Inhibition of glycogen synthase kinase 3β ameliorates D-GalN/LPS-induced liver injury by reducing endoplasmic reticulum stress-triggered apoptosis.

Glycogen synthase kinase 3β(GSK3β) is a ubiquitous serine-threonine protein kinase that participates in numerous cellular processes and disease pathophysiology. We aimed to determine therapeutic potential of GSK3β inhibition and its mechanism in a well-characterized model of lipopolysaccharide (LPS)-induced model of acute liver failure (ALF).In a murine ALF model induced by D-GalN(700 mg/kg)/LPS(10 µg/kg), we analyzed GSK3β mechanisms using a specific chemical inhibitor, SB216763, and detected the role of endoplasmic reticulum stress (ERS). Mice were administered SB216763 at 2 h before or after D-GalN/LPS injection, respectively, and then sacrificed 6 h after D-GalN/LPS treatment to evaluate its prophylactic and therapeutic function. The lethality rate, liver damage, ERS, cytokine expression, MAP kinase, hepatocyte apoptosis and expression of TLR 4 were evaluated, respectively. Whether the inhibition of GSK3β activation protected hepatocyte from ERS-induced apoptosis was investigated in vitro.GSK3β became quickly activated (dephosphorylated) upon D-GalN/LPS exposure. Administration of SB216763 not only ameliorated liver injury, as evidenced by reduced transaminase levels, and well-preserved liver architecture, but also decreased lethality. Moreover, GSK3β inhibition resulted in down-regulation of pro-apoptotic proteins C/EBP-homologous protein(CHOP) and caspase-12, which are related to ERS. To further demonstrate the role of ERS, we found that GSK3β inhibition protected hepatocyte from ERS-induced cell death. GSK3β inhibition down-regulated the MAPK pathways, reduced expression of inflammatory cytokines and decreased expression of TLR4.Our findings demonstrate the key function of GSK3β signaling in the pathophysiology of ALF, especially in regulating the ERS, and provide a rationale for targeting GSK3β as a potential therapeutic strategy to ameliorate ALF

The Inhibition of GSK3β prevents ERS-induced hepatocyte apoptosis <i>in vitro</i> and <i>in vivo</i>.

<p>(a) LDH activities assay in the hepatocyte culture supernatant. SB216763 was added 2 h before tunicamycin(10 µg/ml, Sigma) which is added into the 24 wells culture plate for 24 hours(n = 3 wells). (b) The hepatocyte proteins expression of caspas-3, cleaved caspase-3 and β-actin were measured by Western blots. SB216763 was added 2 h before tunicamycin(10 µg/ml, Sigma) which was added into the 60 cm culture well for 12 hours, total lysates were subjected to immunoblot assay, and densitometry analysis(mean±SEM) was performed (n = 3). (c) The liver proteins expression of ERS (GRP78, CHOP, caspase-12) were measured by Western blots. Liver samples were harvested from C57BL/6 mice at 6 h after D-GalN/LPS injection that were pretreated with only PBS (Control, n = 5–6), and vehicle (DMSO, n = 5–6/group), or SB216763 (50 mg/kg, n = 5–6/group) before D-GalN/LPS injection. Representative of two experiments is shown. Densitometry analysis of the proteins was performed for each sample (mean±SEM).</p

Active GSK3β promotes TLR4 expression in D-GalN/LPS-induced ALF.

<p>(a) Liver samples were harvested from C57BL/6 mice that were subjected to D-GalN/LPS (Control, 1 h, 3 h and 6 h, n = 5–6/group), the gene expression levels of TLR4 after D-GalN/LPS injection were tested by qRT-PCR. (b) Liver samples were harvested from C57BL/6 mice at 6 h after D-GalN/LPS injection that were pretreated with only PBS (Control, n = 5–6), and vehicle (DMSO, n = 5–6/group), or SB216763 (50 mg/kg, n = 5–6/group) before D-GalN/LPS injection. Hepatic gene and proteins expression of TLR4 were measured by Western blots and qRT-PCR, respectively. For western blot analysis, representative of two experiments is shown. Densitometry analysis of the proteins was performed for each sample (mean±SEM).</p

Liver protection against acute liver failure following GSK3β inhibition is dependent of regulating inflammatory reaction and modulating the MAP kinases signaling.

<p>(a) The gene expression levels of inflammatory IL-12p40, IL-10 at 2 hour after D-GalN/LPS injection, and TNF-α, IL-1β and IL-6 at 6 h after D-GalN/LPS injection(n = 5–6/group). (b) Liver MPO levels at 6 h after D-GalN/LPS injection (n = 5–6/group). (c) The phosphorylated MAP kinases including JNK, ERK and p38, and β-actin were measured by Western blots. Liver samples were harvested from C57BL/6 mice that were subjected to PBS (Control, n = 5–6) or D-GalN/LPS, followed by various times (1, 3, and 6 h, n = 5–6/group). Representative of two experiments is shown. Densitometry analysis of the proteins was performed for each sample (mean±SEM). (d) The MAP kinases are regulated by GSK3β inhibition in SB216763-pretreated acute liver failure in mice. Groups of C57BL/6 mice were treated with vehicle (DMSO, n = 5–6/group), or SB216763 (50 mg/kg, n = 5–6/group), followed by 6 h D-GalN/LPS injection. Representative of two experiments is shown. Densitometry analysis of the proteins was performed for each sample (mean±SEM).</p

The GSK3β inhibition by SB216763 ameliorates liver injury induced by D-GalN/LPS in C57BL/6 mice.

<p>(a) Liver samples, harvested 6 h later, were subjected to Western blot analysis of phosphorylated glycogen synthases. Wild-type mice were pretreated with vehicle (DMSO, n = 3) or SB216763 (10, 25, 50 mg/kg, respectively, n = 3). Representative of one experiment is shown. Densitometry analysis of the proteins was performed for each sample (mean±SEM). (b)GSK3β inhibition enhances the survival rate of mice after D-GalN/LPS injection. SB216763 (10, 25, or 50 mg/kg body weight) or vehicle (DMSO) was intraperitoneally administered at 2 h before D-GalN/LPS injection (DMSO+D-GalN/LPS, SB216763 10 mg/kg+D-GalN/LPS, SB216763 25 mg/kg+D-GalN/LPS, SB216763 50 mg/kg+D-GalN/LPS); and the fifth group is administered SB216763 at 2 hour after D-GalN/LPS(D-GalN/LPS+SB216763 50 mg/kg). (n = 10/group). (c) Wild-type mice (n = 8–12) pretreated SB216763 before D-GalN/LPS injection were analyzed for serum ALT and AST level at 6 h after D-GalN/LPS. (d)Wild-type mice (n = 8–12) treated SB216763 after D-GalN/LPS injection were analyzed for serum ALT and AST level at 6 h after D-GalN/LPS. (e) Representative liver histology (H/E staining at 6 h after D-GalN/LPS) and the group averages of liver Suzuki scores (6 h). Control: DMSO groups, DMSO+D-GalN/LPS: model group, SB+D-GalN/LPS: prophylactic group and D-GalN/LPS+SB: therapeutic group. (n = 5–6/group).</p

The acute liver failure induced by D-GalN/LPS triggers GSK3β phosphorylation and ERS.

<p>Liver samples were harvested from C57BL/6 mice that were subjected to PBS (Control, n = 5–6) or D-GalN/LPS, followed by various times (1, 3, and 6 h, n = 5–6). Representative of two experiments is shown. Densitometry analysis of the proteins was performed for each sample (mean±SEM). (a)The phosphorylated (serine 9) and total GSK3β levels were measured by Western blots. (b)The ERS-related protein including GRP78, CHOP, Caspase-12, and β-actin were measured by Western blots.</p

The Inhibition of GSK3β prevents hepatocyte apoptosis in D-GalN/LPS-induced acute liver failure.

<p>Liver samples were harvested from C57BL/6 mice at 6 h after D-GalN/LPS injection that were pretreated with only PBS (Control, n = 5–6), and vehicle (DMSO, n = 5–6/group), or SB216763 (50 mg/kg, n = 5–6/group) before D-GalN/LPS injection. (a)Caspase-3 activity assay at 6 h after D-GalN/LPS injection, controls were defined as 1.0. (b)Immunofluorescence staining for active caspase-3 (green) in the liver of mice. Representative of one experiment is shown. Original magnification×20. (c)Hepatic proteins expression of caspas-3, cleaved caspase-3 and β-actin were measured by Western blots. Representative of two experiments is shown. Densitometry analysis of the proteins was performed for each sample (mean±SEM). (d) TUNEL staining (red) liver tissue at 6 h after D-GalN/LPS administration. Representative of one experiment is shown. Original magnification×20.</p