基于 miR-223 / NLRP3 轴研究柚皮素对氧诱导视网膜病变中小胶质细胞活化的影响
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郑州大学附属儿童医院,河南省儿童医院,郑州儿童医院眼科,郑州 450000

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R-33


Effects of naringenin on microglial activation in oxygen-induced retinopathy based on the miR-223 / NLRP3 axis
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Department of Ophthalmology, Children’s Hospital Affiliated of Zhengzhou University, Henan Children’s Hospital, Zhengzhou Children’s Hospital, Zhengzhou 450000, China

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    摘要:

    目的 基于微小 RNA(miR)-223 / 核苷酸结合寡聚化结构域样受体蛋白 3(NLRP3)轴探讨柚皮素 (NAR)对氧诱导视网膜病变(OIR)中小胶质细胞活化的影响。 方法 150 只 7 日龄(P7)C57BL/ 6J 幼鼠分为常氧组、OIR 组、NAR 组、NAR+阴性对照组、NAR+miR-223 拮抗剂组,每组 30 只。 除常氧组外,其余各组幼鼠及其母鼠在 P7~ P12 移至氧气体积分数(75±2)%封闭氧箱中,连续 5 d,P12 返回正常氧环境中;常氧组在正常氧环境中常规饲养。 NAR 组幼鼠腹腔注射 100 mg / (kg·d) NAR,NAR+阴性对照组在 NAR 基础上 P12 尾静脉注射 2. 5 mg / kg miR-223 拮抗剂阴性对照,NAR+miR-223 拮抗剂组在 NAR 基础上 P12 尾静脉注射 2. 5 mg / kg miR-223 拮抗剂,常氧组、OIR 组每天腹腔注射等体积 CMC、P12 尾静脉注射等体积生理盐水。实时荧光定量 PCR(RT-qRCR)检测视网膜中 miR-223 水平;幼鼠眼底行荧光素眼底血管造影(FFA);苏木精-伊红(HE)染色观察视网膜形态;免疫荧光检测视 网膜中小胶质细胞标志物钙离子结合蛋白-1(Iba-1)情况;Western blot 检测视网膜中 NLRP3、半胱氨酸天冬氨酸蛋白酶-1(Caspase-1)、白细胞介素(IL)-1β 及 IL-18 蛋白表达情况。 结果 OIR 组出现血管破裂,荧光漏在视网膜中,视网膜泛白,血管出现收缩,视网膜厚度变厚、细胞排列松散,部分出现细胞缺失现象、血管新生现象;NAR 组、NAR+阴性对照组血管破裂现象缓解,视网膜泛白现象减轻,但视网膜细胞仍松散;NAR+miR-223 拮抗剂组血管破裂,荧光漏在视网膜中,视网膜泛白明显,视网膜细胞松散严重、细胞缺失明显。与常氧组相比,OIR 组视网膜中 miR-223 水平降低 (P<0. 05),视网膜中 Iba-1 水平、NLRP3、Caspase-1、IL-1β、IL-18 蛋白水平升高(P<0. 05);与 OIR 组相比,NAR 组、 NAR+阴性对照组视网膜中 miR-223 水平升高(P<0. 05),视网膜中 Iba-1 水平、NLRP3、Caspase-1、IL-1β、IL-18 蛋白水平降低(P<0. 05);分别与 NAR 组、NAR+阴性对照组相比,NAR+miR-223 拮抗剂组视网膜中 miR-223 水平降低(P< 0. 05),视网膜中 Iba-1 水平、NLRP3、Caspase-1、IL-1β、IL-18 蛋白水平升高(P<0. 05)。 结论 NAR 能够升高miR-223 的表达进而抑制 NLRP3 炎症体的表达,从而缓解小胶质细胞过度活化现象,实现对 OIR 的缓解。

    Abstract:

    Objective To investigate the effect of naringenin (NAR) on microglial activation in oxygen-induced retinopathy (OIR) based on the microRNA (miR)-223 / nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3) axis. Methods Seven-day-old (P7) C57BL/ 6J mice ( n= 150) were divided into normoxia, OIR, NAR, NAR + negative RNA control and NAR + miR-223 antagomir groups with 30 mice per group. Except for mice in the normoxic group, the young mice and their mothers were moved to a closed oxygen box with an oxygen volume fraction of (75 ± 2)% for 5 consecutive days from P7~ P12 and subsequently returned to a normoxic environment; the normoxic group was raised in a normoxic environment. The 12-day-old mice in the NAR groups were injected intraperitoneally with 100 mg / (kg ·d) NAR. The mice in the NAR + negative RNA control group were also injected with 2. 5 mg / kg miR-223 negative control RNA via the tail vein, and mice in the NAR + miR-223 antagomir group were injected with 2. 5 mg / kg miR-223 antagomir via the tail vein. Mice in the normoxic and OIR groups were intraperitoneally injected with an equal volume of carboxymethyl cellulose and tail vein-injected with an equal volume of normal saline each day. For evaluation of retinas in the young mice, real time quantitative PCR (RT-qPCR) was used to detect the level of miR-223; fundus fluorescein angiography was performed; hematoxylin eosin staining was used to observe morphology; immunofluorescence was used to detect the expression of the microglia marker calcium binding protein-1 (Iba-1); and Western blot were used to detect the expression NLRP3, cysteinyl aspartate-specific proteinase-1 ( Caspase-1), interleukin ( IL)-1β and IL-18. Results In the OIR group, blood vessels were ruptured, fluorescence leaked in the retina, the retinas were white, blood vessels were contracted, the retinas were thickened, cell arrangement was loose, and some cells were missing and angiogenesis occurred. In the NAR and NAR + negative RNA control groups, vascular rupture and retinal whitening were alleviated, but retinal cells were still loosely arranged. In the NAR + miR-223 antagomir group, vessels were ruptured, fluorescence leaked in the retina, retinal whitening was obvious, and retinal cells were severely loosened and missing. Compared with that in the normoxic group, miR-223 levels in retinas of the OIR mice decreased (P<0. 05), and the retinal levels of Iba-1, NLRP3, Caspase-1, IL-1β, IL-18 increased (P<0. 05). Compared with that in the OIR group, miR-223 levels in the retinas from the NAR and NAR + negative RNA control groups increased (P<0. 05), and retinal levels of Iba-1, NLRP3, Caspase-1, IL-1β and IL-18 decreased (P<0. 05). Compared with that in the NAR and NAR + negative control groups, retinal levels of miR-223 in the NAR + miR-223 antagomir group decreased (P<0. 05), and the levels of Iba-1, NLRP3, Caspase-1, IL-1β and IL-18 increased (P<0. 05). Conclusions NAR increased the expression of miR-223 and inhibited components of the NLRP3 inflammasome, thus alleviating the over-activation of microglia and reducing OIR.

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楚瑞雪,孙先桃,王 惠.基于 miR-223 / NLRP3 轴研究柚皮素对氧诱导视网膜病变中小胶质细胞活化的影响[J].中国比较医学杂志,2022,32(2):46~52.

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  • 收稿日期:2021-03-23
  • 在线发布日期: 2022-04-12
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