2B). Histologically, WT livers showed intense inflammation, massive cell death, and red blood

cell sequestration in sinusoidal spaces, with only a few cells spared periportally (Fig. 2B). KO livers showed mostly healthy hepatocytes and intact liver with only occasional sinusoidal dilation (Fig. 2B). Serum biochemistry revealed a 40-fold increase in serum alanine aminotransferase (ALT) and a 20-fold increase in serum aspartate aminotransferase (AST) in WT livers compared with KO livers (Fig. 2C). Assaying the livers of both genotypes for apoptosis revealed that GalN/LPS-treated KO livers had dramatically fewer hepatocytes with terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive nuclei compared with WT livers (Fig. 2D). Finally, WT and KO livers at 6 hours were assessed for the presence of activated caspases by western blotting (WB) (Fig. 2E) and fluorometric measurement

Tanespimycin of caspase-3 activity (Fig. 2F), which showed WT livers to have significantly greater apoptosis compared with KO livers. To characterize the initiation and progression of liver injury in WT and KO mice after GalN/LPS, livers and plasma were obtained 3, 4, and 5 hours after treatment. TUNEL assay showed few apoptotic cells in either WT or KO livers at 3 hours, whereas at 4 hours KO animals displayed more TUNEL-positive nuclei than WT livers. However, at 5 hours, TUNEL positivity in KO mice had not progressed and may have improved, whereas extensive apoptosis was evident in WT mice (Fig. 3A), which was also confirmed by hematoxylin

Lorlatinib solubility dmso and eosin (H&E) staining (Fig. 3B). Consistent with TUNEL, serum AST was low and comparable at 3 hours, greater in KO livers at 4 hours, and markedly higher in WT livers at 5 hours (Fig. 3C). These observations suggest comparable initiation of liver damage in KO and WT mice after GalN/LPS, and although the damage progresses in WT, it is self-limited in KO mice. To determine the mechanism cAMP of protection in KOs, we examined the expression of NF-κB, a known antiapoptotic mediator of TNF-α injury. Six hours after GalN/LPS treatment, there was a clear increase in total p65 levels in KOs, as analyzed via WB (Fig. 4A). Similarly, we detected the presence of total and transcriptionally active Ser-536-phosphorylated p65 (phospho-p65) protein in hepatocyte nuclei of KO but not WT livers.20 An increase in glycogen synthase kinase (GSK-3β), a known NF-κB activator,21 was also observed in KO livers at 6 hours, suggesting a possible mechanism of p65 phosphorylation (Fig. 4A). Extensive cytoplasmic and nuclear p65 in KO livers was verified via immunohistochemistry (IHC) at 5 hours (Fig. 4B). NF-κB activation in KO livers at 6 hours after GalN/LPS administration was further substantiated by the increase of NF-κB downstream targets Traf-1 and Fas, as well as Stat3, which is a downstream effector of the NF-κB target gene interleukin-6 (Fig. 4C).