High-Throughput mRNA Sequencing Reveals Potential Therapeutic Targets of Febuxostat in Secondary Injury After Intracerebral Hemorrhage

Abstract

Febuxostat is a urate-lowering medication for the treatment of patients with gout. This study was performed to elucidate the effects and underlying mechanisms of febuxostat on neuronal injury induced by intracerebral hemorrhage (ICH) in mice. The results showed that the administration of febuxostat improved neurological severity scores and blood-brain barrier (BBB) permeability. Moreover, febuxostat attenuated neuronal cell death and cytokine levels compared with the ICH group. Next, we conducted a transcriptome analysis of the neuroprotective effects of febuxostat. The overlapping significant differentially expressed genes (DEGs) were identified. Gene ontology (GO) analysis revealed that the overlapping significant DEGs were most enriched in five items. The intersecting DEGs of the aforementioned five pathways were Wisp1, Wnt7b, Frzb, and Pitx2. In addition, GO terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways revealed that DEGs were mainly involved in the wnt signaling pathway. Furthermore, the expression of Wisp1 and Wnt7b in the perihematomal region at 72 h post-ICH was observed. The results showed that both Wisp1 and Wnt7b were increased in the ICH group and were decreased by the administration of febuxostat. Taken together, the study showed that febuxostat protected against secondary brain injury after ICH and the Wnt7b-Wisp1 pathway was closely related to neuroprotective effects.

Keywords: Wisp1; febuxostat; high-throughput mRNA sequencing; intracerebral hemorrhage; neuroprotection.

FIGURE 1

Febuxostat improved neurological dysfunction after ICH: (A) Representative images of coronal brain sections; (B) Modified neurological severity score. (C) Wire hanging test. (D) Beam walking test. (E) Forelimb placing test. Values are mean ± S.E.M; **p < 0.01 (n = 6 mice/group, two-way ANOVA).

FIGURE 2

Febuxostat attenuated neurodegeneration after ICH: (A) Degenerating neurons in the brain sections were labeled with FJB stain. Neuronal degradation was stained green. White arrows indicate colocalization. The scale bar is 50 μm. (B) Apoptotic neurons in the brain sections were labeled with the TUNEL stain. TUNEL-positive cells are shown in green. The scale bar is 100 μm. Sections were stained with DAPI (blue) to show all nuclei.

FIGURE 3

Febuxostat attenuated BBB permeability after ICH: (A) Representative coronal brain sections show Evans blue extravasation. (B) Permeability of the BBB using the Evans Blue (EB) assay. EB is shown in red. CD31 is shown in green. White arrows indicate colocalization. Sections were stained with DAPI (blue) to show all nuclei. The scale bar is 40 μm.

FIGURE 4

Hierarchical cluster analysis of DEGs: (A) Hierarchical cluster analysis showed that the DEGs ultimately clustered into two major branches: upregulated genes (labeled in red) and downregulated genes (labeled in blue). (B) Volcano plot of DEGs. The red and blue points in the plots represent the significantly upregulated and downregulated DEGs. n = 3 mice/group.

FIGURE 5

GO and KEGG analyses of DEGs: (A) GO enrichment of DEGs. (B) KEGG pathway enrichment of DEGs. (C) Venn diagram showing the overlapping significant DEGs from GO terms.

FIGURE 6

Protein–protein interactions (PPI): STRING analysis with the interaction score set to 0.15. Each node represents the relevant protein, the edge indicates the predicted functional associations, and the number of lines represents the strength of predicted functional interactions between proteins.

FIGURE 7

Expression of Wisp1 and Wnt7b in mice: (A) Expression of Wisp1 and Wnt7b in the perihematomal area observed by immunofluorescence labeling. Wisp1 is shown in red and Wnt7b is shown in green. White arrows indicate colocalization. Sections were stained with DAPI (blue) to show all nuclei. The scale bar is 50 μm. (B) Expression of Wisp1 and Wnt7b in the perihematomal area. The scale bar is 50 μm.