Background
Intrauterine growth retardation (IUGR) refers to a fetus that grew slowly in the uterus and failed to achieve the expected weight for gestational age [
1]. IUGR usually results in growth retardation, multiple organ dysfunction, and low birth weight, which can cause fetal distress, neonatal asphyxia, and even perinatal death [
2,
3]. The epidemiological survey has shown that the incidence of IUGR was about 3–9% in socioeconomically developed areas, while it was as high as 30% in economically underdeveloped areas [
4]. Children with IUGR, especially those who had catch-up growth during early life, have a higher risk of susceptibility to various diseases, including metabolic diseases (MS), type 2 diabetes, cardiovascular diseases, and mental diseases in later life [
5‐
8]. Besides, animal studies have revealed that adverse environments during pregnancy (such as xenobiotics exposure and dietary restrictions, etc.) can increase the incidence of IUGR and the susceptibility to many chronic diseases after birth [
9‐
11]. At present, there are still many difficulties in the prevention and treatment of IUGR, mainly related to the unclear mechanism of occurrence and target of intervention.
Glucocorticoids play an essential role in maintaining pregnancy and regulating embryonic tissue morphology and function maturity [
12]. However, overexposure to glucocorticoids caused by various prenatal factors may be a trigger for adverse developmental outcomes (e.g., IUGR), affecting fetal developmental programming and leading to susceptibility to a variety of fetal-originated diseases in adults [
13‐
15]. Maternal glucocorticoid levels in the fetus are mainly regulated by the placental glucocorticoid barrier, including 11β-hydroxysteroid dehydrogenase type 2 (11β-HSD2) and P-glycoprotein (P-gp). The 11β-HSD2 can reduce glucocorticoids from entering fetal circulation by inactivating glucocorticoids [
15]. In our previous studies, through a series of animal experiments, we have established stable IUGR offspring rat models caused by prenatal xenobiotics exposure such as caffeine, nicotine, and ethanol and confirmed that prenatal xenobiotics exposure induced-IUGR was associated with a low expression level of placental 11β-HSD2 [
10,
16,
17]. P-gp is one of the most abundantly expressed proteins of the ATP binding cassette efflux transporter family in the placentas. P-gp in the placental trophoblasts can use ATP to pump glucocorticoids back to the maternal circulation [
18]. However, the effect of P-gp as a placental glucocorticoid barrier on the occurrence of IUGR is unclear.
Clinical and animal studies have found that adverse environments during pregnancy can cause IUGR. Caffeine is the most widely consumed psychoactive drug in the world. According to statistics, 82% of pregnant women have been reported to consume caffeine daily in the USA [
19] and 91% in France [
20]. Epidemiological investigations have proved that prenatal caffeine exposure (PCE) can cause toxic effects of reproduction and fetal development, such as the increased risk of congenital malformations, premature delivery, spontaneous abortion, IUGR, and susceptibility to chronic diseases [
21,
22]. Our animal experiments also demonstrated that PCE could not only increase maternal glucocorticoid levels but also cause fetal overexposure to glucocorticoid by inhibiting placental 11β-HSD2, leading to the occurrence of IUGR and susceptibility to multiple chronic diseases in adult offspring [
16,
23‐
26]. However, whether caffeine causes IUGR by affecting placental P-gp has not been reported yet. Epigenetic changes are critical to gene regulation in the developing placenta. As an essential transporter of syncytiotrophoblast, the epigenetic regulation mechanism of placental P-gp is rarely reported. It has been suggested [
27,
28] that the JNK pathway and Y-box binding protein-1 (YB-1) may be involved in P-gp regulation. It is not known whether the JNK pathway, YB-1, and epigenetic modifications are involved in the regulation of placental P-gp by caffeine.
Here, using clinical specimens and PCE-induced IUGR rat model, we first determined the glucocorticoid barrier function of placental P-gp in the IUGR neonates and fetal rats by analyzing the correlations among the placental P-gp expression levels, placental/fetal glucocorticoid levels, and placental/fetal body weights. Furthermore, by combining in vivo experiments of caffeine, the clinical IUGR samples, and rat IUGR models, we will clarify the epigenetic mechanism of P-gp expression changes in placental trophoblast cells. Next, P-gp inducer sodium ferulate will be used to confirm the intervention target on fetal weight loss caused by PCE. Finally, we will confirm the potential early warning targets of IUGR occurrence and multiple disease susceptibility using clinical specimens. This study will illuminate the underlying mechanism of IUGR from the perspective of placental P-gp and provide a novel idea for analyzing the placental origin of adult diseases, as well as the exploration of early warning targets and potential intervention strategies.
Methods
Chemicals and reagents
Caffeine (C0750) and cortisol (C46329) and DNase I (DN25) were obtained from Sigma-Aldrich Co., Ltd. (St Louis, MO, USA). Rat corticosterone ELISA kits (RE52211) and cortisol ELISA kits (RE52061) were purchased from IBL International (Germany). Dantrolene (B6329), SP600125 (A4604), and Rhodamine 123 (Rho 123, C3140) were purchased from APExBIO Technology LLC (Houston, MA, USA). Trypsin (9002-07-7) and lipofectamine 3000 (L3000015) were purchased from Gibco Co. (Detroit, MI, USA). YB-1 pcDNA3.1( + ) plasmid was constructed by GenePharma Co., Ltd. (Shanghai, China). TRIzol (15596018) reagent was purchased from Invitrogen Co. (Carlsbad, CA, USA). The quantitative real-time PCR (qRT-PCR) reverse transcription (R312) and SYBR qPCR master mix kits (Q711) were purchased from Vazyme Biotech Co., Ltd. (Nanjing, China). The nuclear and cytoplasmic protein preparation kit (P1200) and BCA assay kit (P1511) were purchased from Applygen Co., Ltd. (Beijing, China). Primary antibodies anti-P-gp (ab170904), anti-YB-1 (ab76149), anti-P300 (ab14984), anti-acetyl H3K9 (ab10812), anti-acetyl H3K14 (ab52946), and IgG (ab172730) antibody were purchased from Abcam Technology Co., Ltd. (Cambridge, UK). The anti-JNK (A4867), anti-p-JNK (AP0631), anti-H3 (A2348), and anti-GAPDH (A10868) antibodies were purchased from ABclonal Technology Co., Ltd. (Wuhan, China). The enhanced chemiluminescence kit (ECL, 32209) was obtained from Pierce Biotechnology Inc. (Rockford, IL, USA). Chromatin immunoprecipitation (ChIP) assay kit (17-295) was purchased from Millipore Co., Ltd. (Billerica, MA, USA). DNA purification kit (DP214) was purchased from Tiangen Biotech Co., Ltd. (Beijing, China). The other reagents for experiments were of analytical grade.
Human placental tissue collection
The placentas from IUGR neonates were collected after obtaining informed consent from pregnant women who underwent a cesarean section or natural delivery at the Division of Gynecology and Obstetrics of Zhongnan Hospital, Wuhan University, between June 2017 and June 2018. It was approved by the Medical Ethical Committee of Zhongnan Hospital of Wuhan University (approval number 201606). IUGR was diagnosed according to an intrauterine condition when a baby’s weight is below the 10th centile for gestational age [
29]. The following criteria were required for the inclusion: singleton pregnancy, gestational age within 28–40 weeks, and the weight of the fetus in the control group is adapted to the gestational age, while the weight of the fetus in the IUGR group is lower than the 10th percentile of the gestational age. The fetus with any congenital genetic diseases, fetal structural, genetic, or chromosomal abnormalities was excluded. Pregnancy complications, including pre-eclampsia and gestational diabetes, are also adverse environments during pregnancy since they can cause maternal stress and HPA axis activation [
30,
31], which is consistent with the research goal that we want to study the adverse environments during pregnancy leading to fetal weight loss. Therefore, women with pregnancy complications were not excluded from the IUGR group. Thirty control and 26 IUGR neonatal placentas, 89.3% of cesarean section, and 10.7% (3/30 in the control group, 3/26 in the IUGR group) of natural delivery were finally recruited. The samples of the control group were collected from healthy women, and the samples of the IUGR group were collected from healthy women or women with pregnancy complications (15 cases of pre-eclampsia, 6 cases of gestational diabetes, 2 cases of nuchal cord, and 1 case of oligohydramnios). The placental tissues and umbilical cord blood were collected immediately after delivery. The placental tissue was dissected at the middle zone after the amniotic membranes, decidua, and connective tissues had been removed. Partial placental tissue was fixed with 10% paraformaldehyde, and partial tissue was immediately snap-frozen in liquid nitrogen and stored at -80°C after being washed thoroughly with saline. Fetal cord serum was isolated from umbilical cord blood by centrifugation.
Animals and treatment
Specific pathogen-free Wistar rats and C57BL/6 mice were obtained from the Experimental Center of Hubei Medical Scientific Academy (No. 2017-0018, certification number: 42000600002258, Hubei, China). The animal protocol of rats was reviewed and approved by the Ethics Committee of Wuhan University School of Medicine (No. 14016), and the animal protocol of mice was reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of Wuhan University Center for Animal Experiment (No. WP20210003). All the protocols were conformed to the National Institutes of Health guide for the care and use of laboratory animals.
Details of rat feeding and mating were described as before [
32]. Upon confirmation of mating by the appearance of sperm in a vaginal smear, the day was defined as the gestational day (GD) 0. From GD9 to GD20, the rats in PCE groups were intragastrical administered 30 and 120 mg/kg·d caffeine (dissolved in distilled water) according to the PCE(L) (low dose) and PCE(H) (high dose) groups, the prenatal ethanol exposure (PEE) group was given 4 g/kg·d ethanol (dissolved in distilled water) by gavage administration, while the control group was given the same volume of distilled water. Pregnant rats were injected subcutaneously with 0.8 mg/kg·d dexamethasone (dissolved in saline) for the prenatal dexamethasone exposure (PDE) group or saline for the control group. On GD20, pregnant rats were sacrificed after being anesthetized at 8 am. The sex of the fetal rats was judged by the anogenital distance and then was ensured by the anatomy of the testicles or ovaries. The fetal blood of the same sex in each litter was combined into a single specimen. Maternal and fetal serum was isolated from the blood by centrifugation (3500 rpm) for 15 min. The placental tissue from three different fetal rats from each litter was randomly collected, pooled, and counted as one sample according to gender for further RNA and protein extraction according to methods previously described [
33]. Partial placental tissue was fixed with 4% paraformaldehyde for immunohistochemistry assay. While the other samples were frozen immediately in liquid nitrogen, followed by storage at -80°C for subsequent examination.
$$ {\displaystyle \begin{array}{c}\mathrm{IUGR}\ \mathrm{rate}\ \mathrm{per}\ \mathrm{litter}\ \left(\%\right)=\left(\mathrm{number}\ \mathrm{of}\ \mathrm{IUGR}\ \mathrm{rat}\ \mathrm{fetuses}\ \mathrm{per}\ \mathrm{litter}/\mathrm{the}\ \mathrm{total}\ \mathrm{number}\ \mathrm{of}\ \mathrm{fetal}\ \mathrm{rats}\ \mathrm{per}\ \mathrm{litter}\right)\times 100\\ {}\mathrm{IUGR}\ \mathrm{rate}\ \mathrm{per}\ \mathrm{group}\ \left(\%\right)=\left(\mathrm{the}\ \mathrm{sum}\ \mathrm{of}\ \mathrm{fetal}\ \mathrm{rat}\ \mathrm{IUGR}\ \mathrm{rate}\ \mathrm{per}\ \mathrm{litter}/\mathrm{each}\ \mathrm{group}\ \mathrm{of}\ \mathrm{litter}\ \mathrm{number}\right)\times 100\end{array}} $$
For the intervention study of mice, the pregnant mice were randomly divided into four groups, namely the control group (control), the caffeine group (caffeine), the sodium ferulate group (sodium ferulate), and the caffeine + sodium ferulate group (caffeine + sodium ferulate). From GD9 to GD18, both caffeine (120 mg/kg·d) and sodium ferulate (50 mg/kg·d) were administered by gavage, while the control group was given the same volume of distilled water. On GD18, pregnant mice were sacrificed after anesthetized. IUGR was diagnosed when an animal’s body weight was two standard deviations less than the mean body weight of the control group. The methods of the preservation and treatment of the sample in mice were similar to the previous procedure.
Immunohistochemistry and immunofluorescence measurement
Placental tissues embedded with paraffin were cut into 5-μm-thick slices along the longitudinal axis. Then, the slices were deparaffinized and rehydrated with xylene and a series of grades of alcohol and then were performed using a microwave treatment for 15 min in citrate buffer (pH 6.0). Then, they were soaked in 3% H2O2 for 25 min for immunochemistry or socked in Triton X-100 to penetrate the membrane for 30 min for immunofluorescence. The antigen retrieval sections were then blocked with 10% goat serum at 37°C for 30 min and incubated with a primary anti-P-gp (1:200) or anti-YB-1 (1:150) antibody overnight at 4°C, following by incubation with the secondary antibody at 37°C for 50 min. Slides were stained with DAB and counterstained with hematoxylin for immunochemistry or dyed with DAPI for immunofluorescence. The mean of integrated optic density (IOD) was measured in 6 different fields for each sample, 5 different samples of each group using Image-Pro Plus (version 6.1, Media Cybernetics, Silver Spring, MD, USA).
Isolation of villous trophoblasts
A method of modified Kliman [
34] and Zhang [
35] was used for placental trophoblasts isolation and purification. Briefly, the tissue removed from the mother’s side of the placenta was minced after washing with normal saline and was digested with 0.125% trypsin and 0.03% DNase I four times (20, 20, 15, 10 min), followed by digestion termination with 10% fetal bovine serum. Purified trophoblast cells could be obtained by screening with nylon net (40 μm) after isolation using a 5–65% Percoll. Collected trophoblasts were seeded with a density at 2×10
6 cells were inserted into 6-well plates.
Cell culture and treatment
The BeWo cell line is derived from human placental villous carcinoma, which was purchased from the China Center for Type Culture Collection (Wuhan, China). The BeWo cells or placental trophoblasts were cultured in modified Eagle’s medium supplemented with 10% fetal bovine serum and 0.1% penicillin/streptomycin at 37°C in a 5% CO2 humidified incubator. The cells were treated with different concentrations of caffeine (0, 0.1, 1, 10, and 10 μM) for 48 h to test the effect of caffeine on P-gp expression.
To confirm the signaling pathway, nonspecific and competitive ryanodine receptor (RYR) antagonist (dantrolene), JNK inhibitor (SP600125), and YB-1 overexpression plasmid were used. Briefly, the BeWo cells were plated at a density of 4×105 cells per well in 6-well plates, after reaching 30–50% confluent, dantrolene (10 μM), and SP600125 (10 μM) were added to the cells, respectively. As to the overexpression of YB-1, after being cultured in 6-well plates, BeWo cells were transfected with the plasmid 1 μg per well using Lipofectamine 3000 according to the manufacturer’s protocols. After 48 h transfection, the cells were harvested for the subsequent analysis.
P-gp activity measurement
After incubation with caffeine for 48 h in a 96-well plate, the BeWo cells were washed by PBS (37°C) 3 times and then treated with 10 μM Rho 123 for 1.5 h. To detect the accumulation of Rho 123, cells were perforated with 1% Triton X-100 for 10 min after being washed with cold PBS. The fluorescence of Rho 123 accumulation was measured with a fluorescence plate reader (Molecular Devices, Wokingham, UK) (excitation/emission wavelength: 485 nm/530 nm).
Transport experiment
The BeWo cells were transferred to transwell® polycarbonate membranes (12-mm diameter, 0.4-μm pore size). The cells were plated at a density of 1×10
5 cells/cm
2 with a certain volume of medium in the apical and basal chambers, and the medium was replaced daily. The cells were cultured with different concentrations of caffeine for 48 h. On day 6 of post-seeding, the confluent monolayer was formed according to the study’s findings [
36]. 200 nM cortisol was added to the apical chamber, and the transwell was incubated at 37°C for 120 min. Then, a sample of 0.2 ml was taken from the basolateral compartment for cortisol measurement.
Serum, placenta, and cell culture fluid corticosterone/cortisol measurement
Placental tissues were homogenized with 1ml PBS, then centrifuged at 12000×g at 4°C for 5 min. The levels of rats/mice serum and placental corticosterone were detected by an ELISA kit following the manufacturer’s introductions. The minimum detection of the corticosterone is 0.56 ng/mL. The intra-assay and inter-assay coefficients of variation were 4% and 5.5%. The cortisol levels of the human placenta and cell culture were tested using the ELISA kit.
Total RNA extract and qRT-PCR
The total RNA was isolated using TRIzol reagent. Then, 1 μg of purified RNA was reverse-transcribed into cDNA using the qRT-PCR reverse kit according to the supplier’s instructions. The relative mRNA level of ABCB1 (ATP binding cassette subfamily B member 1), RYRs, and P300 was detected by an SYBR qPCR master mix kit. GAPDH was selected for rat and human and ubiquitin C (UBC) was selected for mouse as the housekeeping gene, and the relative expression levels of these genes were calculated using the 2
-ΔΔCt method. The sequences of primers for each gene are shown in Table
1. All oligonucleotide primers were synthesized by Sangon Biotech Co., Ltd. (Shanghai, China).
Table 1
Oligonucleotide primers in quantitative real-time PCR
Rat |
abcb1a | TAGCAGGAGTGGTTGAAATG | CAAGCTCTGGGCATACATAG | NM_133401.2 |
abcb1b | CATCCAGAACGCAGACTTGA | CAGCCTGAACCATCGAGAAA | NM_012623.3 |
RYR1 | CATCCTTTCATCCGTCACTC | TCATCTTCGCTCTTGTTGTAG | XM_039100851.1 |
RYR2 | AGGGAGAGAGGAAGCCATTA | GGTCACTGAGACCAGCATTT | XM_039096071.1 |
RYR3 | CACTGACAACTCCTTTCTCTAC | GGATCGTCCTCAAGGTCTTA | XM_039106806.1 |
GAPDH | GCAAGTTCAACGGCACAG | GCCAGTAGACTCCACGACA | NM_017008.4 |
Mouse |
abcb1a | CAGCCAGCATTCTCCGTAATA | GTGAGGATCTCTCCAGCTTTG | NM_011076.3 |
abcb1b | TCCCTGTTCTTTCTGGTTATGG | CCCGAGGTTTGCTACATTCT | NM_011075.2 |
UBC | AGGTGGGATGCAGATCTTTG | CCTCCTTGTCCTGGATCTTTG | NM_019639.4 |
Human |
ABCB1 | GGTGGTGTCACAGGAAGAGATT | TCTAACAAGGGCACGAGCTATG | NM_001348945.2 |
RYR1 | GCTCCCTGTGTGTGTGTAAT | CGGATGCTGGTGACATAGTT | NM_000540.3 |
RYR2 | GAGATGGTCCCTCACCAAATAG | CGTCCCAAGAGGTCAATCAA | NM_001035.3 |
RYR3 | GGAGAAGGTCAGCATAGACAAG | TCCAGTCACCACTTCAAACTC | NM_001036.6 |
YB-1 | GCAGCAGACCGTAACCATTAT | TCTCCGATCCCTCGTTCTTT | NM_004559.5 |
P300 | CCAGCCATGCAGAACATGAA | CGGAATTGTGAAGGCATGGT | NM_001429.4 |
GAPDH | GAAATCCCATCACCATCTTCCAG | ATGAGTCCTTCCACGATACCAAAG | NM_002046.7 |
Chromatin immunoprecipitation (ChIP) and re-ChIP assays
To analyze binding levels of YB-1, P300, H3K9ac, and H3K14ac at the promoter region of the ABCB1 gene, we performed ChIP assays according to the manufacturer’s procedures. The samples were resuspended in 0.5 ml lysis buffer containing protease inhibitors and then sonicated. 10 μL lysis buffer of the samples was used for input DNA, and the left lysis buffer was incubated with Protein A/G beads used for immunoprecipitation with 1 μg anti-YB-1, anti-P300, anti-acetyl H3K9, anti-acetyl H3K14, and IgG antibody at 4°C overnight, followed by incubation with BSA-treated Proteinase K at 65°C for 8 h. For re-ChIP, an anti-YB-1 antibody was used for primary IP, followed by anti-P300 for the second IP, and a nonspecific IgG antibody was used as a control. The sheared DNA recovered from cross-linking was extracted with a DNA purification kit. The DNA associated with target proteins was analyzed by quantitative PCR to quantify the amount of ABCB1 DNA associated with these marks. Data were obtained by normalizing 2-ΔΔCt from qRT-PCR. ChIP primers spanning the ABCB1 binding region used are as follows: Human (ABCB1): TGTAGCTGGTTGGTTGGGAT and CTGGCCTTGTGACTTGCTTT (histone acetylation); TCTCGAGGAATCAGCATTCA and AAGAGCCGCTACTCGAATGA (YB-1 and P300); Rat (abcb1a): TTATGAAGTGTGCGGGAGTG and GGACCGTAGCGAGAACAAAT (histone acetylation); Rat (abcb1b): GGAGCGCCATGTAAAATGCA and CGTAGCGAGAACAAATGCCA (histone acetylation).
Homogenate tissues or cells were lysed in RIPA buffer containing 1% Protease Inhibitor Cocktail. For the nuclear and cytoplasm protein fractionation, a nuclear and cytoplasmic protein preparation kit was used following the manufacturer’s protocol. The protein concentration of the samples was determined with a BCA assay kit. Equal amounts of protein were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto a PVDF microporous membrane. The membranes were blocked with 5% skim milk and then incubated with anti-P-gp (1:1000), anti-YB-1 (1:1000), anti-P300 (1:1000), anti-JNK (1:1500), anti-p-JNK (1:1000), anti-H3 (1:5000), or anti-GAPDH (1:2000) antibody at 4°C overnight, followed by incubation with secondary antibodies at room temperature for 1.5 h. The blots were visualized by the ECL kit. For the immunoprecipitation assay, the protein was incubated with 1 μg p-JNK or YB-1 antibody overnight. Then, 40 μl 50% protein A/G agarose beads were added and continued for 4 h. Beads were resuspended and boiled with 2× SDS-PAGE loading buffer for western blotting analysis.
Statistical analysis
Data were presented as mean ± S.E.M. Statistical differences among gene expression in the placentas were calculated and plotted using Graph Pad Prism 6.0 (GraphPad Software, Inc., USA). Comparisons between 2 groups were made by two-tailed Student t tests and >2 groups were analyzed by one-way ANOVA method. The association of different targets was analyzed using Pearson methods. For all tests, a P<0.05 was considered to be significantly different.
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