Editor's Highlight: Farnesoid X Receptor Protects Against Low-Dose Carbon Tetrachloride-Induced Liver Injury Through the Taurocholate-JNK Pathway

Abstract

Hepatotoxicity is of major concern for humans exposed to industrial chemicals and drugs. Disruption of farnesoid X receptor (FXR), a master regulator of bile acid (BA) metabolism, enhanced the sensitivity to liver injury in mice after toxicant exposure, but the precise mechanism remains unclear. In this study, the interconnection between BA metabolism, FXR, and chemically induced hepatotoxicity was investigated using metabolomics, Fxr-null mice (Fxr-/-) and hepatocytes, and recombinant adenoviruses. A single low-dose intraperitoneal injection of carbon tetrachloride (CCl4), an inducer of acute hepatitis in mice, resulted in more severe hepatocyte damage and higher induction of pro-inflammatory mediators, such as chemokine (C-C motif) ligand 2 (Ccl2), in Fxr-/-. Serum metabolomics analysis revealed marked increases in circulating taurocholate (TCA) and tauro-β-muricholate (T-β-MCA) in these mice, and forced expression of bile salt export protein (BSEP) by recombinant adenovirus in Fxr-/- ameliorated CCl4-induced liver damage. Treatment of Fxr-null hepatocytes with TCA, but not T-β-MCA, significantly increased c-Jun-N-terminal kinase (JNK) activation and Ccl2 mRNA levels, and up-regulation of Ccl2 mRNA was attenuated by co-treatment with a JNK inhibitor SP600125, indicating that TCA directly amplifies hepatocyte inflammatory signaling mainly mediated by JNK under FXR-deficiency. Additionally, pretreatment with SP600125 or restoration of FXR expression in liver by use of recombinant adenovirus, attenuated CCl4-induced liver injury. Collectively, these results suggest that the TCA-JNK axis is likely associated with increased susceptibility to CCl4-induced acute liver injury in Fxr-/-, and provide clues to the mechanism by which FXR and its downstream gene targets, such as BSEP, protects against chemically induced hepatotoxicity.

Keywords: CCl4; bile acids; c-Jun-N-terminal kinase; farnesoid X receptor; taurocholate.

Figure 1

Fxr^−/− mice are more susceptible to CCl₄-induced liver injury. Fxr^−/− and WT mice were injected with CCl₄ or vehicle (corn oil) (0.25 ml/kg). Twenty-four hours after single CCl₄ injection, these samples were collected and measured. A, Serum AST and ALT levels. B, H&E staining of liver tissue. C, CYP2E1 protein level in liver. D, Expression of FXR and FXR-regulated genes and pro-inflammatory modulator mRNAs. n=5–8 mice per group. Data are presented as the means±SD and one-way ANOVA with Tukey’s correction was adopted for statistical analysis. **P <.01; ***P<.001

Figure 2

Identification of serum metabolites significantly altered in Fxr^−/− mice 24 h after CCl₄ injection. A, Principal component analysis (PCA) of serum metabolites using SIMCA software for metabolomics analysis. Metabolite profiles were different between WT and Fxr^−/− mice after CCl₄ injection. B, The identities of taurine-conjugated BAs having the highest confidence and greatest contribution to separation in S-plot between CCl₄-treated WT and Fxr^−/− mice. C, Bile acids levels in the serum. Data are presented as the means±SD and two-way ANOVA with Tukey’s correction was adopted for statistical analysis. ***P<.001 versus the other groups. α-MCA, α-muricholate; β-MCA, β-muricholate; CA, cholic acid; CDCA, chenodeoxycholic acid; DCA, deoxycholic acid; HDCA, hyodeoxycholic acid; UDCA, ursodeoxycholic acid; T-, taurine-conjugated; N, not detected.

Figure 3

Hepatocyte BSEP recovery by adenovirus decreases CCl₄-induced hepatotoxicity in Fxr^−/− mice. Adenovirus carrying the mouse Abcb11 cDNA (Ad-Abcb11) expressing BSEP or empty vector (Ad-Ctrl) was injected to Fxr^−/− mice (2.0×10⁹ pfu/mouse) 5 days before CCl₄ injection (0.25 ml/kg). A, Serum AST and ALT levels. B, Serum TCA and T-β-MCA concentrations. C, Hepatic mRNA levels of Abcb11, Fxr, and pro-inflammatory modulators. D, H&E staining of liver tissue. Data are presented as the means±SD and two-tailed Student’s t-test was used for statistical analysis. **P<.01; ***P<.001; ND, not detected.

Figure 4

TCA increases mRNA levels of pro-inflammatory modulators in primary hepatocytes isolated from Fxr^−/− mice. A, Primary hepatocytes isolated from Fxr^−/− and WT mice were treated with DCA (100 μM), TCA (100 μM), T-β-MCA (100 μM), or the same volume of vehicle (dimethyl sulfoxide) for 6 h. Levels of Ccl2, Pai1, and Tnf mRNAs were measured by qPCR. The mRNA levels were expressed as the relative values to those of vehicle-treated WT hepatocytes. B, Primary hepatocytes isolated from Fxr^−/− and WT mice were treated with different concentrations of TCA (10, 30, and 100 µM) or vehicle for 6 h. The mRNA levels were expressed as the relative values to those of vehicle-treated WT hepatocytes. *P<.05; **P<.01; ***P<.001.

Figure 5

JNK activation is important for up-regulation of pro-inflammatory modulators by TCA in Fxr-null hepatocytes. A, Primary hepatocytes isolated from Fxr^−/− and WT mice were treated with 100 μM TCA or vehicle (Veh) for 6 h. Whole cell lysates (20 μg of proteins) were subjected to immunoblot analysis for determining hepatic levels of total (T-) and phosphorylated (P-) JNK, ERK, and p65. The band of GAPDH was used as a loading control. B, Quantification of P-JNK. The band intensities of P-JNK and GAPDH were quantified using ImageJ software, and the ratio of P-JNK to GAPDH was calculated. Data are presented as the means±SD and two-way ANOVA with Tukey’s correction was adopted for statistical analysis. *P<.05; **P<.01. C, Primary hepatocytes isolated from Fxr^−/− and WT mice were treated with 100 µM TCA or vehicle (Veh) with or without 10 µM JNK inhibitor (SP600125) for 6 h. The mRNA levels of pro-inflammatory modulators were measured. n=3 samples per group. Data are presented as the means±SD and two-tailed Student's t-test was used for statistical analysis; ***P<.001.

Figure 6

Pretreatment with a JNK inhibitor attenuates CCl₄-induced hepatotoxicity in Fxr^−/− mice. Fxr^−/− mice were injected with SP600125 (30 mg/kg) or vehicle 30 min before CCl₄ injection (0.25 ml/kg). A, Serum AST, ALT, and TCA levels. B, Hepatic mRNA levels of pro-inflammatory modulators. C, H&E staining of liver tissue. Data are presented as the means±SD and two-tailed Student’s t-test was used for statistical analysis. n=6 mice per group. **P<.01; ***P<.001.

Figure 7

Forced expression of FXR ameliorates CCl₄-induced hepatotoxicity in Fxr^−/− mice. Adenovirus carrying mouse Fxr cDNA (Ad-Fxr) or empty vector (Ad-Ctrl) was injected to Fxr^−/− mice and wild-type (WT) mice (2.0×10⁹ pfu/mouse) 5 days before CCl₄ injection (0.25 ml/kg). A, Serum AST, ALT, and TCA levels. B, Hepatic mRNA levels of genes encoding FXR-related proteins and genes encoding proinflammatory modulators. C, Western blot analysis of JNK. Whole liver lysates (20 μg of proteins) were electrophoresed and transferred to PVDF membrane for determining hepatic levels of total (T-) and phosphorylated (P-) JNK. The band of GAPDH was used as a loading control. D, Quantification of P-JNK. The band intensities of P-JNK and GAPDH were quantified using ImageJ software, and the ratio of P-JNK to GAPDH was calculated. Data are presented as the means ± SD and one-way ANOVA followed by Tukey’s post hoc correction was applied for comparisons. *P < .05; ***P < .001 versus all other groups.

Figure 8

Proposed mechanism of higher susceptibility to CCl₄-induced liver toxicity in Fxr^−/− mice. In mice, hepatocyte FXR protects livers from low-dose CCl₄ toxicity due to maintaining BSEP function, limiting the increase in TCA, and inhibiting JNK activation. However, disruption of FXR in hepatocytes impairs BSEP function and increases TCA levels, even at low-dose CCl₄ exposure. Increased TCA activates JNK in FXR-disrupted hepatocytes, leading to induction of cell injury.