Pivotal Role of GSTO2 in Ferroptotic Neuronal Injury After Intracerebral Hemorrhage

Abstract

Previous research has found that an adaptive response to ferroptosis involving glutathione peroxidase 4 (GPX4) is triggered after intracerebral hemorrhage. However, little is known about the mechanisms underlying adaptive responses to ferroptosis. To explore the mechanisms underlying adaptive responses to ferroptosis after intracerebral hemorrhage, we used hemin-treated HT22 cells to mimic brain injury after hemorrhagic stroke in vitro to evaluate the antioxidant enzymes and performed bioinformatics analysis based on the mRNA sequencing data. Further, we determined the expression of GSTO2 in hemin-treated hippocampal neurons and in a mouse model of hippocampus-intracerebral hemorrhage (h-ICH) by using Western blot. After hemin treatment, the antioxidant enzymes GPX4, Nrf2, and glutathione (GSH) were upregulated, suggesting that an adaptive response to ferroptosis was triggered. Furthermore, we performed mRNA sequencing to explore the underlying mechanism, and the results showed that 2234 genes were differentially expressed. Among these, ten genes related to ferroptosis (Acsl1, Ftl1, Gclc, Gclm, Hmox1, Map1lc3b, Slc7a11, Slc40a1, Tfrc, and Slc39a14) were altered after hemin treatment. In addition, analysis of the data retrieved from the GO database for the ten targeted genes showed that 20 items on biological processes, 17 items on cellular components, and 19 items on molecular functions were significantly enriched. Based on the GO data, we performed GSEA and found that the glutathione metabolic process was significantly enriched in the hemin phenotype. Notably, the expression of glutathione S-transferase omega (GSTO2), which is involved in glutathione metabolism, was decreased after hemin treatment, and overexpression of Gsto2 decreased lipid reactive oxygen species level in hemin-exposed HT22 cells. In addition, the expression of GSTO2 was also decreased in a mouse model of hippocampus-intracerebral hemorrhage (h-ICH). The decreased expression of GSTO2 in the glutathione metabolic process may be involved in ferroptotic neuronal injury following hemorrhagic stroke.

Keywords: Ferroptosis; Hemin; Hippocampal neurons; Transcriptome analysis.

Fig. 1

mRNA-Seq experiment process

Fig. 2

Toxic effects of hemin on hippocampal neuronal HT22 cells. The HT22 cells were treated with hemin (25 µM) for 6 h. A Cell viability was determined using CCK-8. B Cell mortality determined using PI staining (fluorescence profiles and mean fluorescence intensity (MFI)). C Representative images of the live HT22 cells (Calcein-AM, green) exposed to hemin (25 µM); scale bars represent a magnification of 100× . The values are presented as the mean ± S.E.M., n = 3. ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001, versus the control group

Fig. 3

Effects of hemin on lipid peroxides and ferroptosis-related genes in HT22 hippocampal neuron cells. HT22 cells exposed to hemin (25 µM) for 6 h. A and B lipid ROS and cellular ROS measured using flow cytometry after C11-BODIPY staining and DCFDA staining, respectively. C GSH content was detected using flow cytometry. D and E mRNA expressions of GPX4 and Nrf2 measured using quantitative real-time polymerase chain reaction (PCR). F Cell viability determined using CCK-8. The values are presented as mean ± S.E.M., n = 3. ^*P < 0.05, ^***P < 0.001, ^****P < 0.0001, versus the control group; ^#P < 0.05, versus the hemin-treated group

Fig. 4

Analysis of gene expression profiles after the exposure of HT22 cells to hemin. A Results of cluster analysis. The X-coordinate represents the sample name, the Y-coordinate represents the corresponding sample name, and the color represents the correlation coefficient. B Results of principal component analysis (PCA). C Heatmaps of mRNA expressions in the hemin-treated and control groups. Red represents highly expressed RNAs and blue represents mRNAs with low expression. D The volcano plot and statistic of differentially expressed genes (DEGs) in the control and hemin-treated groups. Gray dots represent RNAs that are not significantly different, and red (upregulated mRNAs) and green (downregulated mRNAs) dots represent RNAs that are significantly different

Fig. 5

KEGG network of ferroptosis pathway based on RNA sequence. The genes in red (Acsl1, Ftl1, Gclc, Gclm, Hmox1, Map1lc3b, Slc7a11, Slc40a1) represent upregulated genes, and the genes in green (Slc39a14, Tfrc) represent downregulated genes after hemin treatment

Fig. 6

Changes in gene levels involved in the ferroptosis pathway after hemin exposure (25 µM, 6 h). A–J Relative mRNA levels of ferroptosis-related key genes associated with KEGG pathway “ferroptosis.” The values are presented as mean ± S.E.M., n = 3. ^*P < 0.05, ^**P < 0.01, ^****P < 0.0001, versus control group

Fig. 7

Functional enrichment analysis of DEGs. GO term enrichment analysis of DEGs for the ten target genes involved in the ferroptosis pathway. A BP represents biological process; B CC represents cellular component; C MF represents molecular function

Fig. 8

GSEA against the obtained GO database in hemin-treated HT22 cells and the expression of Gsto2 was detected by western blot. A GSEA plots of the gene sets upon the GO database after hemin treatment. B String PPI analysis of the interaction network of Gsto2. C, D Expression of Gsto2 detected by western blotting. E Level of lipid ROS detected by flow cytometry

Fig. 9

Hemorrhage volume following hemorrhagic stroke and the expression of Gsto2 in the hippocampus. A Representative images of brain sections at 24 h post-injury. B Hemorrhage volume measured in the brain (n = 4). C Expression of Gsto2 in the hippocampus detected by western blotting (n = 4)