Takahashi Hiromizu, Kutasy Balazs, Pes Lara, Paradisi Francesca, Puri Prem
National Children's Research Centre, Our Lady's Children's Hospital, Dublin, Ireland.
Pediatr Surg Int. 2015 Jan;31(1):37-43. doi: 10.1007/s00383-014-3621-8. Epub 2014 Oct 26.
Retinoids are essential for fetal and lung development. Beta-carotene(BC) is the main dietary retinoid source and beta-carotene-15,15'-oxygenase-1 and 2 (Bcmo1,2) is the primary enzyme generating retinoid from BC in adult mammalian tissues. Placenta has a major role in the retinol homeostasis in fetal life: Since there is no fetal retinol synthesis, maternal retinol has to cross the placenta. It has been recently shown that BC can be converted to retinol by Bcmo1,2 in placenta for retinol transfer and moreover, BC can cross the placenta intact. The placental Bcmo1,2 expression is tightly controlled by placental retinol level. In severe retinol deficiency it has been shown that placental Bcmo1,2 expression are increased for generating retinol from dietary maternal BC even when the main retinol transfer is blocked. In recent years, low pulmonary retinol levels and disrupted retinoid signaling pathway have been implicated in the pathogenesis of pulmonary hypoplasia and congenital diaphragmatic hernia (CDH) in the nitrofen model of CDH. Recently, it has been demonstrated that the main retinol transfer in the placenta is blocked in the nitrofen model of CDH causing increased placental and decreased serum retinol level. The aim of our study was to determine maternal and fetal β-carotene levels and to investigate the hypothesis that placental expression of BCMO1 and BCMO2 is altered in nitrofen-exposed rat fetuses with CDH.
Pregnant rats were exposed to either olive oil or nitrofen on day 9 of gestation (D9). Maternal and fetal serum, placenta, liver and left lungs were harvested on D21 and divided into two groups: control (n = 8) and nitrofen with CDH (n = 8). Immunochistochemistry was performed to evaluate trophoblasts by cytokeratin expression and placental Bcmo1,2 expression. Expression levels of Bcmo1,2 genes in fetal lungs and liver were determined using RT-PCR and immunohistochemistry. BC level was measured using HPLC.
Markedly increased decidual Bcmo1,2 immunoreactivity was observed in CDH group compared to controls. There was no difference neither in the trophoblastic Bcmo1,2 immunoreactivity nor in the pulmonary and liver Bcmo1,2 expression compared to controls. There was no significant difference in maternal serum BC levels between control and CDH mothers (2.14 ± 0.55 vs 2.56 ± 1.6 μM/g, p = 0.8). BC was not detectable neither in the fetal serum nor liver or lungs.
Our data show that nitrofen increases maternal but not fetal Bcmo1,2 expression in the placenta in nitrofen-induced CDH group. The markedly increased decidual Bcmo1,2 expression suggests that nitrofen may trigger local, decidual retinol synthesis in the nitrofen model of CDH.
类视黄醇对胎儿和肺部发育至关重要。β-胡萝卜素（BC）是饮食中类视黄醇的主要来源，β-胡萝卜素-15,15'-加氧酶-1和2（Bcmo1、2）是成年哺乳动物组织中从BC生成类视黄醇的主要酶。胎盘在胎儿期视黄醇稳态中起主要作用：由于胎儿不能合成视黄醇，母体视黄醇必须穿过胎盘。最近研究表明，BC可通过胎盘内的Bcmo1、2转化为视黄醇以进行视黄醇转运，此外，BC可完整穿过胎盘。胎盘Bcmo1、2的表达受胎盘视黄醇水平严格控制。在严重视黄醇缺乏时，已表明即使主要视黄醇转运受阻，胎盘Bcmo1、2的表达仍会增加，以便从母体饮食中的BC生成视黄醇。近年来，在先天性膈疝（CDH）的硝呋烯腙模型中，低肺视黄醇水平和类视黄醇信号通路紊乱与肺发育不全和先天性膈疝的发病机制有关。最近已证明，在CDH的硝呋烯腙模型中，胎盘内主要视黄醇转运受阻，导致胎盘视黄醇水平升高而血清视黄醇水平降低。我们研究的目的是测定母体和胎儿的β-胡萝卜素水平，并研究在硝呋烯腙暴露致CDH的大鼠胎儿中，BCMO1和BCMO2的胎盘表达是否改变这一假设。
妊娠第9天（D9），将孕鼠暴露于橄榄油或硝呋烯腙中。在D21收集母体和胎儿的血清、胎盘、肝脏和左肺，并分为两组：对照组（n = 8）和患有CDH的硝呋烯腙组（n = 8）。通过细胞角蛋白表达和胎盘Bcmo1、2表达进行免疫组织化学，以评估滋养层细胞。使用逆转录聚合酶链反应（RT-PCR）和免疫组织化学测定胎儿肺和肝脏中Bcmo1、2基因的表达水平。使用高效液相色谱法（HPLC）测量BC水平。
与对照组相比，CDH组蜕膜Bcmo1、2免疫反应性显著增加。与对照组相比，滋养层Bcmo1、2免疫反应性以及肺和肝脏Bcmo1、2表达均无差异。对照组和患有CDH的母亲之间母体血清BC水平无显著差异（2.14±0.55对2.56±1.6μM/g，p = 0.8）。在胎儿血清、肝脏或肺中均未检测到BC。
我们的数据表明，在硝呋烯腙诱导的CDH组中，硝呋烯腙增加了母体而非胎儿胎盘内Bcmo1、2的表达。蜕膜Bcmo1、2表达显著增加表明，在CDH的硝呋烯腙模型中，硝呋烯腙可能触发局部蜕膜视黄醇合成。
