Untargeted metabolomics and transcriptomics identified glutathione metabolism disturbance and PCS and TMAO as potential biomarkers for ER stress in lung

Zijing Wang,He Tian,Chengqian Yin,Peng Ma,Guifen Qiang,Guanghou Shui,Can Zhou,Bowen Li,Yisa Wang,Biyu Hou,Guanhua Du,Xiuying Yang

doi:10.1038/s41598-021-92779-8

Abstract

Endoplasmic reticulum (ER) stress is a cellular state that results from the overload of unfolded/misfolded protein in the ER that, if not resolved properly, can lead to cell death. Both acute lung infections and chronic lung diseases have been found related to ER stress. Yet no study has been presented integrating metabolomic and transcriptomic data from total lung in interpreting the pathogenic state of ER stress. Total mouse lungs were used to perform LC–MS and RNA sequencing in relevance to ER stress. Untargeted metabolomics revealed 16 metabolites of aberrant levels with statistical significance while transcriptomics revealed 1593 genes abnormally expressed. Enrichment results demonstrated the injury ER stress inflicted upon lung through the alteration of multiple critical pathways involving energy expenditure, signal transduction, and redox homeostasis. Ultimately, we have presented p-cresol sulfate (PCS) and trimethylamine N-oxide (TMAO) as two potential ER stress biomarkers. Glutathione metabolism stood out in both omics as a notably altered pathway that believed to take important roles in maintaining the redox homeostasis in the cells critical for the development and relief of ER stress, in consistence with the existing reports.

Highlights

Abbreviations ER Endoplasmic reticulum unfolded protein response (UPR) Unfolded protein response chronic obstructive pulmonary disease (COPD) Chronic obstructive pulmonary disease LC–MS Liquid chromatography–mass spectrometry RNA-seq RNA sequencing MEK5/ERK5 Mitogen-activated protein kinase 5/extracellular signal-regulated kinase 5 PBS Phosphate buffered saline DMSO Dimethylsulphoxide Ab Antibody CHOP CCAAT/enhancer binding protein (C/EBP) homologous protein GRP78 Glucose-regulated protein 78 C/Ctrl Control
The average body weight of the mice in model group (T) decreased 0.85 g (3.6%, P = 0.043) during the 12 h of ER stress induction by tunicamycin while no significant change in body weight was found in the control group (Fig. 1a), and the organ coefficient for lung was of no significant difference (Fig. 1b)
The variation of phototransduction pathway was found of no statistical significance in this analysis, with a P-value of 0.47 and an false discovery rate (FDR) of 0.63. Data from both metabolomics and transcriptomics methods were analyzed independently and further integrated. Through these aberrantly-regulated metabolites and genes, we could identify pathways or biological processes altered in the target phenotype, ER stress, thereby unveil the effects it had on lung and provide insights into this cellular state

Summary

Introduction

Abbreviations ER Endoplasmic reticulum UPR Unfolded protein response COPD Chronic obstructive pulmonary disease LC–MS Liquid chromatography–mass spectrometry RNA-seq RNA sequencing MEK5/ERK5 Mitogen-activated protein kinase 5/extracellular signal-regulated kinase 5 PBS Phosphate buffered saline DMSO Dimethylsulphoxide Ab Antibody CHOP CCAAT/enhancer binding protein (C/EBP) homologous protein GRP78 Glucose-regulated protein 78 C/Ctrl Control (group). After the induction of ER stress in C57BL/6J mice by intraperitoneal injection of tunicamycin, we performed untargeted metabolomic analysis (by LC–MS) in lung tissues to identify the differentially-regulated metabolites as well as reveal the enriched pathways in follow-up data interpretation. Transcriptomic analysis based on RNA-seq results was carried out in the same tissues to unravel the changes in gene expression and biological processes in response to ER stress. Results of both omics were taken together in hope of finding molecules, pathways or biological processes playing central roles in ER stress. We comprehensively evaluated the genetic and metabolic alteration in ER stressed lung samples aiming to shed light on the complex pathological mechanism as well as identify potential biomarkers of ER stress, which has been proven relative to multiple lung injury and diseases

Methods

Results

Conclusion