PANoptosis exists in liver failure
This study included 187 articles, 19 articles on pyroptosis, 151 articles on apoptosis and 17 articles on necroptosis. Among them, 6 studies on patients with liver failure, 93 studies on rodents (including rats and mice), 14 studies on primary cultured cells or cell lines, and 76 studies on rodents in vitro and in vivo. In cell experiments, most cell experiments were been used paracetamol (APAP), lipopolysaccharide (LPS), D-galactamine (D-GalN), carbon tetrachloride (CCL4), TNF α, TGF β1, multiplicity of infection(MOI), etc. to induce liver failure model. Researchers used cell lines such as primary hepatocytes (human, mouse, rat), HepG2, L02, HL-7702, BNLCL2, Hepa1-6, Hep3B, AML12, BRL-3A, PHH, L929, Huh-7, Chang Liver ,Hepa 1, NMH, IHH, HepaRG, and miHeps. The collected results of cell types, intervention methods, cell death types, evaluation methods, and detection of key proteins in the experiments are shown in Table S1. Among the included animal studies, most of them were based on rodent animals, and liver failure was induced by hepatotoxic drugs, such as APAP, LPS, D-GalN, CCL4, HAS, Con-A, TNF, etc., or surgical simulation of liver failure models, such as cecal ligation puncture (CLP), partial hepatectomy (HP), bile duct ligation (BDL), and there were also some methods using viral infection (HBsAg) induction. Sprague-Dawley rats, Wistar rats, F344 Rats, C57BL/6 mice, BALB/c mice, CD-1 mice, ICR mice, Kunming mice, and CD-1 mice were used to establishment of liver failure models. The animal types, modeling methods, cell death types, evaluation methods, and detection of key proteins in the experiments are shown in Table S2 and Table S3. In the study of patients with liver failure, the main type of liver failure is ACLF. The cell death types, evaluation methods, and detection of key proteins in the experiments are shown in Table S4. These modeling methods to simulate experimental animals of liver failure have been recognized in the research field. According to the results of our analysis, in the same cell models or animal disease models, three kinds of RCD (pyroptosis, apoptosis, and necroptosis) may occur concurrently, which would mean that PANoptosis occurs in liver failure.
Screening of DEGs
According to the screening criteria of | logFC |>1, FDR<0.05, there are 1446 DEGs in the ACLF group and the normal control group in GSE139602, including 829 up-regulated DEGs and 617 down-regulated DEGs. The results are visualized using a volcanic map (Figure 2A).
Pathway enrichment analysis and functional annotation
We performed a GO-BP enrichment analysis of DEGs, the results shows that the biological processes of ACLF mainly included extracellular matrix organization, extracellular structure organization, external encapsulating structure organization, small molecule catabolic process, and steroid metabolic process. The cellular component of ACLF mainly included collagen-containing extracellular matrix,cell-substrate junction , focal adhesion , basement membrane ,and collagen trimer . The molecular function of ACLF mainly included actin binding, extracellular matrix structural constituent, gly cosaminoglycan binding, integrin binding, and actin filament binding( Figure 2B). The upregulated DEGs were mainly engaged in Focal adhesion, PI3K-Akt signaling pathway, Human papillomavirus infection, ECM-receptor interaction, and Pathogenic Escherichia coli infection(Figure 2C and D). Downregulated DEGs were mainly engaged in Biosynthesis of cofactors, Carbon metabolism, Drug metabolism - cytochrome P450, and Peroxisome, Bile secretion(Figure 2E and F).
Functional annotation of GSE139602
All annotated gene information of ACLF and healthy subjects in GSE139602 was uploaded to the GSEA sofware for analysis at a holistic level. Pathways with |NES|≥1.0 and NOM p-value<0.05 were considered as signifcantly enriched gene sets. The GSEA results revealed signifcant enrichment in RDC and immunity signalling pathyways, including pyroptosis, apoptosis, NOD-like receptor signalling pathyway, and necroptosis and reglulated necrosis(Figure 3A and B). To screen out PANoptosis -related DEGs, there were 26 genes were obtained from the intersection of the pyroptosis, apoptosis, necroptosis and DEGs (Figure 3C). And the one intersection gene of is BAX. These 26 genes were represented in a heat map, with yellow representing high expression and blue representing low expression (Figure 3D). Moreover, we conducted correlation analysis on these 26 genes, with positive correlation in red and negative correlation in blue (Figure 3E).
PPI network of DEGs and Correlation analysis of immune infltration and gene modules
We imported 1446 DEGs into Cytoscape.Then, the PPI network was constructed, resulting 7 gene clusters (Figure 4). The genes contained in the first subnetwork are LAMB3,LAMA3,ACRN,LAMC3,LAMA,LAMCI,GIT2,LAMB2,LAMB1,and LAMA2 (Figure 4A). The genes contained in the second subnetwork are CYP2J2,CYP1A2,EPHX1,CYP2E1,CYP2C19,CYP4A22,and CYP3A4 (Figure 4B). The genes contained in the third subnetwork are CAT,SLC27A2, SCP2,HAO2,MPV17,EPHX2,PECR,and ACOX2 (Figure 4C). The genes contained in the fourth subnetwork are THBS2, SPON2, DAMTSL3, THSD7A, CFP, ADAMTS9, and ADAMTSL2 (Figure 4D). The genes contained in the fifth subnetwork are MTHFD2, SHMT1, DHFR, AMT, MTHFD1, MTHFD1L, ALDH1L1 (Figure 4E). The genes contained in the sixth subnetwork are COL4A3, COL4A4,COL4A1,COL4A2,P4HA2,P4HA1,COL6A1,COL6A3,COL1A2,COL3A1,COLIAI,PCOLCE2,LUM, and PCOLCE (Figure 4F). The genes contained in the seventh subnetwork are TCAD,JAM3,TGBS,ANXAS,VCL,TLNI,TAGLN2,ENDOD,TUBAA,FLNA,HIFIA,CCNDI, BATF,RORA,FOXO1,and FOS (Figure 4G).We used the CIBERSORT algorithm to investigate the correlation between immune infltration and gene co-expression modules. The proportion of each immune cell type in each sample is shown in a stacked bar graph (Figure 5). A total of 22 types of immune cells were obtained, and the results showed that P<0.05 was considered to have a significant difference. The results showed that the content of T cells CD8,T cells CD4 memory activated, T cells CD4 naïve, NK cells resting, NK cells activated, Monocytes, Macrophages M0, Macrophages M2, and Dendritic cells resting in ACFL were higher.
Liver Histology Observation
The HE staining results showed that the liver lobule structure of the normal group rats was clear and complete, and the hepatocytes were arranged in order, without degeneration, necrosis cells and obvious inflammatory cell infiltration. In the ACLF group, the hepatocytes were disordered and necrotic, accompanied by a large number of inflammatory cell infiltration, obvious hepatic sinusoid expansion and hemorrhage, and massive and sub-massive necrosis of liver tissue. As shown in Figure6A. The masson staining results showed that the morphology and structure of hepatic lobule and portal area of rats in the normal group were complete, and the hepatic cords were arranged radially. In the ACLF group, the collagen in most portal areas in the liver tissue of rats were significantly proliferative, and it was obvious that some portal areas were bridged with proliferative collagen fibers, the structure of hepatic lobules was destroyed, pseudolobules were widely formed, and liver fibrosis and cirrhosis were obvious. As shown in Figure 6B.
Assessment of blood biochemistry and Levels of inflammatory factors in serum of rats
In order to observe the liver and kidney function of rats in each group, we detected the serum ALT, AST, TBiL, ALB, CRE levels. The levels of ALT, AST, TBiL and CRE in ACLF group were significantly higher than those in normal group (P<0.05, P<0.01).The serum ALB level in ACLF group was significantly lower than that in normal group (P<0.01), as shown in Figure 7A-7E.The results of ELISA showed that The levels of IL-6, IL-1β, IFN- γ, and TNF- α in serum of ACLF group were significantly higher than those of the normal group (P<0.05, P<0.01)as shown in Figure 8A-D.
The expression of key proteins in PANoptosis like cell death in ACLF
In order to observe whether PANoptosis like cell death was activated and involved in the progression of ACLF, we detected the expression of PANoptosis key proteins in the liver of ACLF rats using western blot. The ACLF group increased the expression of NLRP3 and cleaved caspase-1 (C-CASP-1, p10), which is a key protein in the pyroptosis pathway. The ACLF group also upregulated the expression of cleaved GSDMD (GSDMD-N, p29). When the GSDMD was cleaved, producing an active p29 fragment that can form membrane pores to induce pyroptosis, which is a driving factor for pyroptosis. GSDMD can be activated by inflammatory bodies downstream of caspase-1 to release this p29 fragment. Consistent with the production of GSDMD-N, activation of caspase-1 produces C-CASP-1 (Figure 9A-F). The results showed that there was apoptosis in ACLF, as evidenced by the cleavage of caspase-3, caspase-7, and caspase-8 (Figure 10A-H). In addition, compared to the normal group, the expression of BAX, a proapoptotic protein in the ACLF group significantly increased (Figure 10A,10F), which are key proteins in the apoptosis pathway. The increased phosphorylation level of MLKL is a marker of necroptosis, as shown in Figure 11A-C, ACLF significantly upregulated the expression of phospho-MLKL. In summary, these results indicate that the key proteins of pyroptosis, apoptosis, and necroptosis were expressed at the protein level in the liver tissue of the ACLF group, and further indicate the presence of PANoptosis in ACLF.