Activation of Pregnane X Receptor Sensitizes Mice to Hemorrhagic Shock-Induced Liver Injury

Abstract

Hemorrhagic shock (HS) is a life-threatening condition associated with tissue hypoperfusion and often leads to injury of multiple organs including the liver. Pregnane X receptor (PXR) is a species-specific xenobiotic receptor that regulates the expression of drug-metabolizing enzymes (DMEs) such as the cytochrome P450 (CYP) 3A. Many clinical drugs, including those often prescribed to trauma patients, are known to activate PXR and induce CYP3A. The goal of this study is to determine whether PXR plays a role in the regulation of DMEs in the setting of HS and whether activation of PXR is beneficial or detrimental to HS-induced hepatic injury. PXR transgenic, knockout, and humanized mice were subject to HS, and the liver injury was assessed histologically and biochemically. The expression and/or activity of PXR and CYP3A were manipulated genetically or pharmacologically in order to determine their effects on HS-induced liver injury. Our results showed that genetic or pharmacological activation of PXR sensitized wild-type and hPXR/CYP3A4 humanized mice to HS-induced hepatic injury, whereas knockout of PXR protected mice from HS-induced liver injury. Mechanistically, the sensitizing effect of PXR activation was accounted for by PXR-responsive induction of CYP3A and increased oxidative stress in the liver. The sensitizing effect of PXR was attenuated by ablation or pharmacological inhibition of CYP3A, treatment with the antioxidant N-acetylcysteine amide, or treatment with a PXR antagonist. Conclusion: We have uncovered a function of PXR in HS-induced hepatic injury. Our results suggest that the unavoidable use of PXR-activating drugs in trauma patients has the potential to exacerbate HS-induced hepatic injury, which can be mitigated by the coadministration of antioxidative agents, CYP3A inhibitors, or PXR antagonists.

Figure 1.. Genetic activation of PXR and Pxr ablation sensitize mice to and protect mice from HS-induced liver injury, respectively.

(A) WT, VP-PXR transgenic, and Pxr^−/− mice subject to sham surgery or HS/R were analyzed for liver injury by H&E staining. The “N”s within the dotted circle indicate the necrotic areas. Bar is 100 μm. (B-E) Mice are the same as in (A). Shown are quantification of necrotic areas (B), Suzuki scores of the liver damage (C), and serum levels of ALT (D), and hepatic expression of Cyp3a11 and Cyp2b10 (E). (F) Mice in (A) were analyzed for lung injury by H&E staining. n=4~5 for each group. *, p < 0.05; **, p < 0.01, compared to the WT (B and C), or the comparisons are labeled (D and E).

Figure 2.. Pharmacological activation of PXR sensitizes mice to HS-induced hepatic injury.

(A) Schematic representation of the PCN pre-treatment model. WT and Pxr^−/− mice were intraperitoneally injected with PCN (40 mg/kg per day) for two days before receiving the sham surgery or HS/R. (B and C) The hepatic mRNA (B) and protein (C) expression of Cyp3a11 and Cyp2b10 were measured by qRT-PCR and Western blotting, respectively. (D) The liver microsomal productions of 1’-OH midazolam (left) and noroxycodone (right) were measured by UPLC-MS analysis. (E) Liver histology was analyzed by H&E staining. Bar is 100 μm. Shown below are quantification of necrotic areas and Suzuki scores. (F) Serum levels of ALT. n=4~5 for each group. *, p < 0.05; **, p < 0.01, the comparisons are labeled (B, D and F), or compared to the WT+VEH group (E).

Figure 3.. Induction of CYP3A is required for the sensitizing effect of PXR activation on HS-induced hepatic injury.

(A) Schematic representation of the ketoconazole (KET) pre-treatment model. WT and VP-PXR transgenic mice were treated with KET (20 mg/kg per day) for two days before receiving the sham surgery or HS/R. (B and C) Liver histology was analyzed by H&E staining (B, Bar is 100 μm), and quantification of necrotic areas and Suzuki scores (C). (D) Serum levels of ALT. (E) VP-PXR and VP-PXR/Cyp3a^−/− mice subject to the sham surgery or HS/R were analyzed for liver histology by H&E staining. Shown on the right are quantification of necrotic areas and Suzuki scores. (F) Serum levels of ALT. n=4~5 for each group. *, p < 0.05; **, p < 0.01, the comparisons are labeled (C, D, and F), or compared to the VP-PXR group (E).

Figure 4.. Pharmacological activation of hPXR sensitizes the hPXR/hCYP3A4 humanized mice to HS-induced hepatic injury in a CYP3A-dependent manner.

(A) Schematic representation of the rifampicin (RIF) pre-treatment model. WT, hPXR/CYP3A4 and hPXR/Cyp3a^−/− mice were treated with RIF (10 mg/kg per day) for four days before receiving the sham surgery or HS/R. (B) The hepatic mRNA expression of human CYP3A4 and mouse Cyp3a11 was measured by qRT-PCR. (C) The hepatic expression of CYP3A4 was measured by immunostaining. CV, central vein; P, portal vein. (D) Liver histology was analyzed by H&E staining. Bar is 100 μm. Shown below are quantification of necrotic areas and Suzuki scores. (E) Serum levels of ALT. n=4~5 for each group. *, p < 0.05; **, p < 0.01, the comparisons are labeled.

Figure 5.. The sensitizing effect of PXR on HS-induced hepatic injury is accompanied by increased oxidative stress.

(A) Immunostaining of 4-HNE in liver tissues from WT, VP-PXR, WT+PCN, and Pxr^−/− mice subject to the sham surgery or HS/R. Arrowheads indicate positive staining. (B) The level of 4-HNE was measured by Western blotting. (C) Immunofluorescent staining of 8-OHdG in liver tissue from WT, VP-PXR and Pxr^−/− mice. (D) The hepatic expression of Atf3 and Chop was measured by qRT-PCR. (E and F) Immunofluorescent staining for 8-OHdG in liver tissues from WT, VP-PXR and VP-PXR/Cyp3a^−/− mice that were subject to HS/R (E), or VEH- or RIF-treated hPXR/CYP3A4 and hPXR/Cyp3a^−/− mice that were subject to HS/R (F). Bars are 100 μm. n=4~5 for each group. *, p < 0.05; **, p < 0.01, the comparisons are labeled.

Figure 6.. Treatment with the antioxidant NACA attenuates the sensitizing effect of PXR on HS-induced hepatic injury.

(A) Liver histology was analyzed by H&E staining in VEH- or NACA-treated VP-PXR mice that were subject to the sham surgery or HS/R. Bar is 100 μm. (B to D) Mice are the same as in (A). Shown are quantification of necrotic areas (B), Suzuki scores (C), and serum levels of ALT (D) in mice subject to HS/R. (E) Mice are the same as in (A). The level of 4-HNE was measured by immunostaining with the positive staining indicated by arrowheads. Bar is 100 μm. n=4~5 for each group. *, p < 0.05; **, p < 0.01, compared to the VEH group (B and C), or the comparisons are labeled (D).

Figure 7.. Post-hemorrhagic shock activation of PXR sensitizes mice to HS-induced hepatic injury.

(A) Schematic representation of the model of post-HS treatment of PCN or DEX. WT mice were subject to a 2-h HS and intraperitoneally injected with PCN (40 mg/kg) or DEX (20 mg/kg) before the resuscitation, and the mice were sacrificed 22 h after. (B) The hepatic mRNA expression of Cyp3a11 was measured by qRT-PCR. (C) Liver histology was analyzed by H&E staining. Shown on the right are quantification of necrotic areas and Suzuki scores. (D) Serum levels of ALT. (E) Immunofluorescent staining for 8-OHdG. Bar is 100 μm. n=4~5 for each group. *, p < 0.05; **, p < 0.01, the comparisons are labeled (B and D), or compared to the VEH group (C).

Figure 8.. Treatment with the hPXR antagonist SPA70 protects the hPXR/hCYP3A4 humanized mice from HS-induced hepatic injury.

(A) Schematic representation of the SPA70 pre-treatment model. Mice were intraperitoneally injected with VEH, RIF (10 mg/kg) and SPA70 (150 mg/kg) individually or in combination for 3 days before receiving the sham surgery or HS/R. (B) The hepatic mRNA expression of CYP3A4 was measured by qRT-PCR. (C) Liver histology was analyzed by H&E staining. Shown on the right are quantification of necrotic areas and Suzuki scores. (D) Serum levels of ALT. (E and F) The hepatic levels of 4-HNE (arrowheads indicated the positive staining) (E) and 8-OHdG (F) were measured by immunostaining and immunofluorescence, respectively. (G) The hepatic mRNA expression of Atf3 and Chop was measured by qRT-PCR. Bars are 100 μm. n=4~5 for each group. *, p < 0.05; **, p < 0.01, the comparisons are labeled.