Cholesterol-27α-hydroxylase inhibitor nilvadipine can effectively treat cholestatic liver injury in adult offspring induced by prenatal dexamethasone exposure

Abstract

Prenatal dexamethasone exposure (PDE) can increase offspring susceptibility to various diseases. However, the pathogenesis and early prevention for PDE offspring prone to cholestatic liver injury have been unclear. In this study, we collected human umbilical cord blood from neonates with prenatal dexamethasone therapy, showing increased primary unconjugated bile acid levels in utero. PDE increased blood primary bile acid levels, enhanced endoplasmic reticulum stress, and led to cholestatic liver injury in adulthood in rats, which is accompanied by the persistent increase of H3K14ac level in cholesterol 27α-hydroxylase (CYP27A1) promoter and its expression before and after birth. In vitro, dexamethasone activates glucocorticoid receptors, binding to the CYP27A1 promoter, and promotes its transcriptional expression. Through the miR-450b-3p/SIRT1 pathway, it increased the H3K14ac level of the CYP27A1 promoter to enhance its transcription, which continues after birth. Finally, nilvadipine effectively reversed cholestatic liver injury induced by PDE. This study confirmed PDE could cause cholestatic liver injury, and innovatively proposed its early intervention target (CYP27A1) and effective drug (nilvadipine), providing a theoretical and experimental basis for guiding rational drug use during pregnancy, and preventing and treating the fetal-originated cholestatic liver injury.

Keywords: cholestatic liver injury; cholesterol 27α‐hydroxylase; dexamethasone; miR‐450b‐3p; nilvadipine.

FIGURE 1

Changes in neonatal serum targeted bile acid metabolic profiles in clinical with prenatal dexamethasone therapy (PDT). (A, F) Heatmap of female or male serum bile acid metabolic profiles; (B‐E) Differential content of bile acids in female serum. (G–L) Differential content of bile acids in male serum. Data are shown as the mean ± SEM, n = 21 for the experiment. ^* p < 0.05, ^** p < 0.01 vs. control. CA, cholic acid; CDCA, deoxycholic acid; HCA, hyocholic acid; THCA, taurohyocholic acid; GHCA, glycohyocholic acid; TCDCA, taurochenodeoxycholic acid; GCDCA, glycochenodeoxycholic acid; GCA, glycocholic acid; TCA, taurocholic acid; T‐α‐MCA, tauro‐alpha‐muricholic acid; 7_ketoLCA, 7‐ketolithocholic acid; HDCA, hyodeoxycholic acid; LCA_S, lithocholic acid 3‐sulfate; DCA, deoxycholic acid; LCA, lithocholic acid; βUDCA, β‐ursodeoxycholic acid; UDCA, ursodeoxycholic acid; 3_DHCA, 3‐dehydrocholic acid; THDCA, taurohyodeoxycholic acid; TDCA, taurodeoxycholic acid; GUDCA, glycoursodeoxycholic acid; CDCA_3Gln, chenodeoxycholic acid‐3‐beta‐d‐glucuronide; GDCA, glycodeoxycholic acid.

FIGURE 2

Changes in related indicators of endoplasmic reticulum stress and cholestatic liver injury in female and male adult offspring rats at postnatal week 28 with PDE. (A–G) Serum ALT, AST, GGT, TBA, ALP, TBI, and DBI levels. (H) Representative rhodanine staining images(400×); (I) Mean optical density value of rhodanine staining. (J, K) CHOP and GRP78 mRNA expression; (L) CHOP and GRP78 protein expression. (M, N) Representative immunoblots and quantification of GRP78 (200×). Data are shown as the mean ± SEM, n = 3 for rhodanine staining, western blot, and immunofluorescence, n = 6 for other experiments. ^* p < 0.05, ^** p < 0.01 vs. control. PDE, prenatal dexamethasone exposure; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, glutamyl transpeptidase; ALP, alkaline phosphatase; TBI, total bilirubin; DBI, direct bilirubin; TBA, total bile acid; CHOP, C/EBP homologous protein; GRP78, glucose‐regulated protein 78; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

FIGURE 3

Changes in bile acid metabolic profiles in female PDE offspring rats and effects of CDCA on endoplasmic reticulum stress and cholestasis and hepatocyte injury in HepG2 cells. (A, F) Heatmap for serum bile acid metabolic profiles on GD20 and PW12. (D, J) Heatmap for liver bile acid metabolic profiles on GD20 and PW12. (B, C, G–I) Differential content of bile acids in serum on GD20 and PW12. (E, K) Differential content of bile acids in the liver on GD20 and PW12. (L) GRP78 and CHOP mRNA expression after CDCA treatment. (M) GRP78 and CHOP protein expression. (N, O) GRP78 and CHOP protein expression by immunofluorescence (400×). (P) Cholestasis and hepatocyte injury‐related indicators after 4‐PBA treatment. Data are shown as the mean ± SEM. n = 3 for western blot and immunofluorescence, n = 6 for metabolic profile, n = 6 for RT‐qPCR. ^* p < 0.05, ^** p < 0.01 vs. control. ^# p < 0.05, ^## p < 0.01 vs. 200 µM CDCA group. PDE, prenatal dexamethasone exposure; CDCA, deoxycholic acid; GD, gestational day; PW, postnatal week; CA, cholic acid; TCA, taurocholic acid; TCDCA, taurochenodeoxycholic acid; MCA, muricholic acid; UDCA, ursodeoxycholic acid; TUDCA, tauroursodeoxycholic acid; TMCA, tauromuricholic acid; TDCA, taurodeoxycholic acid; THDCA, tauro‐hyodeoxycholic acid; GRP78, glucose‐regulated protein 78; CHOP, C/EBP homologous protein; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; 4‐PBA, 4‐phenylbutyrate acid; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, glutamyl transpeptidase.

FIGURE 4

Changes in liver bile acid synthase expression in female PDE offspring rats and possible epigenetic mechanisms. (A, B) Bile acid series synthase mRNA expression on GD20 and PW12. (C) CYP27A1 and CYP7A1 protein expression on GD20 and PW12. (D, E) Representative immunoblots and quantification of CYP27A1 GD20 and PW12 (200×). Data are shown as the mean ± SEM, n = 3 for Western blot and immunofluorescence, n = 6 for RT‐qPCR. ^* p < 0.05, ^** p < 0.01 vs. control. PDE, prenatal dexamethasone exposure; GD, gestational day; PW, postnatal week; CYP7A1, cholesterol 7α‐hydroxylase; HSD3B7, hydroxy‐delta‐5‐steroid dehydrogenase, 3 beta‐ and steroid delta‐isomerase 7; CYP8B1, cytochrome P450 8B1; AKR1D1, aldo‐keto reductase family 1 member D1; AKR1C6, aldo‐keto reductase family 1, member C6; CYP27A1, cholesterol 27α‐hydroxylase; CYP7B1, cytochrome P450 7B1; CH25H, cholesterol 25‐hydroxylase; CYP46A1, cytochrome P450 family 46 subfamily A member 1; CYP39A1, cytochrome P450 39A1; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase.

FIGURE 5

GR‐mediated CYP27A1 expression and TBA production increases in vitro induced by DEX. (A) CYP27A1 mRNA expression after DEX treatment at different times. (B) TBA level in cell supernatant after DEX treatment with different times. (C, D) CYP27A1 mRNA and protein expression after DEX treatment with different concentrations. (E) TBA levels in cell supernatant after DEX treatment with different concentrations. (F) GR protein expression after transiently transfected with 20 µM Nr3c1 siRNA or 20 µM negative control. (G, H) CYP27A1 mRNA and protein expression in the presence of Nr3c1 siRNA. (I, J) TBA level in cell supernatant in the presence of Nr3c1 siRNA and CYP27A1 siRNA. (K) GR protein expression in cytoplasm and nucleus. (L) GR enrichment in CYP27A1 promoter region. (M) The relative luciferase activities after being treated with RU486. A–F was detected in human WJ‐MSCs derived from normal newborns to differentiate into hepatocyte‐like cells. G–Q was detected in HepG2 cells. Data are shown as the mean ± SEM, n = 3 for western blot and ChIP assay, n = 6 for other experiments. ^* p < 0.05, ^** p < 0.01 vs. control; ^# p < 0.05, ^## p < 0.01 vs. 2500 nM DEX. GR, glucocorticoid receptor; CYP27A1, cholesterol 27α‐hydroxylase; TBA, total bile acid; DEX, dexamethasone; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; H3, histone H3; WJ‐MSCs, Wharton's Jelly derived mesenchymal stem cells; ChIP, chromatin immunoprecipitation.

FIGURE 6

GR/miR‐450b‐3p/SIRT1 pathway mediated dexamethasone‐induced the H3K14ac level of hepatocyte CYP27A1 and its expression. (A) Enrichment of H3K14ac in CYP27A1 promoter region on GD20, PW12, and PW28. (B, C) SIRT1 mRNA and protein expression on GD20. (D) The miRNAs’ expression on GD20. (E) Nr3c1 mRNA expression on GD20. (F–H) The miR‐450b‐3p expression, SIRT1 protein expression, and H3K14ac level in CYP27A1 promoter region after DEX treatment with different concentrations. (I) SIRT1 protein expression after transiently transfected with 2.5 µg pcDNA3.1‐SIRT1 or 2.5 µg negative control. (J–M) H3K14ac level in CYP27A1 promoter region, CYP27A1 mRNA and protein expression, TBA level in cell supernatant in the presence of SIRT1 plasmid. (N–P) SIRT1 protein expression, CYP27A1 mRNA, and protein expression in the presence of miR‐450b‐3p inhibitor. (R) Target validation using luciferase reporters. The relative luciferase activities of 3′UTR reporters containing wild‐type (WT) or mutant (Mut) transcripts were assayed 24 h after co‐transfection with the indicated miRNAs or scrambled NC RNA (NC). (S, T) miR‐450b‐3p and SIRT1 protein expression in the presence of Nr3c1 siRNA. F–I was detected in the human WJ‐MSCs derived from normal newborns to differentiate into hepatocyte‐like cells. J–Y was detected in HepG2 cells. Data are shown as the mean ± SEM, n = 3 for Western blot and ChIP assay, n = 6 for other experiments. ^* p < 0.05, ^** p < 0.01 vs. control; ^# p < 0.05, ^## p < 0.01 vs. 2500 nM DEX group. GR, glucocorticoid receptor; SIRT1, Sirtuin 1; H3K14ac, histone 3 lysine 14 acetylation; CYP27A1, cholesterol 27α‐hydroxylase; GD, gestational day; PW, postnatal week; PDE, prenatal dexamethasone exposure; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; DEX, dexamethasone; TBA, total bile acid; WJ‐MSCs, Wharton's Jelly derived mesenchymal stem cells.

FIGURE 7

Nilvadipine reversed cholestatic liver injury induced by dexamethasone or PDE in female offspring rats with long‐term efficacy. (A) GRP78 and CHOP mRNA expression. (B) GRP78 and CHOP protein expression by western blot. (C, D) GRP78 and CHOP protein expression by immunofluorescence (400×). (E) Cholestatic liver injury‐related indicators. (F) The relative luciferase activities. (G, K) Liver injury‐related indicators level at PW12 and PW28. (H, L) Cholestasis‐related indicators level at PW12 and PW28. (I, M) Liver 27‐hydroxycholesterol contents at PW12 and PW28. (J, N) GRP78 and CHOP protein expression at PW12 and PW28. A–G was detected in human WJ‐MSCs after being treated with DEX and/or nilvadipine. Data are shown as the mean ± SEM, n = 3 for western blot and immunofluorescence, n = 10 for detecting the related serum indicators, n = 6 for other experiments. ^* p < 0.05, ^** p < 0.01 vs. control; ^# p < 0.05 vs. DEX or PDE group. PDE, prenatal dexamethasone exposure; GRP78, glucose‐regulated protein 78; CHOP, C/EBP homologous protein; GAPDH, glyceraldehyde‐3‐phosphate dehydrogenase; PW, postnatal week; DEX, dexamethasone; ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST, aspartate aminotransferase; GGT, glutamyl transpeptidase; TBI, total bilirubin; DBI, direct bilirubin; TBA, total bile acid; WJ‐MSCs, Wharton's Jelly derived mesenchymal stem cells; DEX, dexamethasone.

FIGURE 8

Intrauterine programming mechanisms of cholestatic liver injury induced by prenatal dexamethasone exposure and its drug target. GR, glucocorticoid receptor; SIRT1, Sirtuin 1; CYP27A1, cholesterol 27α‐hydroxylase; Oatp2b1, organic anion transporter polypeptide‐related protein 2b1; BAAT, bile acid‐CoA: amino acid N‐acyltransferase; MUC2x, mucin‐2; BSH, bile salt hydrolase.