Friedmacher Florian, Hofmann Alejandro Daniel, Takahashi Toshiaki, Takahashi Hiromizu, Kutasy Balazs, Puri Prem
National Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, Ireland.
Pediatr Surg Int. 2014 Dec;30(12):1183-90. doi: 10.1007/s00383-014-3605-8. Epub 2014 Oct 21.
Pulmonary hypoplasia (PH), characterized by alveolar immaturity, is one of the leading causes of respiratory insufficiency in newborns with congenital diaphragmatic hernia (CDH). Leptin (Lep) and its receptor (Lep-R) play an important role in fetal lung growth by stimulating alveolar differentiation and maturation. Lep and Lep-R are strongly expressed by alveolar cells during the saccular stage of fetal lung development. Lep-deficient mice exhibit decreased alveolarization with reduced pulmonary surfactant phospholipid synthesis, similar to human and nitrofen-induced PH. Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced PH. Recent studies have demonstrated that Lep and Lep-R expression in developing lungs is regulated by ATRA. We hypothesized that prenatal treatment with ATRA increases pulmonary Lep and Lep-R expression in the nitrofen model of CDH-associated PH.
Time-mated rats received either 100 mg nitrofen or vehicle via oral-gastric lavage on embryonic day 9.5 (E9.5). Control and nitrofen-exposed dams were randomly assigned to either intraperitoneal ATRA (5 mg/kg/d) or placebo administration on E18.5, E19.5 and E20.5. Fetal lungs were harvested on E21.5, and divided into Control+Placebo, Control+ATRA, Nitrofen+Placebo and Nitrofen+ATRA. Alveolarization was assessed using stereo- and morphometric analysis techniques. Surfactant phospholipid synthesis was analyzed by labeling for surfactant protein B (SP-B). Pulmonary gene expression levels of Lep and Lep-R were determined using quantitative real-time polymerase chain reaction. Immunohistochemical staining for Lep and Lep-R was performed to evaluate alveolar protein expression and localization.
In vivo administration of ATRA resulted in significantly increased lung-to-body weight ratio with enhanced radial alveolar count and decreased mean linear intercept compared to placebo treatment. Immunofluorescence analysis demonstrated markedly increased pulmonary SP-B expression in Nitrofen+ATRA compared to Nitrofen+Placebo. Relative mRNA expression of Lep and Lep-R was significantly increased in Nitrofen+ATRA compared to Nitrofen+Placebo. Lep and Lep-R immunoreactivity was markedly increased in interstitial and alveolar epithelial cells of Nitrofen+ATRA compared to Nitrofen+Placebo.
Increased Lep and Lep-R expression after prenatal administration of ATRA in nitrofen-induced PH suggests that ATRA may have therapeutic potential in attenuating CDH-associated PH by stimulating alveolarization and de novo surfactant production.
肺发育不全（PH）以肺泡不成熟为特征，是先天性膈疝（CDH）新生儿呼吸功能不全的主要原因之一。瘦素（Lep）及其受体（Lep-R）通过刺激肺泡分化和成熟在胎儿肺生长中起重要作用。在胎儿肺发育的囊状期，肺泡细胞强烈表达Lep和Lep-R。Lep基因缺陷小鼠表现出肺泡化减少，肺表面活性物质磷脂合成减少，类似于人类和硝基芬诱导的PH。已证明产前给予全反式维甲酸（ATRA）可刺激硝基芬诱导的PH中的肺泡化。最近的研究表明，发育中肺中的Lep和Lep-R表达受ATRA调节。我们假设在CDH相关PH的硝基芬模型中，产前用ATRA治疗可增加肺Lep和Lep-R表达。
在胚胎第9.5天（E9.5），经口胃灌洗给定时交配的大鼠给予100 mg硝基芬或赋形剂。将对照和暴露于硝基芬的母鼠在E18.5、E19.5和E20.5随机分为腹腔注射ATRA（5 mg/kg/d）或安慰剂组。在E21.5收获胎儿肺，并分为对照+安慰剂、对照+ATRA、硝基芬+安慰剂和硝基芬+ATRA组。使用立体和形态计量分析技术评估肺泡化。通过标记表面活性蛋白B（SP-B）分析表面活性物质磷脂合成。使用定量实时聚合酶链反应测定肺中Lep和Lep-R的基因表达水平。进行Lep和Lep-R的免疫组织化学染色以评估肺泡蛋白表达和定位。
与安慰剂治疗相比，体内给予ATRA导致肺与体重比显著增加，径向肺泡计数增加，平均线性截距降低。免疫荧光分析表明，与硝基芬+安慰剂相比，硝基芬+ATRA中肺SP-B表达明显增加。与硝基芬+安慰剂相比，硝基芬+ATRA中Lep和Lep-R的相对mRNA表达显著增加。与硝基芬+安慰剂相比，硝基芬+ATRA的间质和肺泡上皮细胞中Lep和Lep-R免疫反应性明显增加。
在硝基芬诱导的PH中，产前给予ATRA后Lep和Lep-R表达增加，表明ATRA可能通过刺激肺泡化和从头产生表面活性物质而具有减轻CDH相关PH 的治疗潜力。
