Objective To investigate the changes of extracellular histones during the course of acute liver failure in mice as well as its therapeutic potential.Methods WT mice (C57BL/6) were randomly (random number) allocated to inducing acute liver failure by lethal doses of GalN/LPS injected i.p.Hepatic function,apoptosis of hepatocytes and histological indexes were measured at different intervals following GalN/LPS challenge.The levels of extracellular histones were determined by using ELISA and Western blot methods.Meanwhile,GalN/LPS-treated mice were administered with anti-histone H3 and antihistone H4 neutralized antibodies,respectively.Results Administration of GalN/LPS to mice caused acute liver failure,characterized by significant elevation of plasma ALT levels and massive hepatocyte apoptosis or necrosis.All mice died within 9-12 hours.The levels of nucleosomes and extracellular histones H3 and H4 were increased considerably in a time-dependent manner.The survival rates in GalN/LPS-treated mice were improved remarkably following administration of anti-histone H3 and H4 neutralized antibodies (P =0.037,P =0.025),likely due to the significant inhibition of TNF-production.Conclusions Extracellular histones are an important mediator implicated in the pathogenesis of acute liver failure.Anti-histones show promising potential in the treatment of acute liver failure,which deserves further investigation in the future.

Objective To analyze the role of a key intracellular signaling molecule GSK3β in hepatocyte apoptosis induced by endoplasmic reticulum stress (ERS).Methods Using mouse hepatoma cell lines(Hepa 1) as cell apoptosis model triggered by tunicamycin,an endoplasmic reticulum stress inducer.One hour before Hepa 1 apoptosis induced by tunicamycin,SB216763 specifically inhibited the activity of GSK3β.Living cells/apoptotic cells were detected using acetoxymethyl (AM)/propidium iodide (PI) staining; Furthermore,the measurement of lactate dehydrogenase(LDH) of cell culture supernatant to evaluate the apoptosis.We detect p-GSK3β,GSK3β,the ERS-related protein(GRP78,CHOP and caspase-12) and caspase-3,cleaved caspase-3 protein expression using Western blot.Results Endoplasmic reticulum stress induced by tunicamycin promotes GSK3β activity; Inhibition of GSK3β activity alleviates endoplasmic reticulum stress:the expression of GRP78,CHOP and caspase-12 expression are inhibited.At the same time,GSK3β activity inhibition significantly reduced the endoplasmic reticulum stress-induced apoptosis:compared to cell apoptosis model group,the intervention group of SB216763 showed that the level of LDH decreased significantly,and PI staining of apoptotic cells was also significant reduction.Western blot results showed that the inhibition of GSK 3 β activity reduced reactive cleaved caspase-3 protein.Conclusion GSK3β is an important signaling molecule in the apoptosis pathway induced by endoplasmic reticulum stress ;Endoplasmic reticulum stress promotes hepatocyte apoptosis by mediating GSK3β.

OBJECTIVETo study the role of endoplasmic reticulum stress (ERS) in acute liver failure (ALF) using a mouse model of D-Galactosamine/lipopolysaccharide (D-GalN/LPS)-induced ALF.

METHODSThe ALF model was established by administering intraperitoneal (i.p.) injections of D-Ga1N (700 mg/kg) and LPS (10 mug/kg) to six C57BL/6 mice. Three of the modeled mice were also administered 4-phenylbutyrate (4-PBA; 100 mg/kg i.p.) at 6 hours before the onset of ALF and served as the intervention group. Non-modeled mice served as controls. All mice were analyzed by western blotting and qRT-PCR to determine the expression levels of ERS-related proteins in liver tissue. Liver function was assessed by measuring levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum. Extent of injury to the liver tissue was assessed by hematoxylin-eosin staining and histological analysis. qRT-PCR was also used to detect differences in expression of inflammation-related genes, and western blotting was also used to detect differences in expression of the apoptosis related protein Caspase-3.The extent of apoptosis in liver tissue was assessed by TUNEL assay.

RESULTSThe ERS markers GRP78 and GRP94 showed increased expression at both the gene and protein levels which followed progression of ALF. The ERS effector proteins XBP-1, ATF-6 and IRE 1 a involved in the unfolded protein response were activated in the early stages of ALF, and the ERS-induced apoptosis regulators Caspase-12 and CHOP were activated in the late stage of ALF. Inhibition of ERS by 4-PBA intervention protected against injury to liver tissue and function, as evidenced by significantly lower levels of serum ALT and AST and a remarkably decreased extent of histological alterations. Furthermore, the inhibition of ERS suppressed expression of the proinflammatory cytokines TNFa, IL-6 and IL-1 β, and reduced the extent of hepatocyte apoptosis.

CONCLUSIONERS is activated in the mouse model of D-GalN/LPS-induced ALF. Inhibition of ERS may be protective against liver injury and the mechanism of action may involve reductions in inflammatory and apoptotic factors and/or signaling. Therefore, inhibiting ERS may represent a novel therapeutic approach for treating ALF.

Animals ; Apoptosis ; Disease Models, Animal ; Endoplasmic Reticulum Stress ; Galactosamine ; adverse effects ; Lipopolysaccharides ; adverse effects ; Liver Failure, Acute ; chemically induced ; metabolism ; pathology ; Male ; Mice ; Mice, Inbred C57BL