The mechanism of the Nfe2l2/Hmox1 signaling pathway in ferroptosis regulation in acute compartment syndrome

Abstract

Acute compartment syndrome (ACS) is a life-threatening orthopedic emergency, which can even result in amputation. Ferroptosis is an iron-dependent form of nonapoptotic cell death. This study investigated the mechanism of ferroptosis in ACS, explored candidate markers, and determined effective treatments. This study identified pathways involved in the development of ACS through gene set enrichment analysis (GSEA), Gene Ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG), and GSEA of heme oxygenase 1 (Hmox1). Bioinformatics methods, combined with real-time quantitative polymerase chain reaction, western blot analysis, and iron staining, were applied to determine whether ferroptosis was involved in the progression of ACS and to explore the mechanism of nuclear factor erythroid-2-related factor 2 (Nfe2l2)/Hmox1 in ferroptosis regulation. Optimal drugs for the treatment of ACS were also investigated using Connectivity Map. The ferroptosis pathway was enriched in GSEA, KEGG of DEGs, and GSEA of Hmox1. After ACS, the reactive oxygen species content, tissue iron content, and oxidative stress level increased, whereas glutathione peroxidase 4 protein expression decreased. The skeletal muscle was swollen and necrotized; the number of mitochondrial cristae became fewer or even disappeared, and Nfe2l2/Hmox1 expression increased at the transcriptional and protein levels. Hmox1 was highly expressed in ACS, indicating that Hmox1 is a possible marker for ACS. we could predict 12 potential target drugs for the treatment of ACS. In conclusion, Hmox1 was a potential candidate marker for ACS diagnosis. Ferroptosis was involved in the progression of ACS. It was speculated that ferroptosis is inhibited by the Nfe2l2/Hmox1 signaling pathway.

Keywords: Nfe2l2/Hmox1 signaling pathway; RNA-sequencing; acute compartment syndrome; bioinformatics analysis; ferroptosis.

Figure 1

Flow diagram of the study. ceRNA, competitive endogenous RNA; DEGs, differentially expressed genes; GO, Gene Ontology; GSEA, gene set enrichment analysis; HE, Hematoxylin–Eosin; KEGG, Kyoto Encyclopedia of Genes and Genomes; PPI, protein–protein interaction; ROS, reactive oxygen species; RT‐qPCR, real‐time quantitative polymerase chain reaction; TEM, transmission electron microscopy; WB, western blot.

Figure 2

Analysis results of DEGs. (A) Schematic diagram of the structure of the animal model: (1) rat, (2) thigh root ligation, (3) scalp needle, (4) disposable infusion sets, (5) tee tubes, (6) pressure transducer, (7) physiological saline, (8) ECG monitor cables, and (9) ECG monitor. (B) Result of PCA. (C) Hmox‐1 is upregulated in ACS (FDR = 1e − 09, log FC = 4.42). (D) Volcano maps of the ACS data set. (E) A heatmap of 25 most upregulated and 25 most downregulated genes. ACS, acute compartment syndrome; DEGs, differentially expressed genes; ECG, electrocardiography; FC, folding change; FDR, false discovery rate; PCA, principal component analysis.

Figure 3

Results of GSEA. (A) BP enriched in ACS. (B) CC enriched in ACS. (C) MF enriched in ACS. (D) KEGG pathways enriched in ACS. ACS, acute compartment syndrome; BP, biological process; CC, cellular component; GSEA, gene set enrichment analysis; KEGG, Kyoto Encyclopedia of Genes and Genomes; MAP, mitogen‐activated protein; MF, molecular function.

Figure 4

Results of functional and pathway enrichment analyses. (A) Results of BP enrichment analysis of DEGs. (B) Results of CC enrichment analysis of DEGs. (C) Results of MF enrichment analysis of DEGs. (D) Results of KEGG pathway analysis of DEGs. BP, biological process; CC, cellular component; DEGs, differentially expressed genes; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function.

Figure 5

Results of PPI networks and the significant gene module. (A) PPI of DEGs and (B) significant gene module in the PPI networks. DEGs, differentially expressed genes; PPI, protein–protein interaction.

Figure 6

Results of the significant gene module analysis. (A) Results of BP enrichment analysis of the significant gene module. (B) CC enriched in the significant gene module. (C) MF enriched in the significant gene module. (D) KEGG pathways enriched in the significant gene module. AGE‐RAGE, advanced glycation endproduct–receptor for AGE; BP, biological process; CC, cellular component; GO, Gene Ontology; KEGG, Kyoto Encyclopedia of Genes and Genomes; MF, molecular function; miRNAs, microRNAs; NF, nuclear factor.

Figure 7

Results of Functional similarity analysis. The horizontal axis represents the similarity score, and the vertical axis represents genes. Gpx4, glutathione peroxidase 4; Hmox1, heme oxygenase 1; Nfe2l2, nuclear factor erythroid‐2‐related factor 2.

Figure 8

Establishment of a ceRNA network, and GSEA of Hmox1. (A) Establishment of a ceRNA network. (B) GSEA of Hmox1. ceRNA, competitive endogenous RNA; GSEA, gene set enrichment analysis; Hmox1, heme oxygenase 1; IL‐17, Interleukin‐17; KCNQ1OT1, potassium voltage‐gated channel subfamily Q member 1 opposite strand 1; TNF, tumor necrosis factor.

Figure 9

Results of RT‐qPCR, western blot (WB) analysis, and HE staining. (A) RT‐qPCR detection of the Nfe2l2mRNA level. (B) RT‐qPCR detection of the Hmox1mRNA level. (C) WB for detection of Nfe2l2 and Hmox1 protein expressions, with β‐actin protein as a standard control. (D) Quantitative analysis of the Nfe2l2 protein level. (E) Quantitative analysis of the Hmox1 protein level. (F) WB for determining the expression of Gpx4 protein (with β‐actin protein as a standardized control). (G) Quantitative analysis of the Gpx4 protein level. (H) HE staining of the flexor digitorum longus muscle, with arrows indicating skeletal muscle necrosis (*indicates p < 0.05 compared with the control group). Gpx4, glutathione peroxidase 4; Hmox1, heme oxygenase 1; Nfe2l2, nuclear factor erythroid‐2‐related factor 2; RT‐qPCR, real‐time quantitative polymerase chain reaction.

Figure 10

Results of DHE staining, Iron staining, and TEM. (A) DHE staining to detect ROS. (B) Quantitative analysis of the ratio of the DHE staining positive fluorescence area. (C) Iron staining of the flexor digitorum longus. (D) Quantitative analysis of the ratio of the iron staining positive area. (E) TEM of the flexor digitorum longus (*indicates p < 0.05 compared with the control group). DHE, dihydroethidium; ROS, reactive oxygen species; TEM, transmission electron microscopy.