Comparative pathophysiological modeling: the advantage of "two-hit" over "one-hit" in acute liver failure studies

Acute liver failure (ALF), a life-threatening condition marked by rapid hepatocyte death and systemic inflammation, poses significant clinical challenges due to its high mortality. The crosstalk between necrotic hepatocytes and infiltrating immune cells is hypothesized to drive disease progression. To investigate this interplay, we developed a sequential "two-hit" murine model using concanavalin A (Con A) challenges and compared its pathophysiological outcomes with the conventional single-dose "one-hit" approach. The results demonstrated that the "two-hit" model induced more severe hepatic coagulation dysfunction, extensive hepatocellular necrosis, destruction of liver lobular architecture, and inflammatory responses. Furthermore, serum levels of alanine transaminase (ALT) and aspartate transaminase (AST) were markedly elevated in the "two-hit" group. Inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) were also significantly elevated. Moreover, substantial recruitment of macrophages was observed in the "two-hit" model, indicating that these cells are key determinants in the interaction with dying hepatocytes for the progression of ALF. Complementary ex vivo experiments revealed that Raw264.7 cells subjected to a "two-hit" stimulation with Con A and hepatocyte debris produced a robust inflammatory response through the classical NF-κB signaling pathway.