脂氧素 A4 通过上调 N- myc 下游调节基因-1 减轻脂多糖诱导的 A549 细胞损伤。
Lipoxin A4 Ameliorates Lipopolysaccharide-Induced A549 Cell Injury through Upregulation of N-myc Downstream-Regulated Gene-1.
机构信息
Department of Anesthesiology, 2nd Clinical Medical College of Jinan University; Department of Anesthesiology, Shenzhen People's Hospital, Shenzhen, Guangdong 518020, China.
Research and Development Department, Shenzhen Acen Regenerative Medicine, Shenzhen, Guangdong 518122, China.
出版信息
Chin Med J (Engl). 2018 Jun 5;131(11):1342-1348. doi: 10.4103/0366-6999.232788.
BACKGROUND
Lipoxin A4 (LXA4) can alleviate lipopolysaccharide (LPS)-induced acute lung injury (ALI) and acute respiratory distress syndrome through promoting epithelial sodium channel (ENaC) expression in lung epithelial cells. However, how LXA4 promote ENaC expression is still largely elusive. The present study aimed to explore genes and signaling pathway involved in regulating ENaC expression induced by LXA4.
METHODS
A549 cells were incubated with LPS and LXA4, or in combination, and analyzed by quantitative real-time polymerase chain reaction (qRT-PCR) of ENaC-α/γ. Candidate genes affected by LXA4 were explored by transcriptome sequencing of A549 cells. The critical candidate gene was validated by qRT-PCR and Western blot analysis of A549 cells treated with LPS and LXA4 at different concentrations and time intervals. LXA4 receptor (ALX) inhibitor BOC-2 was used to test induction of candidate gene by LXA4. Candidate gene siRNA was adopted to analyze its influence on A549 viability and ENaC-α expression. Phosphoinositide 3-kinase (PI3K) inhibitor LY294002 was utilized to probe whether the PI3K signaling pathway was involved in LXA4 induction of candidate gene expression.
RESULTS
The A549 cell models of ALI were constructed and subjected to transcriptome sequencing. Among candidate genes, N-myc downstream-regulated gene-1 (NDRG1) was validated by real-time-PCR and Western blot. NDRG1 mRNA was elevated in a dose-dependent manner of LXA4, whereas BOC-2 antagonized NDRG1 expression induced by LXA4. NDRG1 siRNA suppressed viability of LPS-treated A549 cells (treatment vs. control, 0.605 ± 0.063 vs. 0.878 ± 0.083, P = 0.040) and ENaC-α expression (treatment vs. control, 0.458 ± 0.038 vs. 0.711 ± 0.035, P = 0.008). LY294002 inhibited NDRG1 (treatment vs. control, 0.459 ± 0.023 vs. 0.726 ± 0.020, P = 0.001) and ENaC-α (treatment vs. control, 0.236 ± 0.021 vs. 0.814 ± 0.025, P < 0.001) expressions and serum- and glucocorticoid-inducible kinase 1 phosphorylation (treatment vs. control, 0.442 ± 0.024 vs. 1.046 ± 0.082, P = 0.002), indicating the PI3K signaling pathway was involved in regulating NDRG1 expression induced by LXA4.
CONCLUSION
Our research uncovered a critical role of NDRG1 in LXA4 alleviation of LPS-induced A549 cell injury through mediating PI3K signaling to restore ENaC expression.
背景
脂氧素 A4（LXA4）可以通过促进肺上皮细胞上皮钠通道（ENaC）的表达来减轻脂多糖（LPS）诱导的急性肺损伤（ALI）和急性呼吸窘迫综合征。然而，LXA4 如何促进 ENaC 的表达在很大程度上仍不清楚。本研究旨在探讨调节 LXA4 诱导的 ENaC 表达的相关基因和信号通路。
方法
用 LPS 和 LXA4 或两者联合处理 A549 细胞，并用定量实时聚合酶链反应（qRT-PCR）检测 ENaC-α/γ的表达。通过 A549 细胞的转录组测序探索受 LXA4 影响的候选基因。用不同浓度和时间间隔的 LPS 和 LXA4 处理 A549 细胞，通过 qRT-PCR 和 Western blot 分析验证关键候选基因。用 LXA4 受体（ALX）抑制剂 BOC-2 测试 LXA4 诱导候选基因的情况。用候选基因 siRNA 分析其对 A549 细胞活力和 ENaC-α表达的影响。用磷酸肌醇 3-激酶（PI3K）抑制剂 LY294002 探讨 PI3K 信号通路是否参与 LXA4 诱导候选基因表达。
结果
构建了 A549 细胞 ALI 模型，并进行了转录组测序。在候选基因中，N- MYC 下游调节基因-1（NDRG1）通过实时 PCR 和 Western blot 得到验证。LXA4 呈剂量依赖性增加 NDRG1 mRNA，而 BOC-2 拮抗 LXA4 诱导的 NDRG1 表达。NDRG1 siRNA 抑制 LPS 处理的 A549 细胞的活力（处理与对照，0.605±0.063 与 0.878±0.083，P=0.040）和 ENaC-α 表达（处理与对照，0.458±0.038 与 0.711±0.035，P=0.008）。LY294002 抑制 NDRG1（处理与对照，0.459±0.023 与 0.726±0.020，P=0.001）和 ENaC-α（处理与对照，0.236±0.021 与 0.814±0.025，P＜0.001）表达和血清和糖皮质激素诱导激酶 1 磷酸化（处理与对照，0.442±0.024 与 1.046±0.082，P=0.002），表明 PI3K 信号通路参与调节 LXA4 诱导的 NDRG1 表达。
结论
我们的研究揭示了 NDRG1 在 LXA4 缓解 LPS 诱导的 A549 细胞损伤中的关键作用，通过调节 PI3K 信号通路恢复 ENaC 表达。
Zotero 插件