Department of Paediatrics, Ruian People's Hospital, Ruian Ciy, Wenzhou City, Zhejiang Province, 325200, China.
Acta Biochim Pol. 2021 Mar 22;68(2):151-158. doi: 10.18388/abp.2020_5397.
The aim of this study was to investigate whether the effects of miR-490 on acute lung injury (ALI) induced by sepsis in vitro and in vivo were through targeting multi-drug resistance-associated protein 4 (MRP4). MiR-490 agomir/NC agomir was injected into mice before cecal ligation and puncture (CLP). Pulmonary microvascular endothelial cells (PMVECs) were transfected with or without miR-490 agomir/NC agomir/MRP4/empty vector before lipopolysaccharide (LPS) stimulation. Histopathology, injure score, and Wet/Dry (W/D) of lung tissues were assessed. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β level in bronchoalveolar lavage fluid (BALF) were measured. The levels of caspase-3, Bcl-2, TNF-α, and IL-1β were measured in MPVECs. Dual-luciferase reporter assay was used to analyze the relationship between MRP4 and miR-490. When compared to the sham group, in CLP mice, the alveolar lung tissue showed significantly hyperemic, alveolar collapse, the W/D ratio was increased, and the injury index was increased. The number of neutrophils, macrophages and total cells, total protein concentration, TNF-α and IL-1β levels were significantly increased in BALF from CLP mice. The levels of TNF-α and IL-1β were significantly increased in lung tissue from CLP mice. Overexpression of miR-490 alleviated lung injury caused by CLP and inhibited inflammation in mice. The levels of TNF-α, IL-1β and caspase-3 were significantly increased, but the level of Bcl-2 was significantly decreased in MPVECs treated with LPS compared to the control group. Overexpression of miR-490 also reversed the increase of TNF-α, IL-1β, cleaved caspase-3 and Bcl-2 caused by LPS in MPVECs. Dual-luciferase reporter assay confirmed that the target gene of miR-490 was MRP4. Besides, overexpression of MRP4 upregulated TNF-α, IL-1β, and cleaved caspase-3, but downregulated the increase of Bcl-2 induced by miR-490 agomir transfection. These data suggested that miR-490 could relieve sepsis-induced acute lung injury in neonatal mice via targeting MRP4.
本研究旨在探讨 miR-490 是否通过靶向多药耐药相关蛋白 4（MRP4）来影响体外和体内脓毒症诱导的急性肺损伤（ALI）。在盲肠结扎和穿孔（CLP）前，向小鼠注射 miR-490 激动剂/NC 激动剂。用 miR-490 激动剂/NC 激动剂/MRP4/空载体转染肺微血管内皮细胞（PMVEC），然后用脂多糖（LPS）刺激。评估肺组织的组织病理学、损伤评分和干湿（W/D）比。测量支气管肺泡灌洗液（BALF）中的中性粒细胞、巨噬细胞和总细胞数、总蛋白浓度、TNF-α 和 IL-1β 水平。测量 PMVECs 中的半胱天冬酶-3、Bcl-2、TNF-α 和 IL-1β 水平。双荧光素酶报告基因分析用于分析 MRP4 和 miR-490 之间的关系。与假手术组相比，CLP 小鼠的肺泡肺组织明显充血，肺泡塌陷，W/D 比值增加，损伤指数增加。CLP 小鼠 BALF 中的中性粒细胞、巨噬细胞和总细胞数、总蛋白浓度、TNF-α 和 IL-1β 水平显著升高。CLP 小鼠肺组织中的 TNF-α 和 IL-1β 水平显著升高。miR-490 的过表达减轻了 CLP 引起的肺损伤并抑制了小鼠的炎症反应。与对照组相比，LPS 处理的 PMVECs 中 TNF-α、IL-1β 和半胱天冬酶-3 的水平显著升高，而 Bcl-2 的水平显著降低。miR-490 的过表达也逆转了 LPS 引起的 PMVECs 中 TNF-α、IL-1β、cleaved caspase-3 和 Bcl-2 的增加。双荧光素酶报告基因分析证实，miR-490 的靶基因是 MRP4。此外，MRP4 的过表达上调了 TNF-α、IL-1β 和 cleaved caspase-3，但下调了 miR-490 激动剂转染引起的 Bcl-2 增加。这些数据表明，miR-490 可以通过靶向 MRP4 来缓解新生小鼠脓毒症引起的急性肺损伤。
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