miR-410 通过 PI3K-AKT-VEGF 信号通路调节卵清蛋白诱导的哮喘中的辅助性 T 细胞分化。

miR-410 Regulates Helper T Cell Differentiation in Ovalbumin-Induced Asthma through the PI3K-AKT-VEGF Signaling Pathway.

机构信息

Department of Ophthalmology, Qingdao Municipal Hospital, Qingdao, China.

Department of Pediatrics, The Affiliated Hospital of Qingdao University, Qingdao, China.

出版信息

Int Arch Allergy Immunol. 2024;185(1):1-9. doi: 10.1159/000531493. Epub 2023 Sep 19.

DOI:10.1159/000531493

PMID:37725935

Abstract

INTRODUCTION

Asthma has been attributed to Th1/Th2 imbalance and inappropriate Th2 responses to environmental allergens. MicroRNAs (miRNAs), 21 to 23 RNA molecules, are first found in mammals and have been implicated in various biological activities. Our previous study found that miR-410 effectively ameliorates airway inflammation in the ovalbumin (OVA)-induced asthma murine model. However, the role of miR-410 in regulating helper T (Th) cell differentiation is not clear. In the present study, we aimed to explore the regulatory effects of miR-410 on the differentiation of Th cells through both in vivo and in vitro studies.

METHODS

Dual-luciferase reporter assay was used to find if miR-410 has any direct binding position with VEGF mRNAs. PBMC and CD4+ T cells were isolated and stimulated with OVA. The miR-410 mimics and inhibitors were transfected into CD4+ T cells. The differentiation of Th cells was evaluated by enzyme-linked immunosorbent assay (ELISA) for the concentration of IL-4, IFN-γ, and TGF-β levels in supernatants. Western Blot was used to detect protein expression and phosphorylation of PI3K and AKT. BALB/c mice were kept in a specific pathogen-free condition and received sterile OVA-free food and water. OVA-induced asthmatic mice model was established. ELISA was used to measure the bronchoalveolar lavage fluid (BALF) concentrations of IL-4, IFN-γ, TGF-β, and VEGF. Hematoxylin and eosin staining and immunohistochemical staining were conducted to analyze inflammatory cell infiltration, pathological changes, and the expression of VEGF.

RESULTS

Dual-luciferase reporter assay showed that miR-410 has no direct binding position with VEGF mRNAs. In the OVA-primed mononuclear cells compared to normal cells, IFN-γ and TGF-β were decreased while IL-4 and VEGF were increased. This change was reversed while miRNA-410 mimics were transfected into CD4+ T cells. Besides, the OVA-primed CD4+ T cells treated with miR-410 decrease the proliferation of cytokine of Th2 cells as well as phosphorylation of PI3K, and AKT. In OVA-induced asthma mice, IFN-γ and TGF-β were decreased in BALF while the IL-4 and VEGF were increased. OVA-induced mice with asthma treated with miR-410 mimics showed marked reductions in the infiltration of inflammatory cells as well as IL-4 and VEGF in BALF. The immunohistochemical staining of the expression of VEGF also decreased in OVA-induced asthma mice with the instillation of miR-410.

CONCLUSIONS

In this study, we revealed that miR-410 could regulate the differentiation of Th cells via the PI3K-AKT-VEGF signaling pathway in asthma.

摘要

简介

哮喘被归因于 Th1/Th2 失衡和对环境过敏原的不适当 Th2 反应。微小 RNA（miRNA）是在哺乳动物中首次发现的 21 到 23 个 RNA 分子，它们与各种生物活性有关。我们之前的研究发现 miR-410 可有效改善卵清蛋白（OVA）诱导的哮喘小鼠模型中的气道炎症。然而，miR-410 调节辅助性 T（Th）细胞分化的作用尚不清楚。在本研究中，我们旨在通过体内和体外研究探索 miR-410 对 Th 细胞分化的调节作用。

方法

双荧光素酶报告基因实验用于寻找 miR-410 是否与 VEGF mRNA 有直接结合位。分离外周血单核细胞和 CD4+T 细胞，并使用 OVA 进行刺激。将 miR-410 模拟物和抑制剂转染到 CD4+T 细胞中。通过酶联免疫吸附测定（ELISA）测量上清液中 IL-4、IFN-γ 和 TGF-β 水平来评估 Th 细胞的分化。Western blot 用于检测 PI3K 和 AKT 的蛋白表达和磷酸化。BALB/c 小鼠在无特定病原体的条件下饲养，接受无菌、无 OVA 的食物和水。建立 OVA 诱导的哮喘小鼠模型。ELISA 用于测量支气管肺泡灌洗液（BALF）中 IL-4、IFN-γ、TGF-β 和 VEGF 的浓度。进行苏木精和伊红染色和免疫组织化学染色，以分析炎症细胞浸润、病理变化和 VEGF 的表达。

结果

双荧光素酶报告基因实验表明，miR-410 与 VEGF mRNA 没有直接结合位。与正常细胞相比，在 OVA 致敏的单核细胞中，IFN-γ 和 TGF-β 减少，而 IL-4 和 VEGF 增加。当将 miRNA-410 模拟物转染到 CD4+T 细胞中时，这种变化得到逆转。此外，用 miR-410 处理 OVA 致敏的 CD4+T 细胞可减少 Th2 细胞细胞因子的增殖以及 PI3K 和 AKT 的磷酸化。在 OVA 诱导的哮喘小鼠中，BALF 中的 IFN-γ 和 TGF-β 减少，而 IL-4 和 VEGF 增加。用 miR-410 模拟物处理的 OVA 诱导的哮喘小鼠中，BALF 中的炎症细胞浸润以及 IL-4 和 VEGF 明显减少。OVA 诱导的哮喘小鼠中 VEGF 的免疫组织化学染色表达也随着 miR-410 的注入而减少。