The circadian rhythm gene BHLHE40 regulates inflammation in asthma patients and promotes ferroptosis through PTGS2 and SERPINB2 in lung fibroblast cells
File(s)
Accepted version
Author(s)
Type
Journal Article
Abstract
Background
Circadian rhythm transcription factor basic helix-loop-helix family member e40 (BHLHE40) is a core regulator in the transcriptional response in immunoglobulin class switching to IgE. We describe here the results of our systematic investigations of BHLHE40 in inflammatory responses in lung epithelial cells and fibroblast cells, and particularly for its role in ferroptosis.
Methods
BHLHE40 levels in serum and transcript levels in human peripheral blood mononuclear cells (PBMC) from asthma patients and healthy controls were quantified. BHLHE40 was silenced by siRNAs in human airway epithelial A549 cells and human lung fibroblast MRC5 cells. RNA sequencing was performed with BHLHE40-silenced MRC5 cells. Gene silencing or overexpression models for BHLHE40, PTGS2 and SERPINB2 were established with MRC5 cells. MRC5 cells were co-stimulated by lipopolysaccharide (LPS) and IL-13. Apoptosis, reactive oxygen species (ROS), Fe2+, lipid peroxide (LPO), malondialdehyde (MDA) and glutathione (GSH) levels were quantified. Levels of HMGB-1, IL-4, IL-6 and TNF-α in supernatants were measured by ELISAs. Expression levels of BHLHE40, GPX4, SLC7A11, PTGS2, and SERPINB2 were determined by qPCRs and Western blotting. RNA immunoprecipitation and dual-luciferase reporter assays were applied to detect gene interactions.
Results
BHLHE40 protein levels in serum and BHLHE40 transcript levels in PBMC were higher in asthma patients compared to the control group (P<0.001 and P<0.0001, respectively). BHLHE40 transcript levels were correlated with neutrophil counts in blood, FEV1%pred, PEF and FeNO levels in asthma patients. Silencing BHLHE40 in A549 and MRC5 cells resulted in a decrease of IL-6 and IL-8 levels after stimulation of IL-1β. Transcriptomic analysis for BHLHE40-silenced MRC5 cells showed 506 up-regulating genes and 512 down-regulated genes, which contained PTGS2 and SERPINB2. PTGS2 and SERPINB2 were highly expressed in asthma samples and positively correlated with BHLHE40 (P<0.0001, respectively). Overexpression of either BHLHE40 or PTGS2 consistently showed promotion of cell inflammation and ferroptosis. RNA immunoprecipitation and dual-luciferase reporter assays confirmed interactions of BHLHE40, PTGS2 and SERPINB2 in MRC5 cells.
Conclusions
BHLHE40 regulates lung inflammation and promotes ferroptosis via PTGS2 and SERPINB2 in fibroblasts. BHLHE40 could serve as a new biomarker and therapeutic target for airway inflammation diseases.
Circadian rhythm transcription factor basic helix-loop-helix family member e40 (BHLHE40) is a core regulator in the transcriptional response in immunoglobulin class switching to IgE. We describe here the results of our systematic investigations of BHLHE40 in inflammatory responses in lung epithelial cells and fibroblast cells, and particularly for its role in ferroptosis.
Methods
BHLHE40 levels in serum and transcript levels in human peripheral blood mononuclear cells (PBMC) from asthma patients and healthy controls were quantified. BHLHE40 was silenced by siRNAs in human airway epithelial A549 cells and human lung fibroblast MRC5 cells. RNA sequencing was performed with BHLHE40-silenced MRC5 cells. Gene silencing or overexpression models for BHLHE40, PTGS2 and SERPINB2 were established with MRC5 cells. MRC5 cells were co-stimulated by lipopolysaccharide (LPS) and IL-13. Apoptosis, reactive oxygen species (ROS), Fe2+, lipid peroxide (LPO), malondialdehyde (MDA) and glutathione (GSH) levels were quantified. Levels of HMGB-1, IL-4, IL-6 and TNF-α in supernatants were measured by ELISAs. Expression levels of BHLHE40, GPX4, SLC7A11, PTGS2, and SERPINB2 were determined by qPCRs and Western blotting. RNA immunoprecipitation and dual-luciferase reporter assays were applied to detect gene interactions.
Results
BHLHE40 protein levels in serum and BHLHE40 transcript levels in PBMC were higher in asthma patients compared to the control group (P<0.001 and P<0.0001, respectively). BHLHE40 transcript levels were correlated with neutrophil counts in blood, FEV1%pred, PEF and FeNO levels in asthma patients. Silencing BHLHE40 in A549 and MRC5 cells resulted in a decrease of IL-6 and IL-8 levels after stimulation of IL-1β. Transcriptomic analysis for BHLHE40-silenced MRC5 cells showed 506 up-regulating genes and 512 down-regulated genes, which contained PTGS2 and SERPINB2. PTGS2 and SERPINB2 were highly expressed in asthma samples and positively correlated with BHLHE40 (P<0.0001, respectively). Overexpression of either BHLHE40 or PTGS2 consistently showed promotion of cell inflammation and ferroptosis. RNA immunoprecipitation and dual-luciferase reporter assays confirmed interactions of BHLHE40, PTGS2 and SERPINB2 in MRC5 cells.
Conclusions
BHLHE40 regulates lung inflammation and promotes ferroptosis via PTGS2 and SERPINB2 in fibroblasts. BHLHE40 could serve as a new biomarker and therapeutic target for airway inflammation diseases.
Date Acceptance
2026-09-16
Citation
BMC Pulmonary Medicine
ISSN
1471-2466
Publisher
BMC
Journal / Book Title
BMC Pulmonary Medicine
Copyright Statement
Copyright This paper is embargoed until publication. Once published the Version of Record (VoR) will be available on immediate open access.
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Publication Status
Accepted