Single-cell RNA sequencing explores the mechanisms underlying the protective effects of myeloid cell-specific Pkm gene knockout in ALF-modeling mice

Acute liver failure (ALF) has an extremely high mortality rate and lacks effective treatment. Previous studies have shown that targeting macrophage pyruvate kinase isoenzyme type M2 (PKM2) may be a new approach for ALF treatment, but the exact mechanism remains to be elucidated. In this project, based on Single-cell RNA-sequencing (scRNA-seq), Pkm myeloid selective knockout (PL) mice and wild-type (WT) mice were used to establish ALF model, analyze the single-cell transcriptomic features of liver tissues of WT mice during ALF pathology, and screen for the differentially expressed genes in liver tissues of PL mice. Potential molecular pathways of PKM2 regulation of ALF in macrophages were explored by bioinformatics analysis and validated by molecular biology methods. Result shows that, up-regulated genes in macrophages and neutrophils in mouse liver tissues during ALF were mainly enriched in multiple programmed death, such as pyroptosis, apoptosis and necroptosis, and immune-inflammation pathways. The down-regulated genes in PL mouse liver macrophages and neutrophils were mostly enriched in the above pathways. Among them, Pkm knockdown significantly down-regulated the expression of key genes of NOD-like signaling pathway, such as NLRP3, IL-1ß and IL-18, inhibited hepatic macrophage pyroptosis and attenuated ALF. This study will help to elucidate the molecular mechanism of PKM2 regulating macrophage inflammatory response involved in ALF, suggesting that PKM2 may be a potential therapeutic target for ALF, and providing a theoretical basis for further research on new methods of targeting PKM2 in the treatment of ALF. Overall design: To investigate the function of PKM2 in ALF, we establish ALF model by intraperitoneal injection of D-galactosamine and LPS in Pkm myeloid selective knockout (PL) mice and wild-type (WT) mice. Same volume of PBS was intraperitoneal injected in PL and WT mice to establish negative control (NC) group. Three hours after ALF modeling, mice were sacrificed and hepatic parenchymal and nonparenchymal cells were extracted from liver tissue. After counting, the hepatic parenchymal and non-parenchymal cells were mixed at a ratio of 2:8 and then uploaded for scRNA-seq. We then performed single-cell transcriptomic analysis using data obtained from scRNA-seq of 4 groups.