The protective role of pregnane X receptor in lipopolysaccharide/D-galactosamine-induced acute liver injury

Abstract

The pregnane X receptor (PXR) is a nuclear receptor transcription factor regulating drug-metabolizing enzymes and transporters that facilitate xenobiotic and endobiotic detoxification. Recent studies show that PXR is important in abrogating intestinal tissue damage. This study examines the role of PXR in lipopolysaccharide (LPS)/D-galactosamine (GalN)-induced acute liver injury using wild-type and PXR-null mice. LPS/GalN-treated PXR-null mice had greater increases of alanine transaminase (ALT), hepatocyte apoptosis, necrosis, and hemorrhagic liver injury than wild-type mice. LPS/GalN-mediated phosphorylation of JNK1/2 and ERK1/2 was differentially regulated in wild-type and PXR-null mice. Importantly, LPS/GalN-induced hepatic Stat3 survival signaling was impaired and early activation of Jak2 was delayed in PXR-null mice. Expression levels of pro-survival proteins Bcl-xL and heme oxygenase-1 (HO-1), which are downstream of Stat3, were substantially lower in PXR-null than wild-type mouse livers after LPS/GalN treatment. Autophagy is also involved in LPS/GalN-induced liver injury. Lack of PXR resulted in a significant reduction of LC3B-I, -II as well as Beclin-1 protein levels after LPS/GalN treatment. In addition, PXR is implicated in hepatocytes homeostasis. Taken together, PXR is a critical hepatoprotective factor. Increases of LPS/GalN-induced hepatocyte apoptosis and liver injury in PXR-null mice are due to deregulated mitogen-activated protein (MAP) kinase activation as well as delayed Jak2/Stat3 activation, which lead to a compromise in defense mechanisms that involve Bcl-xL-, HO-1, and autophagy-mediated pathways.

Figure 1

PXR-null mice are sensitive to LPS/GalN-induced liver injury. Mice were i.p. injected with LPS (100 μg/kg) and GalN (700 mg/kg) or saline. (A) ALT levels. Data represent means ± SD. (n=5/group/time point). (B) Representative H&E-stained liver sections of wild type and PXR-null mice 5 h post LPS/GalN injection. Sporadically, apoptotic hepatocytes (triangle) and neutrophil infiltrations (arrow) were observed in liver sections of saline-treated PXR-null mice. * P < 0.05, compared with wild type mice of 0 h; # P < 0.05, compared within same time point.

Figure 2

Phosphorylation of hepatic MAP kinases is differentially altered by LPS/GalN in wild type and PXR-null mice. Mice were i.p. injected with LPS (100 μg/kg) and GalN (700 mg/kg) or saline. (n=5/group/time point). Livers were removed at the time point as indicated. Liver proteins were immunoblotted with indicated antibodies and representative samples are shown in the figure.

Figure 3

Activation of Stat3 by LPS/GalN is delayed in the livers of PXR-null mice. Mice were i.p. injected with LPS (100 μg/kg) and GalN (700 mg/kg) or saline. (n=5/group/time point). Livers were removed at the time point as indicated. Liver proteins were immunoblotted with indicated antibodies and representative samples are shown in the figure.

Figure 4

Loss of PXR impairs LPS/GalN-induced activation of Jak2 in the livers of PXR-null mice. Mice were i.p. injected with LPS (100 μg/kg) and GalN (700 mg/kg) or saline. (n=5/group/time point). Livers were removed at the time point as indicated. Hepatic cytosal fractions were immunoblotted with indicated antibodies. Representative samples are shown in the figure.

Figure 5

LPS/GalN-induced activation of p65 is augmented in the livers of PXR-null mice. Mice were i.p. injected with LPS (100 μg/kg) and GalN (700 mg/kg) or saline. (n=5/group/time point). Livers were removed at the time point as indicated. Liver proteins were immunoblotted with indicated antibody and representative samples are shown in the figure.

Figure 6

LPS/GalN-induced hepatocyte apoptosis is augmented in PXR-null mice. Mice were i.p. injected with LPS (100 μg/kg) and GalN (700 mg/kg) or saline. (n=5/group/time point). (A) Representative TUNEL staining of liver sections of wild type and PXR-null mice i.p. injected with LPS/GalN or saline for 5 h. (B) Quantification of TUNEL-positive hepatocytes of liver sections of wild type or PXR-null mice 5 h post LPS/GalN injection. * P < 0.05, compared with wild type mice of 0 h; # P < 0.05, compared within same time point. (C) Immunoblot analysis of cleaved caspase-3. Representative samples are shown in the figure.

Figure 7

Loss of PXR decreases the levels of anti-apoptotic proteins and alters the expression of proteins involved in autophagy pathways after LPS/GalN injection. Mice were i.p. injected with LPS (100 μg/kg) and GalN (700 mg/kg) or saline. (n=5/group/time point). Livers were removed at the time point as indicated. Liver proteins were immunoblotted with indicated antibodies. Representative samples are shown in the figure.