The GSK3β Activation/inactivation Profile and ERS in GalN/LPS-induced Acute Liver Failure
To dissect the role of GSK3β in the pathogenesis of liver failure, we first determined in vivo whether the GSK3β phosphorylation/dephosphorylation is triggered in acute liver injury. Liver tissues were harvested in 1, 3, and 6 h, respectively, after D-GalN/LPS injection. Compared with those in the negative control, the phosphorylated (serine 9) GSK3β level in the liver tissues was promptly reduced in 1 h and 3 h, and then was restored after 6 h, suggesting dephosphorylation of GSK3β is an early event in the acute phase (). As total GSK3β levels were unchanged among the different time points, our result indicates that the liver GSK3β activity, rather than its protein, was increased during early phase of liver failure induced by D-GalN/LPS, but declined to the basal state thereafter. On the other hand, phosphorylation at tyrosine-216 on GSK3β and the phosphorylation of GSK3α were not detected in the acute liver failure (data not shown). Thus, the GSK3β activation, as measured by dephosphoryaltion at serine-9, is triggered in the early phase of the D-GalN/LPS induced liver failure.
The acute liver failure induced by D-GalN/LPS triggers GSK3β phosphorylation and ERS.
To evaluate the role of ERS in acute liver failure induced by D-GalN/LPS, expression of GRP78, CHOP, and caspase-12 in the liver of D-GalN/LPS-treated mice was determined by Western blot analysis. The expression of GRP78, CHOP, caspase-12, was low in the control liver, but all increased significantly from 3 to 6 h after D-GalN/LPS administration (). The increased expression of GRP78, CHOP and caspase-12 suggested that endoplasmic reticulum stress took place in a delayed fashion in the D-GalN/LPS-induced acute liver failure. These results suggest that ERS may play an important role in D-GalN/LPS-induced acute liver failure.
Active GSK3β is Critical for the Development of ALF Induced by D-GalN/LPS
To address the functional significance of GSK3β in liver injury, we treated mice with a GSK3β specific chemical inhibitor, SB216763, 2 hours prior to or after the onset of liver failure. The inhibition of liver GSK3 activity in vivo was confirmed by reduced phosphorylation of glycogen synthase, a downstream substrate of GSK3β ().
The GSK3β inhibition by SB216763 ameliorates liver injury induced by D-GalN/LPS in C57BL/6 mice.
For mortality analysis, four treatment groups, all challenged with D-GalN/LPS, were examined. The mice in group I (control) received only vehicle, and the mice in groups II to IV were pretreated with three doses of SB216763 before D-GalN/LPS challenge; and the mice in group V were administered SB216763 in 2 hours after onset of ALF by D-GalN/LPS. The survived mice of all groups were sacrificed at 48 h. In the D-GalN/LPS control group, the mice began to die 6 h after D-GalN/LPS injection, and all mice were dead in 20 h after D-GalN/LPS injection (mortality 100%). However, pretreatment with SB216763 reduced the lethality in a dose-dependent manner. Briefly, in 48 h after D-GalN/LPS administration, the lethality rates were 80%, 60%, and 20% (survival of 2, 4, and 8 of 10 mice) in the mice pretreated with 10, 25, and 50 mg/kg SB216763, respectively; For the therapeutic treatment of SB216763(50 mg/kg), the lethality rate was 40% (survival of 6 of 10 mice), as showed in . SB216763 (50 mg/kg) showed significantly protection from liver failure. Therefore, we selected 50 mg/kg of SB216763 for the following study.
To address the effect of inhibiting activity of GSK3β on hepatic injury, we investigated whether SB216763 regulates serum AST and ALT levels in D-GalN/LPS-induced liver failure. After 6 h of D-GalN/LPS-induced liver failure, sALT and sAST levels reached 2195.3±450.5 and 3705.3±632.8 IU/L, which were significantly higher than those in single SB216763 treated ones (961.1±356 and 2709.9±423.9), while vehicle-treated normal mice levels were 28.7±7 and 33.7±9 IU/L, respectively ( ). For therapeutic function of SB216763, as compared with D-GalN/LPS-treated positive groups, livers in animals receiving SB216763 also suffered less acute liver failure injury, evidenced by distinct lower sALT levels (, sALT:1491.2±622.9 versus 2787.52±1066.89; P<0.05), but the sAST level had no statistics difference (2274.5±651 versus 2538.13±588.9; P>0.05). Liver histology was normal in the vehicle-treated normal mice, while hepatic architecture in 6 h after D-GalN/LPS administration was disrupted with the appearance of extensive areas of hemorrhage and coagulative necrosis. However, little hemorrhage and no evidence of necrosis were observed in livers from SB216763 treated mice at this time, better preserved liver architecture by histology and Suzuki grading (, .1±0.2, 3.5±0.6 and 3.6±0.8, P<0.05). Taken together, these results demonstrate not only the prophylactic, but also the therapeutic application of SB216763, being used an agent to protect the liver from the D-GalN/LPS-induced ALF in the mice.
Active GSK3β promotes TLR4 expression in D-GalN/LPS-induced ALF.
Active GSK3β Facilitates Cytokine Programs, Neutrophil Infiltration and Regulates the MAPK Pathways
LPS can trigger inflammatory cascades involving the induction of pro-inflammatory cytokines including TNF-α, IL-1β and IL-6, which are essential for inflammation and consequent liver damage in D-GalN/LPS-treated mice. In order to determine the impact of GSK3β inhibition on the induction of these cytokines by ALF, the livers were harvested at 4 h after D-GalN/LPS injection. Indeed, inhibition of GSK3β attenuated the expression of pro-inflammatory cytokines including TNF-α, IL-1β, and IL-6, and conversely increased immune regulatory cytokines such as IL-12 and IL-10 in the acute phase (). These results also suggest a positive role of GSK3β in regulation of pro-inflammatory responses. To investigate D-GalN/LPS-induced neutrophil infiltration, we assayed MPO activities. As shown in , treatment with SB216763 significantly decreased the injury related MPO activities (3.94±0.58), as compared with the D-GalN/LPS control (6.425±1.24, p<0.05). Meanwhile, we analyzed to which extent macrophages are involved in the inflammatory response of the D-GalN/LPS exposed livers by immunofluorescence staining to detect the PE anti-mouse F4/80. The results showed that there are no difference among all groups (data not shown). Thus, inhibition of GSK3β activity suppressed expression of pro-inflammatory cytokine, and reduced neutrophil activation in D-GalN/LPS induced liver failure.
Liver protection against acute liver failure following GSK3β inhibition is dependent of regulating inflammatory reaction and modulating the MAP kinases signaling.
We then assessed the downstream events of the pro-inflammatory cytokine receptors, namely activation of MAP kinases including ERK, JNK, and p38. As shown in , these three MAP kinases in livers showed different pattern of activation dynamics. Phosphorylation of ERK and p38 was significantly increased in 1 h and maintained at a significantly high level for 3 h after D-GalN/LPS injection, while phosphorylation of JNK proteins was apparently in oscillation with a prompt increase after 1 h, followed by a down turn, and another momentum at 6 h. These results suggest that the MAP kinases which involved in diverse functions were differently regulated in D-GalN/LPS induced ALF. Next, we examined whether the MAPK pathways were involved in the protective effect in D-GalN/LPS–induced ALF by inhibition of GSK3β. As shown in , compared with the control, the pretreatment with SB216763 decreased the levels of phospho-ERK and phospho-p38 MAPK in 3 h and the level of phospho-JNK in 6 h after D-GalN/LPS treatment. These results indicated that the GSK3β may act upstream or modulate the inflammation related three MAPK pathways.
Active GSK3β Promotes TLR4 Expression in Liver
Toll-like receptors (TLRs) regulate systemic acquired immunity and local infammatory responses, the studies have revealed that TLRs involve in the initiation, progression and recovery of various liver diseases. At here, our results showed that the expression of TLR4 mRNA evidently increased at the 3 hours and decreased at 6 hours after D-GalN/LPS stimulation (). The inhibition of GSK3β decreased the expression of TLR4 mRNA and TLR4 proteins at 3 hours detected by qRT-PCR and western blot analysis, respectively(showed as ). So, the active GSK3β signaling promotes the TLR4 status in D-GalN/LPS-induced ALF.
GSK3β Signaling Promotes Hepatocyte Apoptosis during ALF
As D-GalN/LPS-induced liver injury is characterized by apoptosis of hepatocytes, the expression and activity of pro-apoptotic molecules were examined. In animal model, capase-3 activity, active caspase-3 and its maturation/process of the proenzyme was markedly increased in D-GalN/LPS induced liver failure (). Treatment with SB216763 significantly inhibited the caspase-3 activity, active caspase-3 and the cleavage of procaspase-3. Similar results were also observed in liver tissue samples by applying TUNEL assay (). Therefore, GSK3β inhibition was capable of inhibiting hepatocyte apoptosis in hepatic failure induced by D-GalN/LPS.
The Inhibition of GSK3β prevents hepatocyte apoptosis in D-GalN/LPS-induced acute liver failure.
Inhibition of GSK3β Prevents ERS-induced Hepatocyte Apoptosis
As showed above, ERS play an important role in the mechanism of D-GalN/LPS-induced ALF, so we investigated the role of GSK3β in the intrinsic potential of hepatocyte death triggered by ERS pathway. First, we determined the impact of GSK3β inhibition on ERS-induced mouse Hepa 1 cell death in vitro. GSK3β inhibition did protect hepatocytes from tunicamycin-induced ERS and cell death, as measured by the LDH assay and Western blotting for cleaved caspase-3. There was significant decrease in LDH activities in the hepatocyte culture supernatant by the cells pretreated with SB216763, as compared with controls, incubated with tunicamycin (). Western blot analysis showed that SB316763 inhibited the expression of cleaved caspase-3 compared with controls (). By in vivo animal model, Western blot analysis revealed that treatment with SB216763 inhibited the expression of CHOP and caspase-12 compared with controls in 6 hours (). Therefore, GSK3β inhibition was capable of inhibiting hepatocyte death against ER stress.
The Inhibition of GSK3β prevents ERS-induced hepatocyte apoptosis in vitro and in vivo.