Dexamethasone counteracts hepatic inflammation and oxidative stress in cholestatic rats via CAR activation

Abstract

Glucocorticoids (GCs) are currently used for the therapeutic management of cholestatic diseases, but their use and molecular mechanism remain controversial. The aims of this study were 1) to assess the therapeutic effect of a 2-week treatment with the GC dexamethasone on hepatic damage in bile duct-ligated rats; 2) to investigate its effect on the activation of the nuclear receptors (NRs) pregnane X receptor (PXR), constitutive androstane receptor (CAR) and GC receptor (GR), and NF-kB, as well as on oxidative stress and bile acid (BA) hepatic composition. Cholestasis was induced by ligation of bile duct (BDL animals) in 16 male Wistar-Kyoto rats, and eight of them were daily treated by oral gavage with 0.125 mg/ml/kg DEX for 14 days. Eight Sham-operated rats were used as controls. Severity of cholestasis was assessed histologically and on plasma biochemical parameters. The nuclear expression of NF-kB (p65), GR, PXR and CAR was measured in hepatic tissue by Western Blot. Oxidative stress was evaluated by measuring malondialdehyde, carbonylated proteins, GHS and ROS content in rat livers. LC-MS was used to measure the plasma and liver concentration of 7 BAs. Histological findings and a significant drop in several markers of inflammation (p65 nuclear translocation, mRNA expressions of TNF-α, IL-1β, IL-6) showed that DEX treatment reversed cholestasis-induced inflammation, and similar results have been obtained with oxidative stress markers. The nuclear expression of p65 and CAR were inversely correlated, with the latter increasing significantly after DEX treatment (p<0.01 vs vehicle). Hepatic BA levels tended to drop in the untreated cholestatic rats, whereas they were similar to those of healthy rats in DEX-treated animals. Plasma BAs decreased significantly in DEX-treated animals with respect to untreated cholestatic rats. In conclusion, DEX reduces inflammation and oxidative stress in BDL rats, and probably CAR is responsible for this effect. Therefore, this NR represents a promising pharmacological target for managing cholestatic and inflammatory liver diseases.

Fig 1. Liver histology of cholestatic rats.

Representative photomicrographs of rat liver tissue from cholestatic untreated rats on H&E staining (A) ductular reaction is highlighetd by CK7 immunostain (B) and is associated with collagen deposition as shown by Van Gieson stain (C).

Fig 2. Liver histology of healthy and DEX-treated rats.

Representative photomicrographs of rat liver tissue from healthy (A) and BDL rats treated with DEX (B) stained with H&E.

Fig 3. Activation of hepatic stellate cells.

Representative photomicrographs of rat liver tissue from untreated (A) and DEX-treated (B) BDL rats stained with ASMA, a marker of HSCs activation.

Fig 4. NR activation in rat livers.

Densitometric analysis of the immunoreactive bands of: (A) GR nuclear protein expression; (B) PXR nuclear protein expression; and (C) CAR nuclear protein expression. A representative Western blot experiment showing NR nuclear protein expression is shown on the right of each graph. HDAC was used as loading control. Data are expressed as mean ± SEM. ANOVA followed by the Neuman-Keuls post-hoc test. *P < 0.05, **P < 0.01 vs healthy rats; #P < 0.05 vs cholestatic rats treated with vehicle.

Fig 5. Immunofluorescence staining of CAR in HepG2 cells.

Immunostaining for the CAR after incubation in basal condition (control), with 10 μg/ml LPS for 24h, 0.2 μM DEX + 10 μg/ml LPS for 24h and 2 μM DEX + 10 μg/ml LPS for 24h. Values are means ± S.E.M. (n = 10). The histograms report the intensity of fluorescence (arbitrary units). Representative images from five independent experiments are shown. Cell nuclei are stained blue with DAPI. ANOVA followed by the Dunnett post-hoc test. *P < 0.05; ***P < 0.001 vs control.

Fig 6

A: p65 nuclear protein expression in rat livers. A representative Western blot experiment showing p65 nuclear protein expression is shown on the right of the graph. Data are expressed as mean ± SEM. B: IκBαcytosolic protein expression in rat livers. A representative Western blot experiment showing IκBαprotein expression in the cytosolic fraction is shown on the right of the graph. Data are expressed as mean ± SEM. ANOVA followed by the Neuman-Keuls post-hoc test. *P<0.05, **P < 0.01 vs healthy rats; ^# P<0.05, ^##P < 0.01 vs cholestatic rats treated with vehicle.

Fig 7. Inflammatory cytokines in rat livers.

mRNA levels of (A) TNFα, (B) IL-6, and (C) IL-1β. Data are presented as means ± SEM. ANOVA followed by the Neuman-Keuls post-hoc test. *P<0.05, **P < 0.01, ***P < 0.01 vs healthy rats; ^###P < 0.001 vs cholestatic rats treated with vehicle.

Fig 8. Oxidative stress in rat livers.

A: malondialdehyde (MDA) levels; B: carbonylated proteins levels; C: reduced glutathione (GSH) content. D: ROS content. Data are presented as means ± SEM. ANOVA followed by the Neuman-Keuls post-hoc test. *P<0.05, **P < 0.01 vs healthy rats; #P < 0.05 vs cholestatic rats treated with vehicle.

Fig 9. Hepatic BA levels.

CA: cholic acid, DCA: deoxycholic acid, TCA: taurocholic acid, TCDCA: taurochenodeoxycholic acid, TDCA: taurodeoxycholic acid, GCA: glycocholic acid, GDCA: glycodeoxycholic acid. Data are means ± SEM. ANOVA followed by the Neuman-Keuls post-hoc test. ** P<0.01 vs healthy rats; ^# P<0.05 vs vehicle.

Fig 10. Plasma BA levels.

CA: cholic acid, DCA: deoxycholic acid, GCA: glycocholic acid. Data are means ± SEM. ANOVA followed by the Neuman-Keuls post-hoc test. ** P<0.01, ***P<0.001 vs healthy rats; ^# P<0.05, ^### P<0.001 vs vehicle.

Fig 11. CYP3A gene and protein expression in rat livers.

A: CYP3A1 gene expression. B: CYP3A2 gene expression. C: CYP3A1 protein expression in microsomal fraction. D: CYP3A2 protein expression in microsomal fraction. Western blot experiment showing NR nuclear protein expression is shown on the right of C and D graphs. Calnexin was used as loading control. Data are means ± SEM. ANOVA followed by the Neuman-Keuls post-hoc test. **P < 0.01, ***P < 0.001, vs healthy rats; ^#P < 0.05 vs cholestatic rats treated with vehicle.