Phospholipase D regulates ferroptosis signal transduction in mouse spleen hypoxia response

Abstract

High-altitude hypoxia exposure can lead to phospholipase D-mediated lipid metabolism disorder in spleen tissues and induce ferroptosis. Nonetheless, the key genes underlying hypoxia-induced splenic phospholipase D and the ferroptosis pathway remain unclear. This study aimed to establish a hypoxia animal model. Combined transcriptomic and proteomic analyses showed that 95 predicted target genes (proteins) were significantly differentially expressed under hypoxic conditions. Key genes in phospholipase D and ferroptosis pathways under hypoxic exposure were identified by combining Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis techniques. Gene set enrichment analysis (GSEA) showed that the differential gene sets of the phospholipase D and ferroptosis signaling pathways were upregulated in the high-altitude hypoxia group. The genes in the phospholipase D signalling pathway were verified, and the expression levels of KIT and DGKG were upregulated in spleen tissues under hypoxic exposure. Subsequently, the mRNA and protein expression levels of genes from the exogenous pathway such as TFRC, SLC40A1, SLC7A11, TRP53, and FTH1 and those from the endogenous pathway such as GPX4, HMOX1, and ALOX15 differentials in the ferroptosis signalling pathway were verified, and the results indicated significant differential expression. In summary, exposure to high-altitude hypoxia mediated phospholipid metabolism disturbance through the phospholipase D signalling pathway and further induced ferroptosis, leading to splenic injury.

Figure 1. Expression of hypoxia-inducible factor (HIF)-1α in spleen tissues of mice in the high-altitude spleen test (HST) group and in the plain spleen control (PSC) group. A, Western blot analysis of HIF-1α protein expression. B, Quantitative analysis of HIF-1α protein expression with the β-actin as standard. C, mRNA expression analysis of HIF-1α. Data are reported as means±SD (n=3). *P<0.05, ****P<0.0001 compared with PSC (Student's t-test). D, HE staining of mouse spleen tissue (scale bar 20 μm).

Figure 2. Transcriptomic sequencing of samples from the high-altitude spleen test (HST) group and in the plain spleen control (PSC) group. A, Sequencing data error rate distribution. B, GC content distribution. C, Sample sequencing data filtering. D, Sample gene expression distribution. E, Differential gene Venn diagram. F, Peptide length range distribution map. G, Parent ion mass tolerance distribution. H, Distribution map of the number of unique peptides in identified proteins. I, Protein molecular weight distribution. GC content: ratio of quinine and cytosine among the four bases of DNA.

Figure 3. Gene expression analysis of mice in the high-altitude spleen test (HST) group and in the plain spleen control (PSC) group. A, Venn diagram of transcriptome and proteome expression regulation. all_tran represents all genes obtained from transcriptome, diff_tran represents differentially expressed genes identified from transcriptome, all_prot represents all proteins identified from proteome, diff_prot represents differentially expressed proteins identified from proteome. B, Bubble diagram of 95 prediction target gene. C, Association analysis of transcriptome and proteome expressions, each point in the graph represents a protein, green points represent proteins with significant differences in expression, blue points represent proteins with no significant differences in expression. D, Gene Ontology functional enrichment association analysis. E, Heat map of KEGG pathway enrichment clustering.

Figure 4. A-J, Relative expression of key genes in phospholipase D pathway and iron death pathway in mice in the high-altitude hypoxia (HST) group and in the plain normal oxygen (PSC) group (n=5/group). *P<0.05, **P<0.01, ***P<0.001 (Student's t-test). K, Enrichment plot: PHOSPHOLIPASE_D_SIGNALING_PATHWAY(MMU04072) Profile of the Running ES Score & Positions of GeneSet Members on the Rank Ordered List. L, Kegg advanced network diagram: purple circles represent pathways, red circles represent genes, black lines represent positive correlation. M, Enrichment plot: FERROPTOSIS(MMU04216) Profile of the Running ES Score & Positions of GeneSet Members on the Rank Ordered List.

Figure 5. A-C, Quantitative analysis of mRNA expression, protein expression levels, and protein expression of KIT and DGKG in phospholipase D signaling pathway with β-actin as standard in mice in the high-altitude spleen test (HST) group and in the plain spleen control (PSC) group (n=3/group). D-F, Quantitative analysis of mRNA expression, protein expression levels, and protein expression of key genes in the ferroptosis signaling pathway, using β-actin as the standard (n=3/group). *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001 (Student's t-test). G, Ferroptosis signaling pathway.