Cholic acid (CA), pregnenolone-16-α-carbonitrile (PCN) and Dex were purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany). An anti-GR antibody was purchased from Epitomics (Abcam, Cambridge, UK) and an anti-GAPDH antibody was purchased from GenScript (Piscataway, NJ, USA).

All animal studies were approved by the Institutional Animal Care and Use Committee of the Wadsworth Center (State University of New York, Albany, NY, USA) or the Animal Ethics Committee of the Fujian Agriculture and Forestry University (Fuzhou, China). In total, 120 adult male C56BL/6J mice (~25 g; 2–3 months old, 3 mice per group; breeding and feeding in the laboratory) were used for all experiments. Animals were maintained at 22°C, 50% relative humidity and a 12-h light/dark cycle in a specific pathogen-free clean room with food and water ad libitum. Dex dissolved in corn oil was administered to the mice at various doses via intraperitoneal (i.p.) injection. For dose-response experiments, mice were treated with Dex at three different doses (0.8, 8 and 80 mg/kg) for 24 h. Animals were sacrificed at the indicated timepoints following treatment with Dex for tissue collection. Additionally, the weights of the mice and tissue, and tissue morphology were examined to evaluate the toxicity of treatment with Dex. PCN (25 mg/kg, dissolved in corn oil) was administered via oral gavage, and mice were sacrificed at 24 h after treatment with PCN for tissue Collection.

For the co-treatment of CA and Dex, Dex at 80 mg/kg or vehicle (corn oil) was administered by i.p. injection. Mice fasted for 2 h prior to administration of CA (200 mg/kg in PBS by oral gavage). After another 4 h, ileal epithelial cells were collected for RNA preparation.

The small intestine was removed and flushed with ice cold PBS. The small intestine was divided into three segments designated the duodenum (proximal 5 cm), jejunum (middle 5 cm) and ileum (distal 5 cm). The three segments were cut open longitudinally and the epithelial cells were removed by gentle scraping. The collected cells were washed once in cold PBS and subsequently lysed in radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing Protease & Phosphatase Inhibitor (Thermo Fisher Scientific, Inc.) and 5 mM EDTA, and homogenized using a polytron homogenizer. The homogenates were mixed for 10 min at 4°C, and subsequently centrifuged at 14,000 g and 4°C for 15 min. The supernatant was collected and the protein concentration was determined by the bicinchoninic acid method (Pierce; Thermo Fisher Scientific, Inc.).

Proteins (50 µg) were resolved on 10% NuPAGE Bis-Tris-gels (Invitrogen; Thermo Fisher Scientific, Inc.) and subsequently transferred to nitrocellulose membranes. For immunodetection, subsequent to blocking with 5% fat-free milk at room temperature for 1 h, pre-cut membranes were incubated with monoclonal rabbit anti-GR (Epitomics; Abcam; cat. no. EPR4595; 1:4,000) and goat anti-GAPDH (GenScript; cat. no. A00191-40; 1:40,000) antibodies at 4°C overnight. Subsequent to washing, the membranes were incubated with goat anti-rabbit immunoglobulin G (IgG) heavy and light chain (H&L) horseradish peroxidase (HRP)-conjugated antibod (GenScript; cat. no. A00098; 1:8,000) or donkey anti-goat IgG H&L HRP-conjugated antibody (GenScript; cat. no. A00178; 1:8,000) according to the host species of the primary antibody at room temperature for 1 h. Pierce™ Enhanced Chemiluminescent Western Blotting Substrate (Thermo Fisher Scientific, Inc.) was used for the detection of HRP on the immunoblots. Immunoblot quantification was performed using a Bio-Rad ChemiDoc XRS+ System (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with Image Lab 3.0 (Bio-Rad Laboratories, Inc.).

Immediately after harvesting the intestine, 5 cm of the terminal ileum was flushed with PBS to remove the contents and was subsequently cut into small segments (1×3 mm). The segments were cultured at 37°C for 4 h in Dulbecco's modified Eagle's medium (Thermo Fisher Scientific, Inc.) supplemented with 10% heat-inactivated fetal bovine serum (Thermo Fisher Scientific, Inc.), 100 units/ml penicillin and 100 µg/ml streptomycin. Dex (1 µM final concentration) or vehicle (ethanol), and CA (100 µg/ml) or PBS were added to the culture medium. The epithelial cells were collected at the end of 4-h culture for RNA preparation.

Total RNA was prepared from the enterocytes of individual mice, with the use of TRIzol^® reagent (Invitrogen; Thermo Fisher Scientific, Inc.), as previously described (15). RNA concentration and purity were determined spectrally, and the integrity of the RNA samples was assessed by ethidium bromide staining following agarose gel electrophoresis. RT-PCR was performed using 3 µg total RNA and SYBR Green PCR Master PCR Mix (Applied Biosystems; Thermo Fisher Scientific, Inc.), as previously described (16). The comparative 2^−∆∆Cq method was used for quantifying the relative mRNA expression levels (17,18). PCR primers for FXR (19), PXR (20), Fgf15 (3), small heterodimer partner (SHP) (3), glucocorticoid-induced leucine zipper (GILZ) (21), apical sodium dependent bile acid transporter (ASBT) (22), ileum bile acid binding protein (IBABP) (22) and cytochrome P450, family 3, subfamily a, polypeptide 11 Cyp3a11 (23) were as follows: FXR, 5′-CCAACCTGGGTTTCTACCC-3′ (forward) and 5′-CACACAGCTCATCCCCTTT-3′ (reverse); PXR, 5′-CAAGGCCAATGGCTACCA-3′ (forward) and 5′-CGGGTGATCTCGCAGGTT-3′ (reverse); Fgf15, 5′-GAGGACCAAAACGAACGAAATT-3′ (forward) and 5′-ACGTCCTTGATGGCAATCG-3′ (reverse); SHP, 5′-CGATCCTCTTCAACCCAGATG-3′ (forward) and 5′-AGGGCTCCAAGACTTCACACA-3′ (reverse); GILZ, 5′-CAGCAGCCACTCAAACCAGC-3′ (forward) and; 5′-ACCACATCCCCTCCAAGCAG-3′ (reverse); ASBT, 5′-TGGGTTTCTTCCTGGCTAGACT-3′ (forward) and 5′-TGTTCTGCATTCCAGTTTCCAA-3′ (reverse); IBABP, 5′-AGATCATCACAGAGGTCCAGC-3′ (forward) and 5′-GGTAGCCTTGAACTTCTTGCC-3′ (reverse); Cyp3a11, 5′-GACAAACAAGCAGGGATGG-3′ (forward) and 5′-AATGTGGGGGACAGCAAAG-3′ (reverse); and U36B4, 5′-CGTCCTCGTTGGAGTGACA-3′ (forward) and 5′-CGGTGCGTCAGGGATTG-3′ (reverse). The levels of the target mRNAs in various total RNA preparations were normalized by the level of U36B4 mRNA (8).

All experiments were repeated at least three times. Data are presented as the mean ± standard deviation. Statistical analysis was performed using Student's t-test with SPSS 11.5 software (SPSS, Inc., Chicago, IL, USA). For multiple comparisons, one-way analysis of variance followed by Fisher's Least Significant Difference test was used. P<0.05 was considered to indicate a statistically significant difference.

To examine the effect of treatment with Dex on ileal Fgf15 expression, the mRNA expression levels of Fgf15 were compared prior to and following treatment with Dex. Mice were treated with Dex at three different doses (0.8, 8 and 80 mg/kg) for 24 h. The ileal Fgf15 expression decreased in a dose-dependent manner following treatment with Dex (Fig. 1A). At 80 mg/kg Dex, the expression level of FGF15 mRNA was decreased to an undetectable level two days later (Fig. 1B). The downregulation of ileal Fgf15 expression by treatment with Dex was maintained when the mice were treated with Dex for 3 consecutive days (Fig. 1B). These results suggested that treatment with Dex was able to suppress ileal Fgf15 expression at the RNA level. At the same time, although the highest dosage of Dex used, 80 mg/kg, in the present study was effective, the potential cytotoxicity of Dex with this dosage for 3 days requires consideration (data not shown). The phenomenon of tissue hypertrophy was observed (data not shown), which may be due to the compensation for the cytotoxicity of treatment with Dex with high dosage for 3 days.

To investigate whether this downregulation of Fgf15 expression is mediated by FXR, the critical transcriptional factor for regulating Fgf15 expression (3), the effect of treatment with Dex on the FXR transcriptional activity was examined in vivo. Mice were pre-treated with Dex or corn oil, and subsequently FXR was activated with CA. After a 4 h treatment with CA, as presented in Fig. 1C, the mRNA expression level of Fgf15 was upregulated. However, the upregulation of Fgf15 expression by treatment with CA was significantly inhibited in mice pre-treated with Dex (Fig. 1C; P<0.001), suggesting that Dex may interfere with FXR activation. Whether this suppression of Fgf15 expression by Dex directly occurred in ileal enterocytes, using an ex vivo culture system, was further investigated. As presented in Fig. 1D, CA induced Fgf15 expression in the ex vivo culture system, whereas, Dex interfered with the CA-induced FGF15 upregulation in ileal cells.

GR may be activated by Dex, to trigger the transactivation or transrepression of GC-responsive genes (24–26). Therefore, GR protein expression levels in the small intestine were examined by western blot analysis. As presented in Fig. 2A, GR (~86 kDa) was expressed throughout the entire small intestine, between the duodenum and the ileum. Notably, GR expression in the small intestine exhibited a gradual increase between the duodenum and the ileum. The expression level of GR in ileum was ~3-times higher compared with the duodenum and ~2-times higher compared with the jejunum (Fig. 2B), suggesting that GR may have an important function in the ileum. Subsequently, it was determined whether ileal GR may be activated by treatment with Dex at a concentration of 80 mg/kg. It was identified that the mRNA expression levels of two selected GR target genes, GILZ and ASBT (27–29), were upregulated by ~6 and ~2.5-times, respectively, in the ileum following treatment with Dex for 24 h (Fig. 2C). These results demonstrated that GR was highly expressed in the ileum and may be activated by Dex, and thus may potentially mediate the repression of Fgf15 expression by Dex.

As Dex is able to activate GR and PXR, it was further examined if the effect of Dex on FGF15 expression is mediated by PXR. As presented in Fig. 3A, the suppression of Fgf15 expression was observed following treatment with Dex. Since Fgf15 is the downstream gene for FXR activation, the expression of SHP and IBABP, another two classical FXR target genes (3,30,31), was further examined in the ileum following treatment with Dex. As expected, SHP and IBABP expression were additionally significantly downregulated following treatment with Dex (Fig. 3A; P<0.01). This downregulation of Fgf15, SHP and IBABP gene expression reflects the inhibition of FXR activity; however, not FXR expression, as there was no significant difference in FXR expression.

In addition to activating GR, Dex may additionally activate PXR to induce CYP3A expression (32,33). As presented in Fig. 3A, the expression of Cyp3a11, the target gene of PXR, was significantly increased in the ileum following treatment with Dex; however, there was no alteration in PXR expression, a result suggesting that the increased CYP3A expression was due to increased PXR activation. To confirm whether the suppression of Fgf15 expression by Dex is mediated by PXR activation, PCN, a typical PXR agonist, was used to treat mice in the same way as Dex. As presented in Fig. 3B, PCN induced the expression of Cyp3a11, a PXR target gene; however, it had no effect on the expression of either FXR or PXR, or FXR target genes, Fgf15 and IBABP, in the ileum. This result suggested that PXR activation did not contribute to the suppression of FGF15 expression by treatment with Dex.