Identification and validation of candidate genes dysregulated in alveolar macrophages of acute respiratory distress syndrome.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a common cause of death in ICU patients and its underlying mechanism remains unclear, which leads to its high mortality rate. This study aimed to identify candidate genes potentially implicating in the pathogenesis of ARDS and provide novel therapeutic targets. METHODS: Using bioinformatics tools, we searched for differentially expressed genes (DEGs) in an ARDS microarray dataset downloaded from the Gene Expression Omnibus (GEO) database. Afterwards, functional enrichment analysis of GO, KEGG, GSEA and WGCNA were carried out to investigate the potential involvement of these DEGs. Moreover, the Protein-protein interaction (PPI) network was constructed and molecular complexes and hub genes were identified, followed by prognosis analysis of the hub genes. Further, we performed qRT-PCR, Western Blot and flow cytometry analysis to detect candidate genes of CCR2 and FPR3 in macrophage model of LPS-induced ARDS and primary alveolar macrophages(AMs). Macrophage chemotaxis was evaluated using Transwell assay. RESULTS: DEGs mainly involved in myeloid leukocyte activation, cell chemotaxis, adenylate cyclase-modulating G protein-coupled receptor signaling pathway and cytokine-cytokine receptor interaction. Basing on the constructed PPI network, we identified five molecular complexes and 10 hub genes potentially participating in the pathogenesis of ARDS. It was observed that candidate genes of CCR2 and FPR3 were significantly over-expressed in primary alveolar macrophages from ARDS patients and macrophgae model of LPS-induced ARDS. Moreover, in vitro transwell assay demonstrated that CCR2 and FPR3 down-regulation, respectively, inhibited LPS-triggered macrophage chemotaxis toward CCL2. Finally, a positive correlation between FPR3 and CCR2 expression was confirmed using pearson correlation analysis and Western Blot assay. CONCLUSIONS: Our study identified CCR2 and FPR3 as the candidate genes which can promote macrophage chemotaxis through a possible interaction between FPR3 and CCL2/CCR2 axis and provided novel insights into ARDS pathogenesis.

Silencing of long non-coding RNA MEG3 alleviates lipopolysaccharide-induced acute lung injury by acting as a molecular sponge of microRNA-7b to modulate NLRP3.

We aimed to elucidate the roles of the long non-coding RNA (lncRNA) maternally expressed gene 3 (MEG3)/microRNA-7b (miR-7b)/NLR pyrin domain containing 3 (NLRP3) axis in lipopolysaccharide (LPS)-induced acute lung injury (ALI). Mouse alveolar macrophage NR8383 and mice were administrated with LPS to establish ALI models in vitro and in vivo. NLRP3 was silenced while miR-7b was overexpressed in LPS-induced NR8383 cell model of ALI. The interleukin-18 (IL-18) and IL-1ß, as well as caspase-1, tumor necrosis factor-α (TNF-α) and IL-6 protein levels were assayed. To further investigate the underlying mechanisms of NLRP3 in ALI, lncRNA MEG3 was silenced and miR-7b was overexpressed in LPS-induced NR8383 cell model of ALI, after which in vivo experiments were performed for further verification. NLRP3 was highly expressed in LPS-induced NR8383 cell model of ALI. Silencing NLRP3 or overexpressing miR-7b inhibited IL-18 and IL-1ß, as well as caspase-1, TNF-α and IL-6. LncRNA MEG3 could sponge miR-7b, and lncRNA MEG3 silencing or miR-7b overexpression downregulates NLRP3 expression, thus reducing IL-18 and IL-1ß, as well as caspase-1, TNF-α and IL-6 levels. The in vivo experiments further confirmed the aforementioned findings. Silencing lncRNA MEG3 augments miR-7b binding to NLRP3 and downregulates NLRP3 expression, which ultimately improves LPS-induced ALI.

The involvement of the laminin-integrin α7ß1 signaling pathway in mechanical ventilation-induced pulmonary fibrosis.

INTRODUCTION: The central objective of the study was to determine the possibility and potential mechanism by which the laminin-integrin α7ß1 signaling pathway acts on mechanical ventilation (MV)-induced pulmonary fibrosis in a rat model. METHODS: Fibrosis rat models were established via the mechanical injury method. Ninety rats were recruited and divided into the normal, low tidal volume (LVT), huge VT (HVT), Arg-Gly-Asp-Ser (RGDS), LVT + RGDS and HVT + RGDS groups. On day 0, 3, and 7 after model establishment, the pulmonary hydroxyproline content was measured using alkaline hydrolysis and the pulmonary index was also calculated. All rats in each group were executed on day 0, 3 and 7. The histopathological changes detected in the left pulmonary tissues were observed using hematoxylin-eosin (HE) and Masson staining methods. DISCUSSION: The mRNA and protein expressions of Wnt-5A, ß-catenin, E-cadherin and Collagen I in the Wnt/ß-catenin signaling pathway were detected using both reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blotting methods. Immunohistochemistry was employed to detect the fibronectin (FN) expression in the pulmonary tissues on the 7th day. All indexes in the RGDS and LVT + RGDS groups indicated no explicit differences compared with the normal group. In the LVT, HVT, HVT + RGDS groups, the respective weights of the rats and the expression of E-cadherin on the 7th day exhibited decreases, however the pulmonary index, hydroxyproline, pulmonary alveolar inflammation, pulmonary fibrosis, FN expression, and protein expressions of Wnt-5A, ß-catenin, and Collagen I all displayed increased levels (all P<0.05). The index changes detected in the HVT group were the most blatant results observed in the study. The rat pulmonary index on the 7th day, hydroxyproline (HYP), pulmonary alveolar inflammation, pulmonary fibrosis, FN expression, and protein expressions of Wnt-5A, ß-catenin, and type I-collagen were all down-regulated, in contrast the expression of E-cadherin was up-regulated in the LVT + RGDS and HVT + RGDS groups in comparison with the LVT and HVT groups, respectively (all P<0.05). CONCLUSIONS: The findings of the study suggested that RGDS could act to block the laminin-integrin α7ß1-signaling pathway, ultimately contributing to the inhibition of the progression of MV-induced pulmonary fibrosis.