Transcriptomic profiling of HBV transgenic mouse livers reveals ZBP1-associated necroptotic signaling

Although transcriptome studies have been performed in cell culture models of HBV infection, the in vivo hepatic transcriptional response to persistent HBV expression remains incompletely characterized. In addition, whether HBV is associated with activation of necroptotic signaling in the liver has not been fully clarified. Therefore, this study aimed to: (1) define transcriptomic alterations in livers from an HBV transgenic mouse model; and (2) explore whether ZBP1-associated necroptotic signaling is present in HBV-Tg livers by integrating RNA-seq, bioinformatic analysis, and biochemical validation. We utilized an HBV transgenic mouse model and characterized it by measuring serum HBV DNA, HBsAg, AST, ALT, and TBIL levels and by performing hematoxylin and eosin (H&E) staining. Subsequently, the expression of protein-coding genes in HBV transgenic and control mice was analyzed by next-generation RNA sequencing (RNA-seq). Differentially expressed genes (DEGs) were identified using EdgeR with thresholds of |log2(fold change)| > 1 and P value < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed on the DEGs. A protein-protein interaction (PPI) network was constructed based on the STRING database, and PPI modules were analyzed using the MCODE plugin in Cytoscape. Finally, we evaluated ZBP1-associated necroptotic signaling by examining the protein levels of ZBP1 and phosphorylated RIPK3/MLKL by Western blot. We identified 815 candidate differentially expressed genes, including 412 upregulated and 403 downregulated genes in HBV-Tg livers compared with control livers. KEGG pathway analysis indicated enrichment of immune-related pathways, metabolic pathways, and viral infection-related pathways. Zbp1 drew further attention because it was increased in HBV-Tg livers and located within an interferon/innate immune-related PPI module. At the protein level, ZBP1, p-RIPK3, and p-MLKL were increased in HBV-Tg livers. These findings suggest elevated ZBP1-associated necroptosis-related markers in the HBV-Tg liver model. We profiled gene expression in livers from HBV-Tg and control mice using RNA-seq and identified candidate DEGs, GO terms, and pathway terms associated with HBV-related liver responses. Our data support an association between HBV-Tg liver status and increased ZBP1, p-RIPK3, and p-MLKL expression. However, additional functional studies are required to determine cell specificity, causal direction, and the contribution of this signaling axis to liver injury.