平均上升,而抗炎因子IL-10[(15.34 ± 0.35)比(21.17 ± 0.63) pg/(mg·pro)]水平降低( P 均 < 0.05)。NETosis 相关调节因子MPO( 0.68 ±0.01 比0.27 ± 0.01),NE( 0.50 ± 0.02 比0.18 ± 0.02)、CitH3( 0.89 ± 0.01 比0.31 ± 0.02)和PAD4(0.61 ± 0.01 比 0.15 ± 0.02)相对表达量均升高( P 均 < 0.05)。

结论

模拟微重力环境可使大鼠视网膜结构出现病理改变,炎症水平升高,同时NETs 可能参与微重力后视网膜炎症变化过程。

Objective

To investigate the structural changes of the retina and the variations in inflammatory factors in a rat model simulating microgravity, and verify the effects of NETosis.

Methods

Forty rats were randomly divided into control group and tail-suspension group, with 20 rats in each group.Weightlessness model was established by tail-suspension method with a suspension duration of four weeks.HE staining was performed to observe changes in retinal structure, and OCTA was used to measure retinal perfusion area and thickness.Qualitative assessments were made of the retinal structures and NETosis-related regulatory factors[myeloperoxidase (MPO), citrullinated histone H3 (CIT-H3), and neutrophil elastase (NE)].Quantitative evaluations were conducted on NETosis-related regulatory factors[MPO, peptidylarginine deiminase 4 (PAD4), CIT-H3, and NE],pro-inflammatory factors (IL-1, IL-6, and TNF-α), and anti-inflammatory factors (IL-10) before and after simulating microgravity.Independent samples t-tests were used for statistical analysis of the quantitative results.

Results

Compared with the control group, the retinal perfusion area [0- 6mm:(26.93 ± 0.18) vs (24.96 ± 0.26)mm2] and retinal thickness[0-6mm:(255.42 ± 3.37)vs(230.77 ±4.13)μm] were increased in tail-suspension group (all P < 0.05)with varying degrees of thickening as well as disrupted and disorganized arrangement of retinal cell layers.In addition, the expression levels of pro-inflammatory cytokines, including IL-1β[(10.59 ± 0.31) vs (8.25 ± 0.21)pg/(mg·pro)],IL-6[(32.97 ± 1.11) vs( 22.89 ± 0.87)pg/(mg·pro)], and TNF-α[(102.47 ± 4.01) vs (65.84 ±3.48)pg/ (mg·pro)] as well as NETosis-related regulatory factors MPO (0.68 ± 0.01 vs 0.27 ± 0.01),NE (0.50 ± 0.02 vs 0.18 ± 0.02), CitH3 (0.89 ± 0.01 vs 0.31 ± 0.02), and PAD4 (0.61 ± 0.01 vs 0.15 ± 0.02)were elevated, while the anti-inflammatory cytokine IL-10[(15.34 ± 0.35) vs (21.17 ±0.63)pg/(mg·pro)] was reduced in tail-suspension group(all P< 0.05).

Conclusion

Simulating a microgravity environment induces pathological changes in the retinal structure of rats, elevates inflammation levels, which maybe related with NETosis.

图1 微重力对SD 大鼠视网膜形态学的影响 注:a ~ b 图为眼底照相机对大鼠眼底拍摄数字眼底图片(× 10); n = 10 , **P < 0.01
图2 显微镜下观察观察两组大鼠视网膜形态变化 (HE 染色,×20) 注:a 图为正常组,b 图为尾悬吊组,n = 3;GCL 为视神经节细胞层;IPL 为内丛状层;INL 为内核层;OPL 为外丛状层;ONL 为外核层;PISL/POSL为感光细胞层; RPE 为视网膜色素上皮层;绿色箭头指示处显示视神经节细胞层排列稀疏紊乱,黄色箭头指内核层排列稀疏紊乱
图3 两组大鼠视网膜组织炎性因子表达 注:n = 15,**P < 0.01
图4 Western blot 检测 NETosis 相关调节因子 (MPO、NE、CitH3 和PAD4)蛋白表达 注:n = 4,***P < 0.001
图5 显微镜下观察两组大鼠视网膜中NETosis 相关调节因子 (MPO、NE 和CitH3)的表达情况 (免疫组化,×300) 注:n = 3
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