Roles of PANoptosis and related genes in acute liver failure: neoteric insight from bioinformatics analysis and animal experiment verification

Abstract

BACKGROUND: PANoptosis has the features of pyroptosis, apoptosis, and necroptosis. Numerous studies have confirmed the diverse roles of various types of cell death in acute liver failure (ALF), but limited attention has been given to the crosstalk among them. In this study, we aimed to explore the role of PANoptosis in ALF and uncover new targets for its prevention or treatment. METHODS: Three ALF-related datasets (GSE14668, GSE62029, and GSE74000) were downloaded from the Gene Expression Omnibus (GEO) database to identify differentially expressed genes (DEGs). Hub genes were identified through intersecting DEGs, genes obtained from weighted gene co-expression network analysis (WGCNA), and genes related to PANoptosis. Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), protein‍‒‍protein interaction (PPI) analyses and gene set enrichment analysis (GSEA) were performed to determine functional roles. Verification was performed using an ALF mouse model. RESULTS: Our results showed that expression of seven hub genes (B-cell lymphoma-2-modifying factor (BMF), B-cell lymphoma-2-interacting protein 3-like (BNIP3L), Caspase-1 (CASP1), receptor-interacting protein kinase 3 (RIPK3), uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA), uncoordinated-5 homolog B receptor (UNC5B), and Z-DNA-binding protein 1 (ZBP1)) was up-regulated in liver samples of patients. However, in the ALF mouse model, the expression of BNIP3L, RIPK3, phosphorylated RIPK3 (P-RIPK3), UACA, and cleaved caspase-1 was up-regulated, while the expression of CASP1 and UNC5B was down-regulated. The expression of ZBP1 and BMF increased only during the development of ALF, and there was no significant change in the end stage. Immunofluorescence of mouse liver tissue showed that macrophages expressed all seven markers. Western blot results showed that pyroptosis, apoptosis, and necroptosis were always involved in lipopolysaccharide (LPS)/ d-galactosamine (d-gal)‍-induced ALF mice. The ALF cell model showed that bone marrow-derived macrophages (BMDMs) form PANoptosomes after LPS stimulation. CONCLUSIONS: Our results suggest that PANoptosis of macrophages promotes the development of ALF. The seven new ALF biomarkers identified and validated in this study may contribute to further investigation of diagnostic markers or novel therapeutic targets of ALF.

Keywords: Acute liver failure; Biomarker; Gene Expression Omnibus (GEO); PANoptosis; Therapeutic target.

Fig. 1. Flow diagram of the study process. DEGs: differentially expressed genes; WGCNA: weighted gene co-expression network analysis; ALF: acute liver failure; GO: Gene Ontology; GSEA: gene set enrichment analysis; TSA: tyramide signal amplification.

Fig. 2. Screening for differential genes. (a) Intersection sizes of the three datasets. (b, c) Gene expression level statistics of the datasets before and after de-batching. (d, e) Principal component analysis (PCA) of datasets before and after de-batching. (f) Volcano plot of differentially expressed genes (DEGs): up-regulated DEGs are represented by red nodes, down-regulated DEGs by green nodes, and genes with no significant differential expression by black nodes. (g) Heatmap of DEG expression levels: azure indicates control samples, orange indicates acute liver failure (ALF) samples, red indicates high gene expression, and purple indicates low gene expression.

Fig. 3. Weighted gene co-expression network analysis (WGCNA). (a, b) Analysis of the scale-free fit index for various soft-thresholding powers. (c) Gene trees of co-expression gene modules are shown in various colors. (d) Heatmap of eigengene adjacency. (e) Heatmap of the association between acute liver failure (ALF) and modules. The mediumpurple3 module is shown to be significantly correlated with the ALF. The numbers in the top and bottom triangles represent the correlation coefficient and P value, respectively. (f) Correlation plot between gene significance and module membership of genes included in the mediumpurple3 module. r: correlation coefficient.

Fig. 4. Selection of the PANoptosis-related genes. (a) Venn diagram showing the seven PANoptosis-related genes from the intersection of DEGs, mediumpurple3 module via weighted gene co-expression network analysis (WGCNA), and 75 genes that refer to PANoptosis. (b) The protein‍‒‍protein interaction (PPI) network revealed that the seven hub genes were linked directly or indirectly. The genes in the orange circle are the hub genes, and those in the yellow circle are the bridge genes needed to connect the hub genes. (c) Enrichment analyses of Kyoto Encyclopedia of Genes and Genomes (KEGG) and Gene Ontology (GO) pathways related to the seven hub genes.

Fig. 5. Violin plots of the expression of seven PANoptosis-related genes in the acute liver failure (ALF) and control groups. (a) Expression of B-cell lymphoma-2-modifying factor (BMF). (b) Expression of Caspase-1 (CASP1). (c) Expression of uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). (d) Expression of uncoordinated-5 homolog B receptor (UNC5B). (e) Expression of receptor-interacting protein kinase 3 (RIPK3). (f) Expression of Z-DNA-binding protein 1 (ZBP1). (g) Expression of B-cell lymphoma-2-interacting protein 3-like (BNIP3L). The number at the top of the violin diagram for each gene represents a P-value of <0.0001.

Fig. 6. Gene set enrichment analysis (GSEA) of seven PANoptosis-related genes. (a) GSEA of Caspase-1 (CASP1). (b) GSEA of receptor-interacting protein kinase 3 (RIPK3). (c) GSEA of Z-DNA-binding protein 1 (ZBP1). (d) GSEA of B-cell lymphoma-2-modifying factor (BMF). (e) GSEA of B-cell lymphoma-2-interacting protein 3-like (BNIP3L). (f) GSEA of uncoordinated-5 homolog B receptor (UNC5B). (g) GSEA of uveal autoantigen with coiled-coil domains and ankyrin repeats protein (UACA). H represents high-expression group; L represents low-expression group.

Fig. 7. Immune cell infiltration in acute liver failure (ALF). (a) Proportion of 22 types of immune cells in the ALF group. (b) Correlations among seven PANoptosis-related genes and 22 immune cell types. (c) Comparison of the proportions of 22 kinds of immune cells between the ALF and control groups. ^* P<0.05; ^** P<0.01; ^*** P<0.001; ^**** P<0.0001.

Fig. 8. Verification of the animal model. (a) Hematoxylin and eosin (H&E) staining of liver sections. (b) Levels of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), tumor necrosis factor-‍α (TNF-‍α), interferon-‍γ (IFN-‍γ), interleukin (IL)-6, and IL-10. (c) Changes of the expression levels of nine kinds of proteins with the time of modeling by western blot. (d) Cellular localization analyses of the seven genes through immunofluorescence with tyramide signal amplification (TSA). Data are expressed as mean±standard deviation (n=5). The presence of the same letter between two groups indicates no significant difference between them, and the absence of the same letter indicates a significant difference between them (P<0.05). The golden arrows indicate macrophages expressing BMF or UNC5B. BMF: B-cell lymphoma-2-modifying factor; BNIP3L: B-cell lymphoma-2-interacting protein 3-like; CASP1: Caspase-1; C-CASP1: cleaved CASP1; DAPI: 4',6-diamidino-2-phenylindole; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; L/D: lipopolysaccharide/d-galactosamine; RIPK3: receptor-interacting protein kinase 3; P-RIPK3: phosphorylated RIPK3; UACA: uveal autoantigen with coiled-coil domains and ankyrin repeats protein; UNC5B: uncoordinated-5 homolog B receptor; ZBP1: Z-DNA-binding protein 1.

Fig. 9. Changes in macrophages, pyroptosis, apoptosis, and necroptosis in the liver after LPS/d-gal stimulation (labeled L/D). (a) Representative flow cytometry plots of Kupffer cells (labeled KC) and MoMFs (labeled M) of each group. (b) Percentages of Kupffer cells and MoMFs in hepatic CD45⁺ cells. (c) Immunofluorescence results of macrophages in liver tissue at different time points after LPS/d-gal stimulation. (d) Average fluorescence intensity (AVI) of Ly6C⁺ cells. (e) Trends of biomarkers of pyroptosis, apoptosis, and necroptosis. (f) Immunofluorescence results of PANoptosomes in bone marrow-derived macrophages (BMDMs) after LPS stimulation for 6 h (63× oil immersion objective). The golden arrows show the PANoptosome protein complex. Data are expressed as mean±standard deviation (n=5). The presence of the same letter between two groups indicates no significant difference between them, and the absence of the same letter indicates a significant difference between them (P<0.05). C-GSDMD: cleaved gasdermin D; C-CASP8: cleaved Caspase-8; C-CASP3: cleaved Caspase-3; DAPI: 4',6-diamidino-2-phenylindole; d-gal: d-galactosamine; LPS: lipopolysaccharide; MoMFs: monocyte-derived macrophages.