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.
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. Lipoxin A4通过上调NDRG1表达水平缓解LPS诱导的A549细胞损伤摘要背景：脂氧素A4（LXA4）能够通过促进肺上皮细胞的钠离子通道（ENaC）表达而缓解脂多糖（LPS）诱导的急性肺损伤（ALI）和急性呼吸窘迫综合征（ARDS）。然而，LXA4如何促进ENaC表达的主要机制依旧不明。本研究的目的是探索LXA4诱导ENaC表达的相关基因和信号通路机制。 方法：采用LPS和LXA4，或两者的组合，处理A549细胞，模拟ALI/ARDS治疗，通过real-time PCR分析ENaC-α/γ转录水平。对处理的A549细胞进行转录组测序，探索分析受LXA4影响的候选基因。对经不同浓度LPS和LXA4，以及不同时间间隔处理的A549细胞进行real-time PCR和Western blot检测，验证关键的候选基因表达变化。利用LXA4 受体（ALX）抑制剂BOC-2验证LXA4对候选基因的诱导表达作用。通过siRNA分析沉默候选基因对A549细胞存活率和ENaC-α表达的影响。利用PI3K抑制剂LY294002分析PI3K信号通路是否参与调控LXA4诱导候选基因表达。 结果：成功建立模拟ALI的A549细胞模型，并对其进行转录组测序。在多个候选基因中，通过real-time PCR和Western blot验证了LXA4诱导NDRG1基因表达。NDRG1 mRNA水平随LXA4浓度增加而升高，而BOC-2则拮抗LXA4对NDRG1的诱导作用。NDRG1 siRNA抑制LPS处理的A549细胞存活率（treatment vs. control, 0.605±0.063 vs. 0.878±0.083, P=0.040）和ENaC-α表达（treatment vs. control, 0.458±0.038 vs. 0.711±0.035, P=0.008）。LY294002抑制NDRG1 (treatment vs. control, 0.459±0.023 vs. 0.726±0.020, P=0.001) 和ENaC-α (treatment vs. control, 0.236±0.021 vs. 0.814±0.025, P<0.001)的表达，以及SGK1的磷酸化水平（treatment vs. control, 0.442±0.024 vs. 1.046±0.082, P=0.002），表明PI3K信号通路参与调控LXA4诱导NDRG1的表达。 结论：本研究揭示了NDRG1在介导LXA4保护受LPS损伤的A549细胞关键作用。LXA4经PI3K信号通路上调NDRG1的表达水平，增加ENaC的表达水平，缓解LPS诱导的A549细胞损伤。.
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.