Placental mechanism of prenatal nicotine exposure-reduced blood cholesterol levels in female fetal rats

Accepted Manuscript Title: Placental mechanism of prenatal nicotine exposure-reduced blood cholesterol levels in female fetal rats Authors: Guohui Zhang, Jin Zhou, Wen Huang, Luting Yu, Yuanzhen Zhang, Hui Wang PII: DOI: Reference:

S0378-4274(18)31509-1 https://doi.org/10.1016/j.toxlet.2018.07.022 TOXLET 10280

To appear in:

Toxicology Letters

Received date: Revised date: Accepted date:

24-2-2018 9-7-2018 19-7-2018

Please cite this article as: Zhang G, Zhou J, Huang W, Yu L, Zhang Y, Wang H, Placental mechanism of prenatal nicotine exposure-reduced blood cholesterol levels in female fetal rats, Toxicology Letters (2018), https://doi.org/10.1016/j.toxlet.2018.07.022 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Title page Placental mechanism of prenatal nicotine exposure-reduced blood cholesterol levels in female fetal rats

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Guohui Zhanga,1, Jin Zhou b,1, Wen Huanga, Luting Yub , Yuanzhen Zhanga,c,*, Hui Wanga,b,c,*

Department of Obstetrics and Gynecology, Zhongnan Hospital of Wuhan University, 169 Donghu

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Road, Wuchang District, Wuhan 430071, China

Department of Pharmacology, Basic Medical School of Wuhan University, 185 Donghu Road,

Hubei Provincial Key Laboratory of Developmentally Originated Diseases, 185 Donghu Road,

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Wuchang District, Wuhan 430071, China

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Wuchang District, Wuhan 430071, China

These authors contributed equally to this study.

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Corresponding authors at: H. Wang and YZ. Zhang, Department of Obstetrics and Gynecology,

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Zhongnan Hospital of Wuhan University, China.

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E-mail address: [email protected] (H. Wang); [email protected] (YZ. Zhang).

Highlights

Prenatal nicotine exposure（PNE）induces low cholesterol levels in female fetal blood

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Reduced placental cholesterol transport mediates PNE-induced low cholesterol levels in female fetal

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blood 

nAChR-LXR signaling is involved in PNE-reduced placental cholesterol transport

Abstract

Clinical studies showed that intrauterine growth retardation (IUGR) neonatus had lower cholesterol concentrations in cord blood, which might be associated with increased risk of metabolic syndrome and cardiovascular diseases in adulthood. We previously observed lower blood cholesterol levels in

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prenatal nicotine exposure (PNE)-induced IUGR fetal rats, and this study aimed to elucidate the placental mechanism. Pregnant Wistar rats were subcutaneously injected with nicotine (2.0 mg/kgd)

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on gestational day 9-20. In vivo, PNE increased levels of total cholesterol (TCH), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoprotein-cholesterol (LDL-C) in maternal serum,

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while decreased levels of TCH and LDL-C in female fetal serum. Meanwhile, the expression of

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scavenger receptor class B type 1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1) and ATPbinding cassette transporter G1 (ABCG1) were decreased, and the expression of liver X receptor (LXR)

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 and  were also decreased in female placentas. In vitro, nicotine (0.1-10 M) reduced the expression of LXR, LXRβ, SR-B1, ABCA1 and ABCG1 in a concentration dependent manner, which could be

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annulled by nAChR antagonist and LXR agonist. Taken together, nicotine could inhibit the expression of SR-B1, ABCA1 and ABCG1 via nAChR and LXR / in female placentas, finally leading to

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reduced blood cholesterol levels in fetal rats.

Abbreviations: IUGR, intrauterine growth retardation; PNE, prenatal nicotine exposure; TCH, total cholesterol; HDL-C, high-density lipoprotein-cholesterol; LDL-C, low-density lipoprotein-cholesterol;

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SR-B1, scavenger receptor class B type 1; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; LXR, liver X receptor; nAChR, nicotinic acetylcholine receptor; DOHaD, Developmental Origins of Health and Disease; HMGCR, hydroxymethylglutaryl CoA reductase; SREBP-2, sterol regulatory element-binding protein-2; HPA, hypothalamus-pituitaryadrenal

Keywords: prenatal nicotine exposure; intrauterine growth retardation; cholesterol transport; placenta; nicotinic acetylcholine receptor; liver X receptor (LXR)

1. Introduction

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Cholesterol is an essential component of cell membranes and a precursor for steroid hormones, it is important for fetal development (Brett et al., 2014). Cholesterol in blood circulation includes total

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cholesterol (TCH), high-density lipoprotein-cholesterol (HDL-C) and low-density lipoproteincholesterol (LDL-C) (Zhang et al., 2014). It is reported that during the development of fetus, lack of

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cholesterol induced by various reasons can lead to severe birth defects, such as premature delivery,

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neural tube defects and intrauterine growth retardation (IUGR) (Edison et al., 2007). Since the 1980s,

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when retrospective cohort studies implemented by David Barker and his colleagues indicated that the

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incidence of certain adult diseases such as cardiovascular disease and type II diabetes may be linked to early-age development, the hypothesis "Developmental Origins of Health and Disease (DOHaD)"

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has been established, which believes that apart from hereditary factors, the adverse factors (malnutrition and exposure of maternal adverse environmental, etc.) during the beginning stages of life

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including pregnancy, neonatal period and childhood may increase an individual's risk of developing various chronic diseases in later life (Feng et al., 2015). In addition, clinical and retrospective cohort

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studies found that the fetal umbilical cord blood cholesterol concentration is lower in IUGR neonates as compared to gestational age matched controls (Nieto-Diaz et al., 1996; Pecks et al., 2012), and

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individuals with IUGR have increased risk of related-diseases such as metabolic syndrome and cardiovascular diseases in adulthood (Liu et al., 2014; Kopec et al., 2017). These studies suggested that increased risk of adult diseases in individuals with IUGR originate from fetal period, and may be related to low levels of cholesterol in utero. During pregnancy, foetal cholesterol depends primarily on the maternal cholesterol while

endogenous synthesis in fetus is relatively low (Woollett and Heubi, 2000; Baardman et al., 2012; Zwier et al., 2017). In the placenta, cholesterol transport proteins mediate the transportion of maternal cholesterol to the fetus, including scavenger receptor class B type 1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1) and ATP-binding cassette transporter G1 (ABCG1) (Aye et al., 2010). Clinical studies have shown that the expression of ABCA1 increased in placentas of early-onset

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preeclampsia (Plosch et al., 2010), while decreased expression of ABCA1 and ABCG1 and increased expression of SR-B1 were observed in placentas of women complicated with diabetes (Dube et al.,

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2013). Therefore, when facing adverse maternal conditions during pregnancy, the expression of placental cholesterol transporters will make corresponding changes, but there are few reports on the

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exact factors inducing these changes. In addition, it was reported that the expression of SR-B1, ABCA1

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and ABCG1 were regulated by liver X receptor (LXR) in THP-1 macrophage-derived foam cells (Ma

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et al., 2015).

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Maternal smoking during pregnancy is a serious public health problem. Epidemiological studies showed that the prevalence of active smoking among pregnant women is as high as 25% worldwide

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and that approximately 50% of non-smoking pregnant women are exposed to passive smoking (Higgins, 2002). Exposure to tobacco smoke during pregnancy is an important factor inducing IUGR

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(Romo et al., 2009). A clinical study found that newborns of smoker mothers showed a lower level of HDL-C in blood, as compared with newborns of nonsmoker mothers (Iscan et al., 1997). Nicotine, a

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toxic alkaloid, is one of the main components in cigarettes and mediates some of the deleterious effects of smoking (Yildiz, 2004). Studies reported that nicotine can bind to the alpha-subunits of nicotinic

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acetylcholine receptor (nAChR) in placenta, and affect nutritive absorption, blood flow and vascularisation during placental development by stimulation of nAChR, consequently affect the development of fetus (Lips et al., 2005). These studies indicated that exposure to tobacco smoke or nicotine during pregnancy can induce abnormal cholesterol level in fetus, and may be related to adverse effects of nicotine on placenta.

However, as far as we know, there are no studies about the effect of tobacco smoke or nicotine exposure during pregnancy on placental cholesterol transport. This study aimed to investigate the placental mechanism of prenatal nicotine exposure-induced abnormal blood cholesterol levels in fetal rats. This study will provide important theoretical and experimental basis for further understanding of

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nicotine’s effects on placenta and fetus, as well as insights into the fetal origin of adult diseases.

2. Materials and methods

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2.1 Drugs and reagents

Protease Inhibitor Cocktail (P-8849-1ML) and nicotine (N3876-25ML) were obtained from

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Sigma-Aldrich Co., Ltd. (St. Louis, MO, USA). Vecuronium bromide (ab120536, Abcam), GW 3965 hydrochloride (ab141299, Abcam) and primary antibodies SR-B1 (ab217318), ABCA1 (ab18180),

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ABCG1 (ab52617), LXRα (ab41902) and LXR (ab76983) were purchased from Abcam (Cambridge,

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UK). Primary antibody GAPDH (1:5000, AC002) was purchased from ABclonal (Boston, Massachusetts, USA). TCH assay kit was obtained from Sangon Biotech Co., Ltd. (Shanghai, China).

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HDL-C and LDL-C assay kits were purchased from Chuang-Ye Biotech Co., Ltd. (Zhejiang, China). Trizol was obtained from Omega Bio-Tek (Doraville, Georgia, USA). Real-time quantitative PCR (RT-

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qPCR) kit was purchased from Takara Biotechnology Co., Ltd. (Dalian, China) and primers were

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synthesized by Sangon Biotech Co., Ltd. (Shanghai, China). RIPA lysis buffer and BCA Assay kits were purchased from Beyotime (Shanghai, China). The other reagents for experiments were of

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analytical grade.

2.2 Animals and treatment Specific pathogen-free (SPF) Wistar rats (females weighing 200–240 g; males weighing 260–300 g) were purchased from the Experimental Center of Hubei Medical Scientific Academy (No. 2012-2014, Hubei, China). Animal experiments were conducted in the Center for Animal Experiment of Wuhan

University (Wuhan, Hubei, China), which is accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). Details for raising and mating Wistar rats have been described previously (Ao et al., 2015). The day when a vaginal plug presented was designated as gestational day (GD) 0. Subsequently, pregnant rats were randomly assigned to control group and nicotine group, each group had 14 pregnant rats. The pregnant rats were

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housed 2 per cage and had an ad libitum access to a standard diet and water. The pregnant rats in the nicotine group were injected subcutaneously with nicotine tartrate (2.0 mg/kg·d) from GD9 to GD20,

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we chose the neck skin of the pregnant rats as a subcutaneous injection site, while rats in the control group were administered with the same volume of saline. On GD20, the pregnant rats were

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anesthetized with isoflurane and then sacrificed. To avoid the influence of litter size on fetal body

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weight to a certain extent, we had excluded pregnant rats that litter size was smaller than 8 and larger

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than 15, finally there were 11 pregnant rats selected with 8-15 live fetuses in each group. Maternal and

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female fetal serum were collected from blood by centrifugation at 1800 × g, 4°C for 15 min. Female placentas were quickly collected. All samples were immediately transferred to liquid nitrogen, and

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subsequently stored at 80°C for further total RNA and protein extraction.

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2.3 Determination of serum cholesterol concentration

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The concentrations of serum TCH, HDL-C and LDL-C were determined using biochemical assay kits in accordance with the manufacturer's protocols.

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2.4 Cell culture and treatment The human BeWo cell line was purchased from China Center for Type Culture Collection (Wuhan,

China). The cells were cultured in cell culture flasks (25 cm2, Corning, USA) with F12K medium that contained 10% fetal bovine serum (Gibco, USA) and 0.1% penicillin/streptomycin, in a 5% CO2 cell incubator at 37°C. To test the effect of nicotine, the cells were treated with different concentrations

(0.1, 1 and 10 M) of nicotine for 24 h. In order to verify the signaling pathway, the cells were respectively treated with 10 μM nicotinic acetylcholine receptor (nAChR) antagonist (vecuronium bromide) and 2 μM LXR agonist (GW 3965 hydrochloride) for 24 h.

2.5 Total RNA extract and RT-qPCR

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Total RNA extraction, reverse transcription and RT-qPCR were performed as previously described (Tan et al., 2012). 50 mg tissue samples were collected from the same position of placentas for total

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RNA extraction. Total RNA of placental tissues and BeWo cells was extracted using Trizol reagent. The mRNA expression of hydroxymethylglutaryl-CoA reductase (HMGCR), sterol regulatory

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element-binding protein-2 (SREBP-2), SR-B1, ABCA1, ABCG1, LXRα and LXRβ were determined

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by RT-qPCR. We designed the primers of above genes using Premier 6.0, and the sequences were shown below (Table 1). RT-qPCR was conducted in 384-well plates using the ABI Step One PlusTM

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real-time PCR System (Applied Biosystems, Foster City, CA, USA) in a total volume of 10 l which containing 1 μl cDNA template, 0.2 μl forward primer and 0.2 μl reverse primer, 5 μl SYBR Green

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and 3.6 μl DEPC-H2O. The PCR cycling conditions were as follows: pre-denaturation, 95°C for 30 s and then 40 cycles of denaturation, 95°C for 5 s; annealing conditions for each gene are shown in Table

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1. GAPDH mRNA level was measured simultaneously and applied as quantitative control to normalize the relative expression of HMGCR, SREBP-2, SR-B1, ABCA1, ABCG1, LXRα and LXRβ. Relative

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amplicon expression was calculated using the 2−ΔΔCt method.

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2.6 Total protein extract and western blotting assay Methods for western blotting were detailedly described previously (Wen et al., 2014). Briefly

speaking, cells or 50 mg minced tissues were lysed for 30 min at 4°C in RIPA lysis buffer that contained Protease Inhibitor Cocktail. The concentrations of the total protein were determined using BCA Assay kit, and the protein concentration in each group was adjusted to be consistent. 30 g total protein was

loaded to each lane. The proteins with different molecular weights were separated by electrophoresis in 10% SDS-PAGE gel and then were blotted onto PVDF membranes (Millipore, MA, USA). Subsequently, membranes were blocked in 5% fat free milk for 1 h at room temperature. After blocking, membranes were incubated with primary antibodies overnight at 4°C, including SR-B1 (1:2000), ABCA1 (1:300), ABCG1 (1:5000), LXRα (1:2000), LXRβ (1:2500) and GAPDH (1:5000). Then they

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were incubated with horseradish peroxidase(HRP)-conjugated secondary antibody for 1 h at room temperature, and visualized with ECL HRP substrate (PerkinElmer Inc. Boston, Mass). Signals of

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antibody binding were detected using Chemi-doc Image Analyzer (Bio-Rad, Hercules, California). Protein band intensities were analyzed using Image J (National Institutes of Health, Bethesda,

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Maryland) from 3 independent bands. Relative protein levels were standardized with GAPDH protein

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level.

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2.7 Statistical methods

Prism 6 (GraphPad Software, La Jolla, CA, USA) and SPSS 20.0 (SPSS Science Inc., Chicago,

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Illinois, USA) were used for statistical analysis. All data were showed as means ± standard errors of the means (S.E.M.). Student’s two-tailed t-test was performed on one factor of prenatal nicotine

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treatment (control or nicotine). For the data from in vitro studies, one-way ANOVA followed by a post hoc Dunnett-t-test or a post hoc Bonferroni–t-test were used to perform the multiple comparisons.

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Statistical significance was set at P<0.05.

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3. Results

3.1 In vivo 3.1.1 Effects of PNE on maternal and fetal serum cholesterol levels Pregnant Wistar rats were subcutaneously injected with nicotine (2.0 mg/kgd) on gestational day 9-20, and the cholesterol concentrations in maternal and fetal serum were detected respectively. We

found that compared with the control group, TCH, HDL-C and LDL-C levels in maternal serum of PNE group were increased (P<0.05, P<0.01, Fig. 1A), while TCH and LDL-C levels in fetal serum were decreased (P<0.05, Fig. 1B), indicating that PNE could lead to abnormal cholesterol levels in

3.1.2 Effects of PNE on placental cholesterol synthesis and transport

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maternal and fetal blood.

HMGCR is a rate-limiting enzyme while SREBP-2 is a key regulatory factor for cholesterol

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synthesis (Liu et al., 2015). The results showed that there was no significant difference in the mRNA expression of HMGCR and SREBP-2 in placentas of the PNE group as compared with the control (Fig. 2A), whereas the mRNA and protein levels of placental cholesterol transporters (SR-B1, ABCA1 and

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ABCG1) were decreased significantly (P<0.05 or P<0.01, Fig. 2B-D). It has been reported that LXRα

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is involved in the upregulation of SR-B1, ABCA1 and ABCG1 expression in THP-1 macrophagederived foam cells (Ma et al., 2015). Our results showed that both the mRNA and protein levels of

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LXRα and LXRβ in placentas of PNE group were reduced (P<0.05 or P<0.01, Fig. 2B-D,). These

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results suggested that PNE didn’t affect placental cholesterol synthesis but inhibited placental

3.2 In vitro

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cholesterol transport, which may be related to decreased expression of LXRα and LXRβ.

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3.2.1 Effects of nicotine on cholesterol transporters in human BeWo cells The human BeWo cell line is a human placental villus trophoblastic cell, in which most of

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transporters in placenta are expressed, and it has been a well-known in vitro model for studying the transcellular transport of multiple nutrients and compounds, including cholesterol (Bode et al., 2006; Kamper et al., 2017; Szilagyi et al., 2017). BeWo cells were treated with 0.1, 1, 10 M nicotine for 24 h. We found that nicotine did not affect the expression of SREBP-2 and HMGCR (Fig. 3A, 3B), however reduced both the mRNA and protein expression levels of SR-B1, ABCA1 and ABCG1 in the

concentration-dependent manner (P<0.05 or P<0.01, Fig. 3C-E, 3H, 3I). Meanwhile, decreased mRNA and protein expression levels of LXRα and LXRβ were also observed (P<0.05 or P<0.01, Fig. 3F-I). These results indicated that nicotine could inhibit the expression of cholesterol transporters in

3.2.2 Effects of LXR agonist on cholesterol transporters in human BeWo cells

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human BeWo cells, which might be related to the decrease in expression of LXRα and LXRβ.

To further confirm whether LXR mediates the inhibitory effect of nicotine on the expression of

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cholesterol transporters, GW 3965 hydrochloride, a potent LXR agonist acting on hLXRα and hLXRβ, was used to treat the BeWo cells. The results showed that GW 3965 hydrochloride annulled the

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inhibitory effect of nicotine on the expression of SR-B1, ABCA1 and ABCG1 both at the mRNA

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(P<0.01, Fig. 4A-C) and protein (P<0.01, Fig. 4D, 4E) levels, suggesting that LXR was involed in the

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inhibition of cholesterol transporters expression by nicotine.

3.2.3 Effects of nAChR antagonist on cholesterol transporters in human BeWo cells

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Considering that nicotine exerts its pharmacological action via acting on nAChR, vecuronium bromide (Jonsson et al., 2002), a nAChR nonselective antagonist, was used to treat the BeWo cells to

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verify whether nAChR was involved in the inhibitory effect of nicotine on the expression of SR-B1, ABCA1, ABCG1 or even LXRα and LXRβ. The results showed that vecuronium bromide reversed

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the inhibitory effect of nicotine on the expression of LXRα, LXRβ, SR-B1, ABCA1 and ABCG1 both at the mRNA (P<0.05 or P<0.01, Fig.5A-D) and protein (P<0.01, Fig. 5E, 5F) levels. These results

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indicate that nicotine might inhibit the expression of LXR and cholesterol transporters by acting on nAChR.

4. Discussion Exposure to tobacco smoke during pregnancy is associated with an increased risk of IUGR (Blatt

et al., 2015). According to the work by NL Benowitz (Benowitz and Jacob, 1990), average nicotine intake per cigarette is 1.48 mg, therefore, based on the dose conversion between humans and rats (human : rat 1 : 6.17 by body surface area comparison) (Reagan-Shaw et al., 2008), the dose of 2.0 mg/kgd used to treat pregnant rats in this study is approximately equivalent to 15.3 cigarettes/day for a pregnant woman weighing approximately 70 kg, calculated as following: 2 mg/kg/d×1/6.17×70

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kg/1.48 mg/cigarette = 15.3 cigarettes/d. It is reported that heavy smokers can consume up to 25 cigarettes/day during pregnancy (Delpisheh et al., 2006; Bao et al., 2016), so the dose we used is

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reachable for heavy smokers as reported. Moreover, according to our previous studies (Chen et al., 2007; Xu et al., 2012; Feng et al., 2014; He et al., 2017), we could establish a stable low birth weight

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rat model by using the dose of 2.0 mg/kgd in this study, which could be used to conduct further studies

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on developmental toxicity.

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A clinical study found decreased cholesterol levels in umbilical vein blood of IUGR neonates (Bon

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et al., 2007), and it is reported that exposure to tobacco smoke during pregnancy could lead to lower levels of blood cholesterol in the newborns (Adam et al., 1993). In the present study, PNE decreased

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the levels of blood TCH and LDL-C in female fetal rats. According to our previous study, PNE could cause IUGR, increased blood TCH level in adulthood (Liu et al., 2012), and induced poor articular

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cartilage quality and susceptibility to osteoarthritis in adult offspring rats fed a high-fat diet, which

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indicated that PNE-induced low level of blood cholesterol in utero might be involved in increased risk of related adult diseases in the IUGR offspring. Cholesterol is essential for embryonic development, playing an important role in the development

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of neural tube, limbs and heart. During pregnancy, maternal cholesterol serves as a major source of fetal cholesterol due to insufficient fetal cholesterol synthesis (Woollett and Heubi, 2000; Baardman et al., 2012; Zwier et al., 2017). It is reported that decreased cholesterol transport from maternal circulation to fetal circulation that induced by various reasons, could result in poor development of the fetus (Baardman et al., 2013). SR-B1, ABCA1 and ABCG1 are important cholesterol transporters in

placenta. SR-B1 is mainly responsible for uptaking cholesterol from maternal blood to placenta, while ABCA1 and ABCG1 are mainly responsible for discharging cholesterol from placenta into fetal circulation, and they together accomplish the transportion of cholesterol from the mother to the fetus (Larque et al., 2013). It was found in studies that disruption of ABCA1 or SR-B1 could reduce the maternal-fetal cholesterol transport by 29% and 31% respectively (Lindegaard et al., 2008), suggesting

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the critical role of placental cholesterol transporters in obtaining adequate cholesterol for the fetus. In this study, PNE increased TCH, HDL-C and LDL-C levels in maternal blood, but induced lower levels

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of them in fetal blood. We further found that PNE significantly inhibited the expression of placental cholesterol transporters SR-B1, ABCA1 and ABCG1. Meanwhile, it was confirmed that nicotine could

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inhibit the expression of SR-B1, ABCA1 and ABCG1 in human BeWo cells in a concentration

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dependent manner. In addition, no changes of placental cholesterol synthesis mainly manifested as the

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expression of HMGCR and SREBP-2 were observed in PNE placentas and nicotine-treated BeWo cells.

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Taken together, nicotine could affect maternal cholesterol to fetal circulation via inhibition of cholesterol transporters’ expression in placenta, and eventually induced a low level of blood cholesterol

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in the fetus. Besides, based on our previous study (Feng et al., 2014), we speculated that PNE-elevated maternal cholesterol levels may result from an increased maternal glucocorticoid levels by activating

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hypothalamus-pituitary-adrenal (HPA) axis.

Researchers have reported that the expression of SR-B1, ABCA1 and ABCG1 are regulated by

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multiple nuclear receptors and transcription factors (Ahmed et al., 2009; Calkin and Tontonoz, 2012). LXR is a nuclear receptor activated by ligand, which has two subtypes of LXRα and LXRβ, and they

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are expressed in all tissues, including placental trophoblast cells (Willy et al., 1995; Janowski et al., 1996; Peet et al., 1998). Activated LXR can bind to the retinoic acid X receptor to form a dimer and further activates the transcription of downstream genes such as ABCA1 and ABCG1 (Janowski et al., 1996; Janowski et al., 1999). The study of THP-1 macrophage derived-foam cells showed that downregulation of the expression of LXRα was involved in reduced expression of SR-B1, ABCA1 and

ABCG1 (Tang et al., 2012). Another study (Lindegaard et al., 2008) has found that LXR agonist could double increase the expression of ABCA1 and ABCG1 in mouse placenta, and the maternal-fetal cholesterol transport was up 25%. These studies indicated that LXR plays a key role in regulating the expression of SR-B1, ABCA1 and ABCG1. In the present study, in vivo and in vitro experiments showed that PNE inhibited the expression of

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LXRα and LXRβ in placentas, while nicotine inhibited the expression of them in BeWo cells. Meanwhile, LXR agonist GW 3965 hydrochloride (simultaneously activating LXR α and LXR β) can

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compensate for the inhibitory effect of nicotine on the expression of SR-B1, ABCA1 and ABCG1, suggesting that LXR mediated the inhibitory effect of nicotine on cholesterol tranport in placentas.

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Besides, we also found that the positive effects of GW 3965 hydrochloride on the expression of

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ABCA1 and ABCG1 were greater than that on SR-B1 expression, and we speculated that it was

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probably due to direct binding of LXR to the promoter region of ABCA1 and ABCG1 genes, which could induce transcriptional activation rapidly (Plosch et al., 2007). However, it remains unclear how

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LXR upregulate the expression of SR-B1. In addition, although the responses of LXRα and LXRβ to

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nicotine were similar in this study, LXRα might play a more important role in the regulation of SRB1, ABCA1 and ABCG1 expression, compared with LXRβ, according to other’s studies (Zhang et al.,

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2012; Ma et al., 2014).

Nicotine exerts its pharmacological effect by acting on nAChR. We also confirmed that nAChR

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antagonist vecuronium bromide could attenuate the inhibitory effect of nicotine on the expression of SR-B1, ABCA1, ABCG1, LXRα and LXRβ, suggesting the important role of nAChR in nicotine’s

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regulation of placental cholesterol transport. Nevertheless, there are multiple subtypes of nAChR and we did not demonstrate which nAChR subunit is specifically involved in the regulatory role of nicotine in this study, which is a limitation of this study.

5. Conclusion

PNE induced a low level of blood cholesterol in female fetal rats. The mechanism (Fig. 6) is that nicotine could inhibit the expression of LXRα and LXRβ by acting on nAChR, which caused reduced expression of cholesterol transporters (SR-B1, ABCA1 and ABCG1) in the placentas and then decreased cholesterol transport from maternal blood to fetal blood, eventually resulted in hypocholesterolemia in the fetuses. The present study has elucidated the placental mechanism of PNE-

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induced a low level of blood cholesterol in fetuses, providing an important theoretical and experimental

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basis for resolving fetal-originated chronic diseases in adulthood.

Conflict of interest

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The authors declare no conflicts of interest.

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Acknowledgements

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This work was supported by grants from the National Key Research and Development Program of China (2017YFC1001300), the National Natural Science Foundation of China (Nos. 81430089,

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(No. WJ2017C0003).

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81673524, 81771543) and Hubei Province Health and Family Planning Scientific Research Project

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Figure legends Fig. 1. Effects of prenatal nicotine exposure (PNE) on maternal and fetal serum cholesterol levels. (A) maternal serum total cholesterol (TCH), high-density lipoprotein-cholesterol (HDL-C) and lowdensity lipoprotein-cholesterol (LDL-C); (B) female fetal serum TCH, HDL-C, LDL-C. Data are presented as Mean ± S.E.M., n=11 serum samples from 11 pregnant rats in each group. *P<0.05, **

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P<0.01 vs control.

Fig. 2. Effects of prenatal nicotine exposure (PNE) on placental cholesterol synthesis and transport. (A) the mRNA expression of hydroxymethylglutaryl-CoA reductase (HMGCR) and sterol regulatory element-binding protein-2 (SREBP-2) in placentas were measured by real-time quantitative PCR (RT-qPCR); the mRNA (B) and protein (C and D) levels of placental scavenger receptor B1 (SRB1), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1),

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liver X receptor (LXR)  and  were measured by RT-qPCR and western blotting respectively. Data are presented as Mean ± S.E.M., n=11 placentas from 11 pregnant rats in each group and n=3 for

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protein assay in each group. *P<0.05, **P<0.01 vs control.

Fig. 3. Effects of nicotine on cholesterol transporters in BeWo cells. (A and B) the mRNA expression of sterol regulatory element-binding protein-2 (SREBP-2) and hydroxymethylglutaryl-CoA reductase (HMGCR) was measured by real-time quantitative PCR. The mRNA (C-G) and protein (H and I) expression of scavenger receptor B1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), liver X receptor (LXR)  and  were measured by

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real-time quantitative PCR (RT-qPCR) and western blotting respectively. Data are presented as mean

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± S.E.M., n=6 for RT-qPCR and n=3 for protein assay in each group. *P<0.05, **P<0.01 vs control.

Fig. 4. Effects of liver X receptor (LXR) agonist on cholesterol transporters in BeWo cells. The mRNA (A-C) and protein (D and E) expression of scavenger receptor B1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1) were measured by real-time quantitative PCR (RT-qPCR) and western blotting respectively. Data are presented as mean

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± S.E.M., n=6 for RT-qPCR and n=3 for protein assay in each group. *P<0.05, **P<0.01 vs control.

Fig. 5. Effects of nicotinic acetylcholine receptor (nAChR) antagonist on cholesterol transporters in BeWo cells. The mRNA (A-D) and protein (E and F) expression of scavenger receptor B1 (SR-B1), ATP-binding cassette transporter A1 (ABCA1), ATP-binding cassette transporter G1 (ABCG1), liver X receptor (LXR)  and  were measured by RT-qPCR and western blotting respectively. Data are presented as mean ± S.E.M., n=6 for RT-qPCR and n=3 for protein assay in each group. *P<0.05, **

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P<0.01 vs control.

Fig. 6. Placental mechanism of prenatal nicotine exposure-induced a low level of blood cholesterol in female fetuses. LXR: liver X receptor; nAChR: nicotinic acetylcholine receptor; SRB1: scavenger receptor B1; ABCA1: ATP-binding cassette transporter A1; ABCG1: ATP-binding

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cassette transporter G1.

Table 1. Human and rat primers used for real-time quantitative PCR. Forward primer(5’-3’)

Reverse primer(5’-3’)

Annealin g

LXRα (human ) LXRβ (human ) SR-B1

CCACTCAGAGCAAGTGTTT

CTTCTCAGTCTGTTCCACTT C

60℃, 30 s

CTTGAAGGACTTCACCTAC AG

AGATGTTGATGGCGATGAG

62℃, 30 s

TTGATGCCCAAGGTGATG

CCTTATCCTTTGAGCCCTTT

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GTCCAGCTTCTCTACCTAGT AA

CATTGGCCTGCTGTACTT

CGAAGAAAGACTCCCATCT C

CAAGAGCACAAGAGGAAG AG

GTTGAGCACAGGGTACTTT A

63℃, 30 s

CCGTGTTTCAGTCCAGTATG

60℃, 30 s

63℃, 30 s

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63℃, 30 s

TTCCTTCCCACTCCTCTAC

CTCTATGGGAGGTAGGAGTT 60℃, 30 AG s CATAGCCATCAGCATCTTCT GGCTCACCAGCTTCATTAG 63℃, 30 C s CTCTCCTACACGAGGATCAA CAGATCTCGGACAGCAAAG 63℃, 30

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(rat) SR-B1 (rat) ABCA1 (rat) ABCG1 (rat) GAPD

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CTGCAATCTCGGCACTTT

(human ) HMGC CTGGTGAGTTGTCCTTGATG R (rat) SREBP -2 (rat) LXRα (rat) LXRβ

60℃, 30 s

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(human ) ABCA1 (human ) ABCG1 (human ) GAPD H

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Genes

CTTCTGGTGCCCATCATTTA

CCTACAGCTTGGCTTCTTG

CTTAAAGGACCTGTCACTAC GCTCTCCCTTCCTTTCATTC AC CTCCTTCCAGACTTCCTTTC GCTCTGTGGAGGTAGTTAAT G GCAAGTTCAACG GCACAG GCCAGTAGACTCCACGACA

s 63℃, s 60℃, s 60℃, s 60℃,

30 30 30 30

H (Rat)

s

LXRα, Liver X receptor α; LXRβ, Liver X receptor β; SR-B1, scavenger receptor class B type 1; ABCA1, ATP-binding cassette transporter A1; ABCG1, ATP-binding cassette transporter G1; GAPDH, glyceraldehyde phosphate dehydrogenase; HMGCR, hydroxymethylglutaryl CoA reductase; SREBP-

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2, sterol regulatory element-binding protein-2.