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. 2024 Dec;56(1):2411015.
doi: 10.1080/07853890.2024.2411015. Epub 2024 Oct 10.

Systematic analysis based on bioinformatics and experimental validation identifies Alox5 as a novel therapeutic target of quercetin for sepsis

Chu-Yun Liu  1 , Yu-Shen Yang  1 , Meng-Qin Pei  1 , Yan Zhang  2 , Wei-Can Chen  1 , Jin-Wei Liang  1 , He-Fan He  1
Affiliations

Systematic analysis based on bioinformatics and experimental validation identifies Alox5 as a novel therapeutic target of quercetin for sepsis

Chu-Yun Liu et al. Ann Med. 2024 Dec.

Abstract

Purpose: This study investigated the molecular mechanism of quercetin in the treatment of sepsis using network pharmacological prediction and experimentation.

Methods: Hub genes were identified by intersecting the differentially expressed genes (DEGs) of the GSE131761 and GSE9960 databases with genes from the hub modules of Weighted Gene Co-Expression Network Analysis (WGCNA), targets of quercetin, and ferroptosis. Subsequently, in order to determine the functional characteristics and molecular link of hub gene obtained above, we redetermined the hub-DEGs in GSE131761 according to high or low hub gene expression. Afterward, the main pathways of enrichment analysis were validated using these hub-DEGs. Finally, an experiment was conducted to validate the findings.

Results: By intersecting 1415 DEGs in GSE131761, 543 DEGs in GSE9960, 5784 key modular genes, 470 ferroptosis-related genes, and 154 quercetin-related genes, we obtained one quercetin-related gene, Alox5. Subsequently, 340 hub-DEGs were further validated according to high or low Alox5 expression. The results of the enrichment analysis revealed that hub-DEGs were mainly associated with inflammation and the immune response. Immune infiltration analysis showed that higher expression of Alox5 was related to macrophage infiltration and could be a predictor of diagnosis in patients with sepsis. The expression pattern of Alox5 was then depicted and the upregulation of Alox5 in the vital organs of septic mice was further demonstrated. In vitro and in vivo experiments showed that upregulation of Alox5 and inflammation-related cytokines induced by sepsis could be inhibited by quercetin (p < 0.05).

Conclusions: Alox5 may be involved in the occurrence and development of multi-organ functional disturbances in sepsis and is a reliable target of quercetin against sepsis.

Keywords: Alox5; inflammation; peripheral blood mononuclear cells; quercetin; sepsis.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Schematic flow chart depicting the general steps taken in this research.
Figure 2.
Figure 2.
Weighted gene co-expression network analysis. (A) Module–trait association. (B–D) Scatter plots of key modules.
Figure 3.
Figure 3.
Identification of DEGs and hub gene. (A) Volcano graph of DEGs in GSE131761 dataset. (B) Heat map of DEGs in GSE131761 dataset. (C) Volcano graph of DEGs in GSE9960 dataset. (D) Heat map of DEGs in GSE9960 dataset. (E) Venn diagram for intersected genes in DEG_GSE131761, DEG_GSE9960, quercetin target, ferr gene, and WGCNA_GSE131761. (F) The ROC curve analysis of Alox5 in GSE131761.
Figure 4.
Figure 4.
The expression profiling of Alox5 in sepsis. (A) Alox5 expression level in blood tissue of septic patients from the GSE131761 dataset. (B) Alox5 expression level in monocytes of septic patients from the GSE9960 dataset. (C) Alox5 expression pattern in the vital organs of septic patients. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 5.
Figure 5.
Differential and enrichment analysis of Alox5 grouping. (A) In GSE131761, differential analysis volcano graph for Alox5 expression. (B) In GSE131761, the differential analysis heat map of Alox5 high and low expression groups. (C) DEGs in GO analysis. (D) DEGs in KEGG analysis.
Figure 6.
Figure 6.
GSEA And GSVA. (A) Mountain range plot showing the GSEA results of the GSE131761 dataset. (B) Volcano map for differential GSVA enrichment analysis of Alox5 expression high and low groups.
Figure 7.
Figure 7.
PPI Network and related functional network analysis. (A) The 104 DEGs in PPI network constructed by the STRING database. (B) CytoHubba calculated 20 hub genes. (C) Hub gene–TF network. Small circle inside represents a hub gene, and big circle outside represents TF. (E) Hub gene–chemical interactive network.
Figure 8.
Figure 8.
Evaluation of immune cell infiltration and correlational analysis based on the CIBERSORT algorithm. (A) Bar plot showing percentage infiltration of 22 immune cells in each sample. (B) Correlation heatmap of 22 immune cell types. (C) The correlation between Alox5 and immune cells is lollipop graph. (D) Box plot showing differential infiltration of the 22 immune cell populations. *p < 0.05; **p < 0.01; ***p < 0.001.
Figure 9.
Figure 9.
Clinical correlational analysis of Alox5. (A) The ROC curve analysis of Alox5 in GSE131761. (B) Nomogram plot for clinicopathological parameters. (C) Calibration curve. (D) The ROC curve analysis of nomogram in GSE131761. (E) Decision curve analysis; (F) Clinical impact curve.
Figure 10.
Figure 10.
Quercetin alleviated sepsis probably by inhibiting the overactivation of macrophages via reducing the expression of Alox5. (A) CCK-8 assay. (B–F) Quercetin intervention could decrease the mRNA level of Alox5 (B), MCP-1 (C), IL-1 (D), IL-6 (E) and TNF (F) in activated THP-1 cell lines by LPS. (G–I) HE stains. (J–M) Quercetin administration could decrease the mRNA level of Alox5 (J) in the brain tissue of septic mice by CLP, with the decreased mRNA level in IL-1 (K), IL-6 (L) and TNF (M). */#p < 0.05; **/##p < 0.01; ***/###p < 0.001.
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