Dexamethasone induces an imbalanced fetal-placental-maternal bile acid circulation: involvement of placental transporters

Abstract

Background: The use of prenatal dexamethasone remains controversial. Our recent studies found that prenatal dexamethasone exposure can induce maternal intrahepatic cholestasis and have a lasting adverse influence on bile acid (BA) metabolism in the offspring. The purpose of this study was to investigate the effects of dexamethasone on fetal-placental-maternal BA circulation during the intrauterine period, as well as its placental mechanism.

Methods: Clinical data and human placentas were collected and analyzed. Pregnant Wistar rats were injected subcutaneously with dexamethasone (0.2 mg/kg per day) from gestational day 9 to 20. The metabolomic spectra of BAs in maternal and fetal rat serum were determined by LC-MS. Human and rat placentas were collected for histological and gene expression analysis. BeWo human placental cell line was treated with dexamethasone (20-500 nM).

Results: Human male neonates born after prenatal dexamethasone treatment showed an increased serum BA level while no significant change was observed in females. Moreover, the expression of organic anion transporter polypeptide-related protein 2B1 (OATP2B1) and breast cancer resistance protein (BCRP) in the male neonates' placenta was decreased, while multidrug resistance-associated protein 4 (MRP4) was upregulated. In experimental rats, dexamethasone increased male but decreased female fetal serum total bile acid (TBA) level. LC-MS revealed that primary BAs were the major component that increased in both male and female fetal serum, and all kinds of BAs were significantly increased in maternal serum. The expression of Oatp2b1 and Bcrp were reduced, while Mrp4 expression was increased in the dexamethasone-treated rat placentas. Moreover, dexamethasone increased glucocorticoid receptor (GR) expression and decreased farnesoid X receptor (FXR) expression in the rat placenta. In BeWo cells, dexamethasone induced GR translocation into the nucleus; decreased FXR, OATP2B1, and BCRP expression; and increased MRP4 expression. Furthermore, GR was verified to mediate the downregulation of OATP2B1, while FXR mediated dexamethasone-altered expression of BCRP and MRP4.

Conclusions: By affecting placental BA transporters, dexamethasone induces an imbalanced fetal-placental-maternal BA circulation, as showed by the increase of primary BA levels in the fetal serum. This study provides an important experimental and theoretical basis for elucidating the mechanism of dexamethasone-induced alteration of maternal and fetal BA metabolism and for exploring early prevention and treatment strategies.

Keywords: Bile acids; Dexamethasone; Farnesoid X receptor; Glucocorticoid receptor; Placenta; Transporters.

Fig. 1

Effects of dexamethasone on human neonates’ bile acid level and placental bile acid transporters’ expression. Total bile acid (TBA) levels in neonatal serum from controls and prenatal dexamethasone-treated pregnant women (a). Immunohistochemistry was applied to detect the protein localization and expression of organic anion transporter polypeptide-related protein 2B1 (OATP2B1), breast cancer resistance protein (BCRP), and multidrug resistance-associated proteins 4 (MRP4) in paraffin-embedded male neonatal placenta tissue sections (5 μm) (b, c). Images were taken at × 400 magnification. Scale bars 50 μm. n = 5 placentas per group and three random areas in each section were scored. The protein expression of OATP2B1, BCRP, and MRP4 in male neonatal placentas (d, e) were measured by western blotting. Data are presented as mean ± SD. *P < 0.05 vs. control. **P < 0.01 vs. control. Dex, dexamethasone

Fig. 2

Effects of dexamethasone on bile acid levels in rat fetal serum, maternal serum, and placenta. Total bile acid (TBA) levels of male and female fetal serum (a), maternal serum (b), and placenta (c) were measured by TBA test kits according to the manufacturer’s protocol. Data are presented as mean ± SD, n = 11 litters and placentas from 11 pregnant rats in each group. *P < 0.05 vs. control. **P < 0.01 vs. control. PDE, 0.2 mg kg⁻¹ day⁻¹ dexamethasone

Fig. 3

Effects of dexamethasone on rat fetal and maternal serum bile acid metabolic profile. Bile acids’ metabolic profile of male fetal serum (a), female fetal serum (b), and maternal serum (c) were determined by liquid chromatography/mass spectrometry (LC-MS). The relative amount of bile acid was displayed as a log intensity of the peak area. Data are presented as mean ± SD, n = 11 per group. *P < 0.05 vs. control. **P < 0.01 vs. control. CA, cholic acid; CDCA, chenodeoxycholic acid; MCA, muricholic acid; TCA, taurocholic acid; TCDCA, tauro-chenodeoxycholic acid; TMCA, tauro-muricholic acid; DCA, deoxycholic acid; UDCA, ursodeoxycholic acid; HDCA, hyodeoxycholic acid; TDCA, tauro-deoxycholic acid; TUDCA, tauro-ursodeoxycholic acid; THDCA, tauro-hyodeoxycholic acid; PDE, 0.2 mg kg⁻¹ day⁻¹ dexamethasone

Fig. 4

Effects of dexamethasone on the expression of fetal rat hepatic BA metabolic enzymes and rat placental bile acid transporters and nuclear receptors. The mRNA abundance of cytochrome P450 family 7 subfamily A member 1 (Cyp7a1), cytochrome P450 family 27 subfamily A member 1 (Cyp27a1), and bile acid-CoA ligase (Bacl) in male (a) and female (b) fetal livers was measured by RT-qPCR. The mRNA expression of organic anion transporter polypeptide-related protein 2b1 (Oatp2b1), breast cancer resistance protein (Bcrp), organic anion transporter polypeptide-related protein 4a1 (Oatp4a1), and multidrug resistance-associated proteins 4 (Mrp4) in male (c) and female (d) placentas were measured by RT-qPCR. Immunohistochemistry for Oatp2b1, Bcrp, and Mrp4 (e) in paraffin-embedded placenta tissue sections (5 μm) from the control group and PDE group (magnification × 400). Scale bars 50 μm. n = 5 placentas from five pregnant rats, and three random fields of each section were scored. The corresponding anti-IgG served as non-specific controls (e), and quantitative fold changes of transporters expression were analyzed by ImageJ version 1.44. The mRNA expression of the glucocorticoid receptor (GR) and farnesoid X receptor (FXR) in male and female (F) fetal placentas were measured by RT-qPCR. The protein expression of GR and FXR in male fetal placentas (g) were measured by western blotting. In each group, n = 11 livers and n = 11 placentas for qRT-PCR, n = 3 for western blotting. Data are presented as mean ± SD. *P < 0.05 vs. control. **P < 0.01 vs. control. PDE, 0.2 mg kg⁻¹ day⁻¹ dexamethasone

Fig. 5

Effects of dexamethasone on expression of bile acid transporters and nuclear receptors in BeWo cells. BeWo cells were treated with dexamethasone (0, 20,100, and 500 nM) for 5 days; cytotoxicity of dexamethasone was measured by MTS assay (a). The mRNA expression of organic anion transporter polypeptide-related protein 2B1 (OATP2B1), breast cancer resistance protein (BCRP), organic anion transporter polypeptide-related protein 4A1 (OATP4A1), and multidrug resistance-associated proteins 4 (MRP4) (b), glucocorticoid receptor (GR), and farnesoid X receptor (FXR) (c) were measured by RT-qPCR. Cells were treated with 500 nM dexamethasone for 5 days; the protein expression of bile acid transporters (OATP2B1, BCRP, and MRP4) (d–f) and GR protein in cytoblast (g) were detected by immunofluorescence (magnification × 400, except “H” × 200). The protein expression of FXR was measured by western blotting, and quantitative fold change of protein level was analyzed by ImageJ version 1.44 (h). Cells were treated with 500 nM dexamethasone for 5 days; after that, CDCA (20 μM) and TCA (20 μM) were added into the upper compartment of a transwell insert, and the concentration of CDCA and TCA (i) in the upper compartment was measured by TBA test kit after 120-min incubation at 37 °C. n = 11 for maternal serum detection, n = 6 for the rest of the experiments. Data are presented as mean ± SD. *P < 0.05 vs. control. **P < 0.01 vs. control. Dex, dexamethasone

Fig. 6

Effects of GR siRNA, RU486, and GW4064 on the expression of bile acid transporters in the human BeWo cells. BeWo cells were transiently transfected with 100 nM GR siRNA, 100 nM negative control (i.e., scrambled) siRNA. After 24-h transfection, cells were treated with or without 500 nM dexamethasone for 3 days. At the end of treatment, mRNA expression of organic anion transporter polypeptide-related protein 2B1 (OATP2B1), breast cancer resistance protein (BCRP), organic anion transporter polypeptide-related protein 4A1 (OATP4A1), and multidrug resistance-associated proteins 4 (MRP4) (a) were measured by RT-qPCR. BeWo cells were treated with 500 nM dexamethasone, 500 nM dexamethasone plus 10 μM RU486, or 500 nM dexamethasone plus GW4064 for 5 days. At the end of treatment, mRNA expression of OATP2B1, ABCG2, and ABCC4 (b, c) were measured by RT-qPCR, and the protein expression of OATP2B1, BCRP, and MRP4 (d–f) were measured by immunofluorescence staining (magnification × 400). Scale bars 50 μm. Quantitative fold changes of transporter expression were analyzed by using ImageJ version 1.44. n = 6 per group. Data are presented as mean ± SD. *P < 0.05 vs. control. **P < 0.01 vs. control. Dex, dexamethasone; NS, no significance

Fig. 7

Placental mechanism in the metabolic changes of fetal serum bile acids induced by prenatal dexamethasone. BAs, bile acids; TBA, total bile acid; GR, glucocorticoid receptor; FXR, farnesoid X receptor; Bcrp, breast cancer resistant protein; Oatp2b1, organic anion-transporting polypeptides 2b1; Mrp4, multidrug resistance protein