[1]屈如意,周梦贤,彭媛,等.miR-223-3p调控NLRP3炎症小体对自身免疫性葡萄膜炎大鼠M1/M2巨噬细胞极化平衡的影响[J].眼科新进展,2023,43(1):007-12.[doi:10.13389/j.cnki.rao.2023.0002]
　QU Ruyi,ZHOU Mengxian,PENG Yuan,et al.Effect of miR-223-3p regulating the expression of Nod-like receptor family pyrin domain containing 3 inflammasome on the balance of M1/M2 macrophage polarization in rats with autoimmune uveitis[J].Recent Advances in Ophthalmology,2023,43(1):007-12.[doi:10.13389/j.cnki.rao.2023.0002]
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miR-223-3p调控NLRP3炎症小体对自身免疫性葡萄膜炎大鼠M1/M2巨噬细胞极化平衡的影响/HTML
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《眼科新进展》[ISSN:1003-5141/CN:41-1105/R]

卷:
43卷
期数:
2023年1期
页码:
007-12
栏目:
实验研究
出版日期:
2023-01-05

文章信息/Info

Title:
Effect of miR-223-3p regulating the expression of Nod-like receptor family pyrin domain containing 3 inflammasome on the balance of M1/M2 macrophage polarization in rats with autoimmune uveitis
作者:
屈如意; 周梦贤; 彭媛; 殷学伟; 郭大东
250014　山东省济南市，山东中医药大学［屈如意（2021级硕士研究生），周梦贤（2020级硕士研究生），彭媛（2022级硕士研究生），殷学伟（2020级博士研究生）］；250002　山东省济南市，山东省中西医结合眼病防治重点实验室，山东省眼病防治研究院（郭大东）
Author(s):
QU Ruyi1; ZHOU Mengxian1; PENG Yuan1; YIN Xuewei1; GUO Dadong2
1.Shandong University of Traditional Chinese Medicine,Jinan 250014,Shandong Province,China
2.Shandong Provincial Key Laboratory of Integrated Traditional Chinese and Western Medicine for Prevention and Therapy of Ocular Diseases；Shandong Academy of Eye Disease Prevention and Therapy，Jinan 250002，Shandong Province，China
关键词:
miR-223-3p; NLRP3炎症小体; 葡萄膜炎; 巨噬细胞极化
Keywords:
miR-223-3p;  Nod-like receptor family pyrin domain containing 3 inflammasome;  uveitis;  macrophage polarization
分类号:
R773
DOI:
10.13389/j.cnki.rao.2023.0002
文献标志码:
A
摘要:
目的　探讨miR-223-3p调控NOD样受体热蛋白结构域相关蛋白3（NLRP3）炎症小体的表达水平对实验性自身免疫性葡萄膜炎（EAU）大鼠M1/M2巨噬细胞极化平衡的影响。
方法　首先构建双荧光素酶报告质粒载体，并验证miR-223-3p对NLRP3基因表达的调控作用。然后将48只健康Lewis雌性大鼠随机分为正常对照（NC）组、EAU组和miR-223-3p慢病毒组，每组16只。EAU组和miR-223-3p慢病毒组大鼠首先用光感受器间维生素A结合蛋白（IRBP）乳糜液诱导EAU模型，同时miR-223-3p慢病毒组每只大鼠双眼玻璃体内注射8 μL携带miR-223-3p的慢病毒，造模12 d后麻醉处死大鼠，分离各组大鼠眼、脾脏和淋巴结组织。实时荧光定量PCR(Q-PCR)检测各组大鼠眼、脾脏和淋巴结组织中miR-223-3p、NLRP3、诱导型一氧化氮合酶（iNOS）和精氨酸酶-1（Arg-1）的基因表达水平； 酶联免疫吸附试验（ELISA）检测各组大鼠眼、脾脏和淋巴结组织中肿瘤坏死因子α（TNF-α）和白细胞介素10（IL-10）以及NLRP3的蛋白表达水平；流式细胞仪检测各组大鼠眼、脾脏和淋巴结组织中M1、M2型巨噬细胞水平。
结果　双荧光素酶表达报告系统检测证实NLRP3是miR-223-3p调控的靶基因。与NC组相比，造模12 d后，EAU组大鼠眼、脾脏和淋巴结组织中miR-223-3p的表达水平均降低（均为P<0.05），而NLRP3和iNOS的mRNA表达水平均升高（均为P<0.05），M1型巨噬细胞相关因子iNOS mRNA和TNF-α蛋白表达水平均升高（均为P<0.05），而M2型巨噬细胞相关因子Arg-1 mRNA和IL-10蛋白表达水平均降低（均为P<0.05）；与EAU组相比，造模12 d后，miR-223-3p慢病毒组大鼠眼、脾脏和淋巴结组织中NLRP3 mRNA和蛋白表达水平均降低，iNOS mRNA和TNF-α蛋白表达水平均降低，而Arg-1 mRNA和IL-10蛋白表达水平均升高（均为P<0.05）。流式细胞仪检测结果显示，与NC组相比，造模12 d后，EAU组大鼠M1型巨噬细胞极化水平升高，而M2型巨噬细胞极化水平降低，M1/M2巨噬细胞比例升高（均为P<0.05）；与EAU组相比，造模12 d后，miR-223-3p慢病毒组大鼠M1型巨噬细胞极化水平降低，而M2型巨噬细胞极化水平升高，M1/M2巨噬细胞比例逐渐恢复平衡(均为P<0.05)。
结论　EAU大鼠体内巨噬细胞极化失衡，提高miR-223-3p水平可抑制NLRP3炎症小体的表达，进而抑制炎症相关信号通路，降低M1型巨噬细胞极化水平以及提高M2型巨噬细胞极化水平，从而发挥对葡萄膜炎大鼠巨噬细胞极化平衡的调控作用。
Abstract:
Objective　To investigate the effect of miR-223-3p regulating the expression level of Nod-like receptor family pyrin domain containing 3 (NLRP3) inflammasome on the balance of M1/M2 macrophage polarization in experimental autoimmune uveitis (EAU) rats.
Methods　The regulatory effect of miR-223-3p on the expression of NLRP3 inflammasome was first investigated by a dual luciferase reporter plasmid vector. Then, 48 healthy female Lewis rats were randomly divided into a normal control (NC) group, an EAU group and a miR-223-3p lentivirus group, with 16 rats in each group. Rats in the EAU group and the miR-223-3p lentivirus group were administered with inter-photoreceptor retinoid binding protein (IRBP) chylous effusion to induce the EAU model, and the vitreous body of both eyes of each rat in the miR-223-3p lentivirus group was injected with 8μL lentivirus carrying miR-223-3p. The rats were sacrificed under anesthesia after 12 d of modeling, and the eyes, spleens, and lymph node tissues were isolated. Next, real-time quantitative PCR was performed to determine the expression levels of miR-223-3p, NLRP3, inducible nitric oxide synthase (iNOS) and Arginase-1 (Arg-1) in the eyes, spleens and lymph nodes of rats in each group; enzyme-linked immunosorbent assay was used to measure the protein levels of tumor necrosis factor-α (TNF-α), interleukin-10 (IL-10) and NLRP3 in the eyes, spleens and lymph node tissues of rats in each group. Meanwhile, the levels of M1 and M2 macrophages in the eyes, spleens and lymph nodes of each group were further determined by flow cytometer.
Results　The results of the dual luciferase assay confirmed that NLRP3 was the target gene regulated by miR-223-3p. Compared with the NC group, the expression levels of miR-223-3p in the eyes, spleens, and lymph nodes of rats in the EAU group were decreased 12 d after modeling (all P<0.05), while the expression levels of messenger ribonucleic acid (mRNA) in NLRP3 and iNOS were increased (both P<0.05); the M1 macrophage-associated factors iNOS mRNA and TNF-α protein expression levels were increased (both P<0.05), while the M2 macrophage-associated factors Arg-1 mRNA and IL-10 protein expression levels were decreased (both P<0.05). Compared with the EAU group, the expression levels of the NLRP3 mRNA and inflammasome, iNOS mRNA and TNF-α protein were decreased, while Arg-1 mRNA and IL-10 protein expression levels were increased in the eyes, spleens and lymph node tissues of rats in the miR-223-3p lentivirus group after 12 d (all P<0.05). The measurement of the flow cytometer showed that the level of M1 macrophage polarization in the EAU group was increased than that in the NC group 12 d after modeling, while the level of M2 macrophage polarization decreased, and the ratio of M1/M2 macrophages increased (all P<0.05). Compared with the EAU group, the polarization level of M1 macrophages was decreased, while the polarization level of M2 macrophages was increased in the miR-223-3p lentivirus group after 12 d (both P<0.05), and the ratio of M1/M2 macrophages was gradually restored to balance.
Conclusion　Macrophage polarization is imbalanced in EAU rats; elevating miR-223-3p level can inhibit the NLRP3 inflammasome expression, which inhibits inflammation-related signaling pathways, reduces the M1 macrophage polarization level and increases the M2 macrophage polarization level, thereby exerting a regulatory effect on the balance of macrophage polarization in rats with uveitis.

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备注/Memo

备注/Memo:
山东省自然科学基金重点项目（编号：ZR2020KC024）; 国家自然科学基金项目（编号：81873163）
更新日期/Last Update: 2023-01-05