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GCH1 reduces LPS-induced alveolar macrophage polarization and inflammation by inhibition of ferroptosis

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Abstract

Objective

GTP cyclohydrolase 1(GCH1) was reported to protect against ferroptosis. However, it is not clear whether GCH1 reduced lipopolysaccharide (LPS)-induced macrophage polarization and inflammation by inhibition of ferroptosis.

Methods

Bioinformatics analysis was used to screen differential expression genes (DEGs) and obtain the different pathways and biological features. Lasso cox regression analysis with ferroptosis related DEGs was established to screen the most relevant genes for disease risk. LPS induced Raw264.7 macrophage polarization model and GCH1-specific siRNA oligos transfection were performed to confirm the function of GCH1. Immunofluorescence staining, western blot and quantitative real-time PCR were performed to detect the expression of iNOS, CD206, GCH1, IL6, SLC2A6, F4/80, IL1β, TNFα, IL10, GPX4, ACSL4, AMPK and p-AMPK in macrophages. The levels of ROS, SOD, MDA and GSH were detected according to the instructions of the reagent kit, respectively.

Results

542 DEGs were screened from GSE40885 microarray. GO and KEGG pathway enrichment analysis showed that the upregulated DEGs induced by LPS in alveolar macrophage were closely associated with inflammatory and immune responses, the downregulated DEGs were related to lipid metabolism, insulin resistance and AMPK signal pathway. Lasso cox regression analysis screened GCH1, IL6, and SLC2A6. Our experimental results showed that the expression of GCH1 and IL6 in the LPS group was higher than that in the control group, but there was no difference in the expression of SLC2A6. Bioinformatics analysis with GSE112720 observed that ferroptosis was enriched in GCHfl/fl + LPS group compared with GCHfl/flTie2cre + LPS group and GCHfl/fl + control group. Silence of GCH1 increased the levels of IL6, TNF-α and IL-1β and decreased IL10 level. Silence of GCH1 increased iNOS level and decreased CD206 level. Moreover, silence of GCH1 raised ferroptosis induced by LPS in macrophages and suppressed the activity of AMPK pathway.

Conclusions

GCH1 inhibited ferroptosis in LPS-stimulated macrophages, reduced macrophage toward to M1 polarization and inflammatory response.

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Availability of data and materials

The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Abbreviations

GCH1:

GTP cyclohydrolase-1

LPS:

Lipopolysaccharide

GPX4:

Glutathione peroxidase 4

ACSL4:

Acyl-coenzyme A synthetase long-chain family member 4

SLC2A6:

6 Solute carrier family 2 (facilitated glucose transporter)

iNOS:

Inducible nitric oxide synthase

CD206:

Human mannose receptor 206

IL6:

Interleukin 6

IL1β:

Interleukin 1 beta

TNFα:

Tumor necrosis factor alpha

IL10:

Interleukin 10

AMPK:

AMP-activated protein kinase

DEG:

Differentially expressed gene

GO:

Gene ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

PCR:

Polymerase chain reaction

DCFH-DA:

2',7'-Dichlorodihydrofluorescein diacetate

ROS:

Reactive oxygen species

MDA:

Malondialdehyde

SOD:

Superoxide dismutase

GSH:

Glutathione

TUNEL:

TdT-mediated dUTP nick end-labeling

DAPI:

4',6-Diamidino-2-phenylindole

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

SDS–PAGE:

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis

PVDF:

Polyvinylidene difluoride

MODS:

Multiple organ dysfunction syndrome

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Acknowledgements

This work was supported by the National Key Research and Development Program of China (No. 2020YFC2005800), the National Natural Science Foundation of China (No. 82100308), the National Natural Science Foundation Excellent Youth Cultivation Project (No. 20202ZDB01017), the Natural Science Foundation of Jiangxi Province (No. 20202BABL216035, No. 20202BAB206042, No. 20212BCD42005 and G/Y2428).

Author information

Author notes

  1. Yuhong Xiao, Ye Yuan, Yuhui Yang have contributed equally to this study and share first authorship.

Authors and Affiliations

  1. Department of Rehabilitation Medicine, The Second Affiliated Hospital of Nanchang University, No. 1 Minde Road, Nanchang, 330006, Jiangxi, China

    Yuhong Xiao, Jun Luo & Lingling Yu

  2. HuanKui Academy of Nanchang University, Nanchang, Jiangxi, China

    Ye Yuan, Yuhui Yang & Zhanyuan Ding

  3. Department of Orthopedics, The Second Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, China

    Bo Liu

  4. Department of Pulmonary and Critical Care Medicine, The First Affiliated Hospital of Nanchang University, No. 1519 Dongyue Avenue, Nanchang, 330006, Jiangxi, China

    Shengsong Chen

  5. Department of Pulmonary and Critical Care Medicine, National Regional Center for Respiratory Medicine, Jiangxi Hospital of China-Japan Friendship Hospital, Nanchang, Jiangxi, China

    Shengsong Chen

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  1. Yuhong Xiao
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Contributions

LLY and SSC designed the study. YHX performed the experiments. YY and YHY collected the data, and YY, YHY, BL and ZYD analyzed and interpreted the data. LLY, SSC and JL prepared the manuscript. All the authors read and approved the final manuscript.

Corresponding authors

Correspondence to Shengsong Chen or Lingling Yu.

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The authors declare that they have no competing interests.

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The study was supported by the Medical Research Ethics Committee of the Second Affiliated Hospital of Nanchang University.

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Responsible Editor: L Li.

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Xiao, Y., Yuan, Y., Yang, Y. et al. GCH1 reduces LPS-induced alveolar macrophage polarization and inflammation by inhibition of ferroptosis. Inflamm. Res. 72, 1941–1955 (2023). https://doi.org/10.1007/s00011-023-01785-1

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