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

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/fl Tie2cre + 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.