Background: Fulminant hepatic failure is a severe and life-threatening acute liver injury (ALI) that is characterized by excessive hepatic apoptosis and inflammation mediated by pro-inflammation cytokines. Cytokine signaling is mediated by mitogen-activated protein kinase (MAPK) pathways, however, the regulatory mechanisms remain unclear. We here focused on Sprouty-related EVH1-domain-containing protein (Spred)-2, the negative regulator of Ras-Raf-ERK-MAPK pathway, and investigated the role in LPS/D-GalN-induced ALI.
Method: ALI was induced in WT and Spred-2-/- mice by an i.p. injection of LPS (20 μg/kg of body weight) and D-GalN (400 mg/kg of body weight). Mice were euthanized at appropriate time points and the sera were obtained. Livers were resected to assess the leukocyte infiltration, cytokine/chemokine production, MAPK activation, and caspase 3/8 activity. In different sets of experiments, mice were i.v. received TNF-α/D-GalN. For further analysis, CD11b+ Kupffer cells were isolated from WT and Spred-2-/- mice and the cells were stimulated with LPS in vitro.
Results: LPS/D-GalN-induced ALI were exacerbated in Spred-2-/- mice compared with WT mice, as determined by increased serum levels of ALT, the numbers of apoptotic cells and infiltrating leukocytes and caspase 3/8 activity in the livers. U0126, a selective MEK/ERK inhibitor, ameliorated LPS/D-GalN-induced ALI in Spred-2-/- mice. TNF-α expression was augmented in Spred-2-/- mice relative to the WT mice, and immunohistochemical analysis showed that Kupffer cells were positive for TNF-α. Upon stimulation with LPS, isolated Kupffer cells from Spred-2-/- mice produced higher levels of TNF-α than those from WT mice, which was inhibited by U0126. Blocking TNF-α by neutralizing antibodies against TNF-α ameliorated LPS/D-GalN-induced ALI in Spred-2-/- mice. Finally, Spred-2-/- mice showed severer liver damage than WT mice, when mice were received TNF-α/D-GalN.
Conclusions: Spred-2 controls LPS/D-GalN-induced ALI through inhibiting TNF-α production by Kupffer cells. Thus, Spred-2 substitution can be a new therapeutic strategy for the treatment of ALI.