Proteogenomic verifies targets underlying erythromycin alleviate neutrophil extracellular traps-induced inflammation

Abstract

Background: Neutrophil Extracellular Traps (NETs) are closely related to the progression of inflammation in Chronic Obstructive Pulmonary Disease (COPD). Erythromycin (EM) has been shown to inhibit inflammation in COPD, but its molecular mechanisms is still unclear. The aim of our study is investigate the molecular mechanisms of EM's anti-inflammatory effects in NETs-induced inflammation.

Methods: Transcriptomics and proteomics data were obtained from U937 cells treated with NETs and EM. Differentially expressed genes (DEGs) and differentially expressed proteins (DEPs) were identified using R software. Pathway enrichment analyses, were employed to identify inflammation-related pathways. Cytoscape were utilized to construct network of hub targets regulated by EM which related with oxidative stress and inflammation. Additionally, Cytoscape and STRING were used to construct protein-protein interaction (PPI) network of key targets regulated by EM. The expression levels of key targets were further confirmed through WB and PCR experiments.

Results: Both transcriptomics and proteomics indicate that EM decrease NETs -induced AKT1 expression. Enrichment analysis of DEGs and DEPs reveal multiple common pathways involved in EM's regulation inflammation, including the PI3K/AKT pathway, response to oxidative stress, IKK/NF-κB signaling and PTEN signaling pathway. Nine key targets in PI3K/AKT-related inflammatory pathways regulated by EM and ten targets of EM-regulated oxidative stress were identified. WB and PCR results confirmed that EM reversing the NETs-induced inflammation by modulating the activity of these targets. Furthermore, clinical samples and vitro experiments confirm that EM alleviates NETs-induced glucocorticoid resistance via inhibiting PI3K/AKT, thereby repressing inflammation.

Conclusions: Our study provides a comprehensive proteogenomic characterization of how EM alleviates NET-related inflammation, and identify PI3K/AKT play a critical role in the mechanism by which EM inhibits inflammation.

Keywords: Erythromycin; Inflammation; NETs; PI3K/AKT; Proteogenomic.

Fig. 1

Identify DEGs and DEPs in each group. A The flow chart show analysis of proteomic and transcriptomic. B Volcano map of DEGs between each group, and AKT1 gene expression between each group (P < 0.05). C Volcano map of DEPs between each group, and AKT1 protein expression between each group (P < 0.05)

Fig. 2

GO and KEGG enrichment of DEGs. A The results of GO enrichment of DEGs induced by NETs (P < 0.05). B KEGG enrichment results of DEGs induced by NETs (P < 0.05). C GO enrichment of DEGs regulated by EM (P < 0.05). D The KEGG enrichment analysis results of DEGs regulated by EM (P < 0.05)

Fig. 3

The results of GSEA. A The GSEA results of DEGs induced by NETs (P < 0.05). B The GSEA results of DEGs between NETs and EM groups (P < 0.05)

Fig. 4

GO and KEGG enrichment of DEPs. A The results of GO enrichment analysis of DEPs induced by NETs (P < 0.05). B KEGG enrichment of DEPs induced by NETs (P < 0.05). C GO enrichment of DEPs regulated by EM (P < 0.05). D KEGG enrichment of DEPs regulated by EM (P < 0.05)

Fig. 5

GSEA result of the DEPs. A GSEA results of DEPs between NETs and control group (P < 0.05). B GSEA results of DEPs between NETs and EM group (P < 0.05)

Fig. 6

Enrichment of co-expressed DEGs-DEPs. A GO enrichment of co-expressed DEGs-DEPs between NETs and control group. B KEGG enrichment of co-expressed DEGs-DEPs between NETs and control group. C GO enrichment of co-expressed DEGs-DEPs between NETs and EM group. D KEGG enrichment of co-expressed DEGs-DEPs between NETs and EM group

Fig. 7

Enrichment of DEGs-DEPs with different expression patterns. GO enrichment (A) and KEGG enrichment (B) of DEGs-DEPs with different expression patterns between NETs and control group; GO enrichment (C) and KEGG enrichment (D) of DEGs-DEPs with different expression patterns between NETs and EM group (P < 0.05)

Fig. 8

Key targets in EM regulation of AKT-related inflammatory pathways and oxidative stress in proteogenomics. A Key genes of EM regulates AKT related inflammation in transcriptomics. B Key genes of EM regulates oxidative stress in transcriptomics. C Key proteins of EM regulates AKT related inflammation in proteomics. D Key proteins of EM regulates oxidative stress in proteomics. E Key targets of EM regulates AKT related inflammation in transcriptomics and proteomics. F Key targets of EM regulates oxidative stress in transcriptomics and proteomics

Fig. 9

Proteins expression of NETs-related inflammation which regulates by EM. A Protein expression of PI3K-δ, AKT, AKT1, p-AKT, and p-AKT1 in each group. B Relative protein expression in each group of cells (Mean ± SD, *P < 0.05; n = 4). C Phosphorylation levels of AKT and AKT1 (mean ± SD, *P < 0.05; n = 4). D Protein expression of NLRP3, mTor and p-mTor in each groups. E Relative protein expression of NLRP3, mTor and p-mTor in each group (mean ± SD, *P < 0.05; n = 4). F mTor phosphorylation level in each group of cells (Mean ± SD, *P < 0.05; n = 4). G Protein expression of STAT3, p-STAT3, c-myc, GSK3β and p-GSK3β in each group of cells. H Relative protein expression of STAT3, p-STAT3, c-myc, GSK3β and p-GSK3β in each group (mean ± SD, *P < 0.05; n = 4). I STAT3 and GSK3β phosphorylation levels (mean ± SD, *P < 0.05; n = 4). J Protein expression of JNK, c-Fos and c-Jun in each group. K Relative protein expression of JNK, c-Fos and c-Jun in each group (mean ± SD, *P < 0.05; n = 4)

Fig. 10

Effect of EM antioxidant stress and regulation on oxidative stress-related proteins. The level of ROS release was determined by fluorescence microscopy. A Control, B NETs, C EM, D the results of statistical analysis of fluorescence intensity (Mean ± SD, *P < 0.05, n = 3, 200×). E The expression of PI3K-δ, NRF2, NLRP3 and HDAC2 mRNA in each group of cells (Mean ± SD, *P < 0.05; n = 3). F The expression of mTor, AKT, AKT1 and GR mRNA in each group (mean ± SD, *P < 0.05; n = 3). G Inflammatory cytokines expression of IL-6, IL-8 and TNF-α in each groups (mean ± SD, *P < 0.05; n = 4). H Protein expression of IKK, NF-κB p65, NF-κB p50, SIRT3, and PPARγ in each group of cells. I Relative protein expression of SIRT3 and PPARγ in each group (Mean ± SD, *P < 0.05; n = 4). J Relative protein expression of IKK, NF-κB p65, and NF-κB p50 in each group of cells (mean ± SD, *P < 0.05; n = 4). K Protein expression of PTEN, SIRT1, KEAP1, Nrf2, and PGC-1α in each group of cells. L Relative protein expression of KEAP1 and Nrf2 in each group of cells (mean ± SD, *P < 0.05; n = 4). M Relative protein expression of SIRT1, PTEN and PGC-1α in each group of cells (mean ± SD, *P < 0.05; n = 4)

Fig. 11

Effect of EM regulates on proteins induced by NETs in inflammation. oxidative stress-related proteins. A The expression of AKT, mTor, GR and HDAC2 mRNA in each group of cells (Mean ± SD, *P < 0.05; n = 3). B The expression of SIRT1 and NF-κB mRNA in each group (mean ± SD, *P < 0.05; n = 3). C Protein expression of GR, AKT, mTor and HDAC2 in each group of cells. D Relative protein expression of GR, AKT, mTor and HDAC2 in each group of cells (mean ± SD, *P < 0.05; n = 4). E Protein expression of SIRT1 and NF-κB in each group of cells. F Relative protein expression of SIRT1 and NF-κB in each group of cells (mean ± SD, *P < 0.05; n = 4). G IL-6 expression in the supernatant of each group (mean ± SD, *P < 0.05; n = 3). H IL-8 expression in the supernatant of each group (mean ± SD, *P < 0.05; n = 3). I TNF-α expression in the supernatant of each group (mean ± SD, *P < 0.05; n = 3)

Fig. 12

EM improved NETs-induce glucocorticoid sensitivity by inhibiting PI3K/AKT activity. A Protein levels of AKT, HDAC2 and GR in each group. B Relative AKT, HDAC2, and GR protein expression in each group (mean ± SD, *P < 0.05; n = 4). C Correlation heat map of clinical features and protein expression. D Protein expression of GR, P-GR and HDAC2 in each group of cells. E Relative intensity of GR, p-GR and HDAC2 protein in each group of cells (Mean ± SD, *P < 0.05; n = 4). F GR phosphorylation level (Mean ± SD, *P < 0.05; n = 4)