| miR-381调节NF-κB信号通路改善缺血性脑卒中的机制研究 |
| 投稿时间：2025-03-04 修订日期：2025-04-03 点此下载全文 |
| 引用本文：张友涛,董利平,李树铁.miR-381调节NF-κB信号通路改善缺血性脑卒中的机制研究[J].医学研究杂志,2025,54(8):94-100 |
| DOI：
10.11969/j.issn.1673-548X.2025.08.016 |
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| 基金项目:河北省医学科学研究课题(20241344) |
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| 中文摘要:目的 探究miR-381在缺血性脑卒中(ischemic stroke,IS)发病过程中的作用及机制。方法 选取2020年1月～2022年12月河北北方学院附属第一医院收治的90例IS患者作为IS组,同期招募110例健康志愿者作为对照组。实时荧光定量聚合酶链反应(real-time fluorescence quantitative polymerase chain reaction,RT-qPCR)法检测两组受试者血清miR-381水平差异,酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)法检测炎性指标肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白细胞介素-6(interleukin-6,IL-6)和白细胞介素-1β(interleukin-1β,IL-1β)水平差异；CCK-8法评估miR-381对人脐静脉内皮细胞(human umbilical vein endothelial cell,HUVEC)活性的影响；TargetScan、Western blot法与荧光素酶报告基因验证κB抑制因子激酶(inhibitor of kappa B kinase, IKK)是否为miR-381调控的靶基因。构建体外体外氧葡萄糖剥夺(oxygen-glucose deprivation,OGD)模型,过表达或抑制miR-381后,RT-qPCR、Western blot法检测miR-381对凋亡和炎症相关蛋白的影响；受试者工作特征(receiver operating characteristic,ROC)曲线评估miR-381对于IS的诊断价值。结果 与对照组比较,IS组血清miR-381显著下调(P＜0.05),中重度IS组与轻度IS组比较,大体积梗死组与小体积梗死组比较,血清miR-381均显著降低(P值分别为＜0.05、＜0.01)。与对照组比较,IS组血清IL-1β、IL-6及TNF-α水平显著高于对照组(P值分别为＜0.01、＜0.05、＜0.01),与轻度IS组或小体积梗死组比较,中重度IS组或大体积梗死组血清IL-1β、IL-6及TNF-α水平显著升高(P值分别为＜0.01、＜0.05)。CCK-8法检测结果显示,miR-381可增加HUVEC活性；双荧光素酶报告基因实验结果显示,IKK是miR-381的直接作用靶标；Western blot法检测结果显示,过表达miR-381可抑制p65及核因子-κB抑制蛋白(inhibitor of NF-κB,IκB)激活,上调抑凋亡的B细胞淋巴瘤因子-2(B-cell lymphoma-2,Bcl-2)蛋白表达,并降低促凋亡的Bcl-2相关X(Bcl-2 associated X-protein,BAX)蛋白表达；RT-qPCR法检测结果显示,过表达miR-381降低了NF-κB与BAX mRNA表达水平,并升高了Bcl-2mRNA表达水平；ELISA法检测结果显示,过表达miR-381降低了血清IL-1β、IL-6及TNF-α(P值分别为＜0.05、＜0.01、＜0.01)；ROC曲线分析结果显示,miR-381诊断IS的敏感度和特异性分别为76.23%和81.25%。结论 miR-381可通过靶向IKK抑制HUVEC中的凋亡和炎性反应,其有潜力成为IS的治疗靶点。 |
| 中文关键词:缺血性脑卒中 miR-381 炎性反应 凋亡 |
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| Study on the Mechanism of miR-381 Ameliorates Ischemic Stroke by Modulating the NF-κB Signaling Pathway. |
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| Abstract:Objective To explore the role and mechanism of miR-381 in the pathogenesis of ischemic stroke (IS). Methods A total of 90 patients with IS treated in the First Affiliated Hospital of Hebei North University between January 2020 and December 2022 were selected as the IS group, while 110healthy volunteers were selected as the control group. Real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) was used to detect the difference of serum miR-381 between the two groups, and enzyme-linked immunosorbent assay (ELISA) was used to measure the differences of inflammatory markers interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) between the groups. The CCK-8 assay was used to evaluate the effect of miR-381 on the activity of human umbilical vein endothelial cell (HUVEC). Use TargetScan, Western blot and luciferase reporter assays to validate whether inhibitor of kappa B kinase (IKK) was a target gene regulated by miR-381. An in vitro model of oxygen-glucose deprivation (OGD) was constructed, and the effects of miR-381 over-expression or inhibition on apoptosis- and inflammation-related proteins were detected by RT-qPCR and Western blot. The diagnostic value of miR-381 for IS was evaluated using receiver operating characteristic curve (ROC). Results Compared with the control group, the serum miR-381 levels in the IS group were significantly down-regulated (P<0.05). In comparison to the mild IS group or small infarct volume group, serum miR-381 levels were significantly lower in patients with moderate to severe IS or large infarct volumes (P were <0.05, <0.01). Additionally, compared with the control group, the serum levels of IL-1β, IL-6 and TNF-α in the IS group were significantly higher (P were <0.01, <0.05,<0.01). Furthermore, compared to the mild IS group or small infarct volume group, the serum levels of IL-1β, IL-6, and TNF-α were significantly elevated in patients with moderate to severe IS group or large infarct volume group (P were <0.01, <0.05). CCK-8 results indicated that miR-381 promoted the viability of HUVEC. Dual-Luciferase reporter gene assay results demonstrated that IKK was a direct target of miR-381. Western blot results showed that over-expression of miR-381 inhibited the activation of p65 and inhibitor of NF-κB (IKB) proteins, promoted the expression of the anti-apoptotic protein B-cell lymphoma 2 (Bcl-2), and suppressed the expression of the pro-apoptotic protein Bcl-2 associated X (BAX). RT-qPCR results showed that over-expression miR-381 reduced the levels of NF-κB mRNA and BAX mRNA, and increased the levels of Bcl-2mRNA. ELISA results indicated that over-expression miR-381decreased the serum levels of TNF-α, IL-6 and IL-1β (P were <0.05, <0.01,＜0.01). ROC curve analysis showed that the sensitivity and specificity of miR-381 for diagnosing IS were 76.23% and 81.25%, respectively. Conclusion miR-381 can inhibit HUVEC apoptosis and inflammatory responses by targeting IKK, indicating its potential as a therapeutic target for IS. |
| keywords:Ischemic stroke miR-381 Inflammatory response Apoptosis |
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