FGF21 attenuates neuroinflammation following subarachnoid hemorrhage through promoting mitophagy and inhibiting the cGAS-STING pathway

Abstract

Background: Subarachnoid hemorrhage (SAH) represents a form of cerebrovascular event characterized by a notable mortality and morbidity rate. Fibroblast growth factor 21 (FGF21), a versatile hormone predominantly synthesized by the hepatic tissue, has emerged as a promising neuroprotective agent. Nevertheless, the precise impacts and underlying mechanisms of FGF21 in the context of SAH remain enigmatic.

Methods: To elucidate the role of FGF21 in inhibiting the microglial cGAS-STING pathway and providing protection against SAH-induced cerebral injury, a series of cellular and molecular techniques, including western blot analysis, real-time polymerase chain reaction, immunohistochemistry, RNA sequencing, and behavioral assays, were employed.

Results: Administration of recombinant fibroblast growth factor 21 (rFGF21) effectively mitigated neural apoptosis, improved cerebral edema, and attenuated neurological impairments post-SAH. Transcriptomic analysis revealed that SAH triggered the upregulation of numerous genes linked to innate immunity, particularly those involved in the type I interferon (IFN-I) pathway and microglial function, which were notably suppressed upon adjunctive rFGF21 treatment. Mechanistically, rFGF21 intervention facilitated mitophagy in an AMP-activated protein kinase (AMPK)-dependent manner, thereby preventing mitochondrial DNA (mtDNA) release into the cytoplasm and dampening the activation of the DNA-sensing cyclic GMP-AMP synthase (cGAS)-stimulator of interferon genes (STING) signaling pathway. Conditional knockout of STING in microglia markedly ameliorated the inflammatory response and mitigated secondary brain injuries post-SAH.

Conclusion: Our results present the initial evidence that FGF21 confers a protective effect against neuroinflammation-associated brain damage subsequent to SAH. Mechanistically, we have elucidated a novel pathway by which FGF21 exerts this neuroprotection through inhibition of the cGAS-STING signaling cascade.

Keywords: FGF21; Microglia; Mitophagy; Subarachnoid haemorrhage; cGAS-STING; mtDNA.

Fig. 1

The effects of rFGF21 treatment on brain injuries after SAH. (A) Quantification of brain water content of mouse in each group (n = 6 per group). (B) Modified Garcia score in different experimental groups (n = 12 per group). (C-D) Representative TUNEL staining images and quantification of TUNEL-positive cells in mouse brain (n = 6 per group). Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 2

FGF21 attenuated neuroinflammation injury after SAH. (A) Venn diagram showed that 1372 shared genes overlapping between those differentially expressed genes were reversed by rFGF21 treatment. (C) Go enrichment analysis of the overlap of genes in the Venn diagram. (C) KEGG enrichment analysis of the overlap of genes in the Venn diagram. (D-G) GSEA plot of the NF − kappa B signaling pathway, Toll − like receptor signaling pathway, Cytokine − cytokine receptor interaction, and NOD-like receptor signaling pathway (SAH + rFGF21 versus SAH)

Fig. 3

FGF21 inhibited microglial pro-inflammatory response after SAH. (A-B) Immunofluorescence staining for Iba1(red) with CD68 (green) revealing the activated microglia levels in each group (n = 18 from 6 mice per group). (C-D) Statistical analysis of total branch length (micrometers), and soma area (square micrometers) of Iba1-positive cells in each group (n = 18 from 6 mice per group). (E-F) qPCR analysis of M1 microglia-specific transcripts (CD86, iNOS) and of M2 microglia-specific transcripts (CD206, Arg1) in isolated microglia using flow cytometry. Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4

rFGF21 inhibits the activation of cGAS-STING pathway following SAH. (A) GSEA plot showing that the activation of cytosolic dna sensing pathway were activated in the brain from SAH mice, which were then alleviated following rFGF21 treatment. (B) Heatmap showing that differently expressed genes involved in cytosolic DNA sensor pathway. (C-H) Western blot analysis of the effects of rFGF21 treatment on the expression level of cGAS and the phosphorylation level of STING, TBK1, IRF3, and NF-κB following SAH operation in mice. (n = 6 per group). Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 5

rFGF21 attenuates SAH-induced neuroinflammation through inhibition of cGAS-STING pathway. (A-B) Immunofluorescence staining for Iba1(red) with CD68 (green) in Sting^fl/fl and Sting ^CKO mice after SAH (n = 6 per group). (C-F) Western blot analysis of the phosphorylation level of TBK1, IRF3, and NF-κB in Sting^fl/fl and Sting ^CKO mice after SAH (n = 6 per group). (G-H) qPCR quantification of TNF and IFNB1 mRNA in Sting^fl/fl and Sting ^CKO mice after SAH (n = 6 per group). Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 6

rFGF21 attenuates SAH-induced brain injuries through inhibition of cGAS-STING pathway. (A-B) Representative TUNEL staining images and quantification of TUNEL-positive cells in Sting^fl/fl and Sting ^CKO mice after SAH. (n = 6 per group). (C) Brain edema statistical analysis in Sting^fl/fl and Sting ^CKO mice after SAH. (D) Modified Garcia score of Sting^fl/fl and Sting ^CKO mice after SAH (n = 12 per group). Data are presented as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 7

FGF21 inhibited activation of cGAS-STING pathway through inducing mitophagy and reducing cytosolic mtDNA. (A) Measurement of the cytosolic level of mtDNA (MT-ND1 and MT-ND2) through qPCR in isolated microglia from SAH mice with or without rFGF21 treatment (n = 6 per group). (B) Measurement of the cytosolic level of DNA corresponding to abundant nuclear LINE1 elements (L1ORF1 and L1ORF2) through qPCR in isolated microglia from SAH mice with or without rFGF21 treatment (n = 6 per group). (C-G) Western blot analysis of mitophagy related proteins in isolated microglia from SAH mice with or without rFGF21 treatment (n = 6 per group). (H-K) Inhibition of AMPK diminished the promotive effects of rFGF21 treatment on mitophagy (n = 6 per group). CC: compound C (inhibitor of AMPK pathway). (L) Inhibition of AMPK reversed the decreased cytosolic level of mtDNA induced by rFGF21 treatment (n = 6 per group). (M-N) Inhibition of AMPK diminished the suppression of rFGF21 treatment on TNF and IFNB1 mRNA expressions (n = 6 per group). Data are presented as means ± SD. *P < 0.05, **P < 0.01, ***P < 0.001