Introduction

Intrauterine growth restriction (IUGR) has a global frequency of roughly 10% and is linked with a higher likelihood of fetal and neonatal mortality and morbidity.1,2 Current evidence suggests that stressful prenatal settings alter the development of lungs and result in lasting abnormalities in the structure of lungs and decreased respiratory performance during postnatal life.3,4,5 Our and other preclinical investigations have shown that IUGR is related to decreased lung development during the neonate and early childhood.6,7 Human investigation has discovered that IUGR increases the chances of having lower lung function in individuals during their developmental years, adolescence, and later in life.8,9,10,11,12 The condition is marked by reduced placental activity and impaired vital nutrients and oxygen transfer to the developing fetus.2,4,13 Previous studies have demonstrated that an impaired placental function may impede the appropriate growth and development of a fetus, and is closely associated with bronchopulmonary dysplasia.14,15 A fetus’s growth rate below its genetically determined potential size per race and sex is characterized as IUGR.16,17 Research conducted both in vivo and in vitro has demonstrated that adipose tissue is capable of synthesizing various amino acids.18 Within adipose tissue, leptin is mainly synthesized and secreted.19,20 Leptin is a hormone approximately 16-kDa in size and possesses pro-inflammatory properties. As an adipokine, it significantly impacts both innate and adaptive immune responses. Leptin performs essential bodily functions such as cellular homeostasis, metabolism, angiogenesis, and regulating the immune system and inflammatory responses. Additionally, studies have shown that it can cause airway smooth muscle cells to produce higher levels of vascular endothelial growth factors (VEGF), which might be linked to IUGR.21,22,23,24 Pathways related to amino acid metabolism, such as the urea cycle, aspartate metabolism, arginine and proline metabolism, and glycine and serine metabolism, demonstrate a significant association with leptin levels. 25,26,27 Nonetheless, leptin and its receptors are present in fat tissue and various organs, such as the lungs.20,28 Leptin levels increase at late gestation and influence postnatal organ development.29 Administering leptin to pregnant rats increased the lung weight/body weight ratio and promoted alveolar type II cell number and maturation, enhancing surfactant protein expression in rat fetuses.30 In a rat model of fetal growth restriction, Chen et al. discovered that administering maternal leptin treatment improved lung maturation and elevated levels of SP-A expression.31 These lung maturational effects of leptin were also found in the ovine fetus.32 In rats, postnatal leptin deficiency persistently impaired lung development and decreased surface area and lung volume from 2 to 10 weeks of age.33

These findings suggest that leptin is critical for postnatal lung remodeling. However, the lung leptin status during the postnatal period was unknown in intrauterine growth-restricted offspring. We hypothesized that if leptin has a significant impact on lung development, UPI would decrease leptin during the postnatal period, and leptin expression would be correlated with lung development in growth-restricted rat offspring. This study aimed to investigate UPI’s effects on lung leptin expression and lung development in rat offspring.

Materials and Methods

Animal model

The animal study conducted at Taipei Medical University was reviewed and approved by the Laboratory Animal Care Committee. Time-dated pregnant Sprague-Dawley rats were kept in separate cages and given laboratory food and water as per their needs. They were exposed to a 12:12-hour light-dark cycle. On the 17th day of gestation, either bilateral uterine artery ligation was performed to induce uteroplacental insufficiency (IUGR group) or sham surgery was done (control group).7 The dams recovered fast from the uterine artery ligation and sham surgeries and were able to resume feeding the next day. On gestation day 22, all newborn rats were born normally. The day of birth for the rats was designated as postnatal day 0. The newborn pups were gathered and redistributed among the mothers within 12 hours of birth. To ensure all pups had equal access to breast milk, the control and IUGR groups’ litters were culled to nine and four pups, respectively, after the pups were euthanized on postnatal days 0 and 7. The pulmonary tissues employed in these experiments were obtained from a previous study to investigate metabolomics, which detected metabolites, and miobiota investigation, which revealed the radial alveolar count (RAC) and the von Willebrand Factor (vWF) in IUGR rats.34,35

Rat tissue collection and processing

On the postnatal days 0 and 7, rats were randomly selected for examination from each group, irrespective of sex. Animals were euthanized with an overdose of isoflurane inhalation, body weights were recorded and blood from the heart was withdrawn to check serum leptin levels, and lungs were harvested for lung leptin and leptin receptor levels, histological and western blot analysis.

Serum leptin levels

Serum leptin levels were measured using the Bio-Plex 200 analyzer (Bio-Rad Laboratories, Hercules, CA) in accordance with the manufacturer’s instructions. In sample diluent, we diluted the serum 1:4. The samples were measured using a low Photomultiplier Tube setting, as specified in the assay manual. The Bio-Plex filter plates and manual vacuum manifold were used for the tests. The Bio-Plex ManagerTM software was used.

Lung morphometry

The volume fraction was calculated by digitizing pictures and selecting five nonoverlapping fields (four corners and one middle region) from each section. At a final magnification of 200×, the images were printed and analyzed. Each animal was observed in 10 different regions at random. The number of points along the alveolar airways and alveolar walls was counted by superimposing 49-point clear grids onto enlarged printed pictures. The volume fraction was equal to Pi/Pt, where Pi was the number of test points that touched the structure of interest and Pt was the total number of points that hit the reference space.36

Western blot analysis of leptin and leptin receptor

Lung tissues were homogenized in ice-cold buffer containing 50 mM TrisHCl (pH 7.5), 1 mM egtazic acid, 1 mM ethylenediaminetetraacetic acid, and a protease inhibitor cocktail (complete minitablets; Roche, Mannheim, Germany). The samples were sonicated and then centrifuged at 500 g for 20 min at 4 °C to remove cellular debris. Proteins (30 μg) were resolved on 12% sodium dodecyl sulfate–polyaylamide gel electrophoresis gels under reducing conditions. Next, they were electroblotted onto a polyvinylidene difluoride membrane (ImmobilonP, Millipore, Bedford, MA). Following blocking with 5% nonfat dry milk, the membranes were incubated with an anti-leptin antibody (1:500, ab117751, Abcam, Boston, MA), an anti-leptin receptor antibody (1:750, ab5593, Abcam, Boston), and an anti-β-actin antibody (1:1000, C4 sc-47778, Santa uz Biotechnology, Santa uz, CA). They were subsequently incubated with horseradish peroxidase-conjugated goat antirabbit IgG or anti-mouse IgG (Pierce Biotechnology, Rockford, IL). The protein bands were detected using the BioSpectrum AC System (UVP, Upland, CA) and VisionWorks LS Software version 8.6 (UVP, Upland).

Immunohistochemistry of proliferating cell nuclear antigen

Proliferating cell nuclear antigen (PCNA) is expressed in cells from late G1 through S-phase. As a result, antibodies targeted towards this particular protein should be used as a probe for immunocytochemically marking the nuclei of proliferating cells.37 By measuring immunostained pulmonary nuclei, we demonstrated the feasibility and reliability of this approach. Furthermore, after deparaffinization in xylene and rehydration in an alcohol series, the 5-μm lung sections were preincubated for 1 hour at room temperature in 0.1 M PBS containing 10% normal goat serum and 0.3% H2O2 to block endogenous peroxidase activity and nonspecific antibody binding. The sections were then incubated for 20 hours at 4 °C with rabbit monoclonal anti-PCNA antibody (1:100, ab92552 Abcam, Cambridge, MA). The sections were followed for one hour at 37 °C with biotinylated goat anti-rabbit immunoglobulin G (1:200, Vector Laboratories, Burlingame, CA), and then reacted with the reagents from an Avidin–Biotin Complex kit (Vector Laboratories). The brown reaction products were visualized after using a diaminobenzidine substrate kit (Vector Laboratories) according to the manufacturer’s recommendations. The immunostained sections were viewed and photographed using a Nikon Eclipse E600 (Nikon, Tokyo, Japan). PCNA-stained vessel density was calculated in an unbiased manner at 200× magnification, with at least five random lung regions selected per animal, according to the method modified by Agard et al.38. The digital images were quantified using the automatic object counting and measuring process to generate a percentage of positive vessels.

Statistical analysis

Data are expressed in terms of the mean ± SD. Age groups were compared using Student’s t-test. Correlations between the serum leptin, the RAC, and the vWF were analyzed using Pearson’s correlation test. Differences were considered statistically significant at p < 0.05 and a strong positive linear relationship at r’s value near 1.39

Results

UPI decreased body weights at birth and on postnatal days 0 and 7

In summary, four sham-operated delivered 39 control pups and three dams with UPI delivered 26 IUGR rats. The IUGR rats’ mean birth weight (5.89 ± 0.74 g) was significantly lower than the control rats (6.36 ± 0.55 g; p < 0.01). These findings indicate that IUGR has been established. We retrieved 12 and 8 pups from the sham-operated and UPI-induced dams for examination on postnatal day 0. On postnatal day 7, we retrieved eight pups from the sham-operated and eight pups from the UPI-induced dams, respectively. On both postnatal days 0 and 7, the IUGR rats had significantly lower body weights than the control rats (Fig. 1a).

Fig. 1: The effect of UPI on rat pup weight and serum leptin.
figure 1

a Body weight and b serum leptin levels (pg/ml) of the control and intrauterine growth restriction (IUGR) groups on postnatal days 0 and 7. Compared with the control rats, the IUGR rats had a significantly lower body weight and serum leptin. Data are expressed in terms of the mean ± standard deviation. Age groups were compared using Student’s t-test (control, n = 12; IUGR, n = 8) (**p < 0.01, ***p < 0.001 vs. control).

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UPI decreased serum leptin levels in growth-restricted newborn rats

The lungs of the IUGR rats exhibited significantly lower serum leptin levels on postnatal day 0 (253.65 pg/ml; p < 0.001) and postnatal day 7 (270.36 pg/ml; p < 0.001) than the control rats on postnatal days 0 (429.41 pg/ml) and 7 (445.40 pg/ml) (Fig. 1b).

UPI decreased lung leptin and leptin receptor expression in growth-restricted newborn rats

Representative Western blots and quantitative data determined using densitometry for lung leptin and leptin receptor are presented in Fig. 2a, b. The IUGR rats had significantly lower leptin and leptin receptor levels on postnatal day 0 (p < 0.01) and 7 (p < 0.05) than the control rats, respectively.

Fig. 2: The effect of UPI on lung leptin and leptin-receptor expression.
figure 2

a Representative western blots of leptin and (b) leptin-receptor in the lung tissue sample of the control and intrauterine growth restriction (IUGR) groups on postnatal days 0 and 7. Compared with the control rats, the IUGR rats had significantly lower leptin and leptin-receptor. Data are expressed in terms of the mean ± standard deviation. Age groups were compared using Student’s t-test (control, n = 12; IUGR, n = 8) (*p < 0.05, **p < 0.01 vs. control).

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UPI altered lung morphometry in growth-restricted newborn rats

Figure 3a presents the representative lung tissue sections stained with hematoxylin and eosin on postnatal days 0 and 7. In both those days, the lungs of rats born to control dams exhibited normal morphology. The lungs of the IUGR rats exhibited a significantly higher volume fraction of alveolar airspace on postnatal day 0 (p < 0.01) and 7 (p < 0.001). The IUGR rats had a significantly lower volume fraction of the alveolar wall on postnatal day 0 (p < 0.05) and 7 (p < 0.001) than the control rats (Fig. 3b).

Fig. 3: The effect of UPI on lung morphometry.
figure 3

a Representative lung sections stained with hematoxylin and eosin, b alveolar space volume fraction, and alveolar wall volume fraction in the lung tissue sample of the control and intrauterine growth restriction (IUGR) groups on postnatal days 0 and 7. Compared with the control rats, the IUGR rats had a significantly higher alveolar space volume fraction and lower alveolar wall volume fraction. Data are expressed in terms of the mean ± standard deviation. Age groups were compared using Student’s t-test (control, n = 12; IUGR, n = 8) (*p < 0.05, **p < 0.01, ***p < 0.001 vs. control).

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UPI decreased lung PCNA expression in growth-restricted newborn rats

Representative immunohistochemical images of PCNA are presented in Fig. 4a and the results of semiquantitative analysis for vascular density determined by PCNA-positive vessels. The control rats exhibited high and similar PCNA immunoreactivity on postnatal days 0 and 7. The PCNA immunoreactivity and vascular density of the IUGR rats were significantly lower on postnatal days 0 (p < 0.0001) and 7 (p < 0.001) than the control group (Fig. 4b).

Fig. 4: The effect of UPI on lung PCNA expression.
figure 4

a Representative immunohistochemistry for proliferating cell nuclear antigen (PCNA) and b semiquantitative analysis for vascular density determined by PCNA-positive vessels in the lung tissue sample of the control and intrauterine growth restriction (IUGR) groups on postnatal days 0 and 7. Compared with the control rats, the IUGR rats had a significantly lower PCNA-stained vessel. Data are expressed in terms of the mean ± standard deviation. Age groups were compared using Student’s t-test (control, n = 12; IUGR, n = 8) (***p < 0.001, ****p < 0.0001 vs. control).

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UPI had high correlations between serum leptin, RAC, vWF, and metabolites

Strong correlations were found between serum leptin and RAC (r = 0.876, p = 4.08 × 10–7), and vWF (r = 0.8911, p = 1.3 × 10–7) on postnatal days 0 (Fig. 5a), serum leptin and RAC (r = 0.617, p = 0.004) and vWF (r = 0.9324, p = 2.1 × 10–9) on postnatal day 7 (Fig. 5b), which metabolite data from IUGR rats’ metabolomics analysis, RAC and vWF data from microbiota research, derived from previous studies [34,35]. Moreover, the correlation coefficient showed high correlations between RAC and metabolites such as LysoPC(20:5(5Z,8Z,11Z,14Z,17Z)/0:0),1-Isopropyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3-yl)methyl)-6-(2-(4-methylpipe razin-1yl)pyridin-4-yl)-1H-inazole-4-carboxamide,Cytosine,1H-Imidazole-4-carboxamide,3-Aminopyrazin-2-ol, Imexon (p < 0.05), and vWF with metabolites such as LysoPC(20:5(5Z,8Z, 11Z,14Z,17Z)/0:0),1-Isopropyl-N-((6-methyl-2-oxo-4-propyl-1,2-dihydropyridin-3yl)methyl) -6-(2-(4-methylpipe-razin-1yl)pyridin-4-yl)-1H-indazole-4-carboxamid, S-Methylmethanesulf inothioate (p < 0.05), Val-Pro-Asp-Pro-Arg, and viloxazine (p < 0.01) on postnatal day 7.

Fig. 5: Strong correlation between serum leptin, RAC, and vWF.
figure 5

a Correlations coefficient between serum leptin, radial alveolar count (RAC), and von Willebrand Factor (vWF) in the lung tissue sample of the control and intrauterine growth restriction (IUGR) groups on postnatal days 0 and b 7. Correlations were analyzed using Pearson’s correlation test (control, n = 12; IUGR, n = 8).

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Discussion

The present study was designed to unravel the effects of uteroplacental insufficiency-induced IUGR on lung leptin expression and lung morphology using an experimental animal model. We found that experimental IUGR-induced altered lung development during early postnatal development in rats. Our in vivo model demonstrated that UPI during the saccular stage of lung development impaired lung and vascular development, as evidenced by a higher volume fraction of the alveolar airspace and lower volume fraction of the alveolar wall, and decreased vascular density. This is consistent with defective alveolarization in newborn rats exposed to uteroplacental insufficiency during lung development.7,40 These UPI effects were associated with decreased serum leptin and decreased lung leptin and leptin receptor expression, suggesting that the leptin pathway is involved in UPI-induced impaired lung development during the postnatal period.

In this study, IUGR was induced by UPI and all sham-operated and UPI-induced dams were fed standard rat chow through the pregnancy and nourished their offspring during the lactation period. We found that UPI affected body weight in IUGR rats. The IUGR rats exhibited significantly lower body weights at birth (p < 0.01) and on postnatal days 7 (p < 0.001) than control rats. These findings indicate that IUGR has been established.

On postnatal days 0 and 7, these histological abnormalities were the most and least significant, respectively. These findings suggest that developmental exposure to uteroplacental insufficiency has the greatest impact on lung maturation during the immediate postnatal period. Decreased alveolar surface area is associated with reduced lung compliance and increased resistance, while a decreased alveolar wall volume fraction is associated with disturbed pulmonary diffusion capacity.41 However, alveolar and capillary structure estimations are typically associated with measurements of gas diffusion.41 Moreover, PCNA has been investigated as a cell proliferation marker in rat offspring lung tissue. In the presence of peroxidase enzymes that evaluate vascular density, PCNA-stained vessels using 3,3’-diaminobenzidine eate a brown reaction result. Interestingly, the assessment of PCNA revealed that the control rats had high and similar PCNA immunoreactivity, the IUGR rats group had a significant decrease on postnatal days 0 and 7, representing the proliferation in the lung. In support, PCNA promotes the proliferation of cells and growth, also known as a proliferative marker in the lungs.42,43,44 On the other hand, previous studies showed PCNA levels were not changed by the effect of IUGR on apoptosis in neonatal and adult rat lungs,45 and another clinical finding in humans showed that PCNA in IUGR with catchup growth group had a significant increase in primary neonatal myofibroblasts from rat lungs of IUGR, supporting the increase in proliferation, indicated airway resistance, and reduced lung compliance.42 Our results were consistent with the latter concept.

Furthermore, we investigated the correlations between serum leptin, RAC, and vWF on postnatal days 0 and 7. Previous research revealed that various cell types in the healthy lung had high amounts of Ob-Rb,46,47 with particular leptin-binding sites detected in both bronchial and alveolar epithelial cells,48,49,50 airway smooth muscle cells, and (infiltrating) inflammatory cells. Notably, leptin significantly correlates with fibrinogen and von Willebrand factor.51,52 These studies support our findings whereas UPI had high correlations between serum leptin with RAC and vWF on postnatal days 0 and 7. On the other hand, we only investigated the correlation coefficient between RAC, vWF, and metabolites on postnatal day 7, whereas lysophosphatidylcholine (Lyso-PC) showed high correlations with both RAC and vWF. Lyso-PC, a minor fraction, plays a central role in phosphatidylcholine (PC) metabolism and substrate generation. Pulmonary surfactants primarily consist of phospholipids and surfactant proteins, with PC being the most abundant.53,54,55 However, Lyso-PC is potentially toxic, impairing alveolar epithelium barrier functions, causing cell swelling, membrane loss, and protein leak due to its wedge-shaped shape. Earlier research stated lyso-PC, a bioactive proinflammatory lipid, has a substantial impact on inflammatory disorders. LysoPC activates a wide variety of cell types, including endothelial cells, smooth muscle cells, monocytes, maophages, and T-cells. According to a recent study, LysoPC may control second messenger systems by using cloned orphan G-protein-coupled receptors, and directly modulates contractile responses in vascular smooth muscle and prevents relaxations that are dependent on the endothelium.54,56 As a result, based on our research, we speculate that lyso-PC is one of many potential metabolites that can foretell changes in lung development. These findings support the study of altered metabolites in metabolomic analysis and impaired lung development in microbiota investigation in IUGR rats.34,35

The limitation of this study is that it mainly concentrated on the lungs of IUGR and the control group, which only demonstrates the knowledge of itself and does not involve the use of other organs that enable the investigation of certain circumstances in the lungs, and the calculation of correlation between variables was only investigated on postnatal day 7.

In conclusion, leptin may be involved in the mechanism of UPI-induced impaired lung development during the postnatal period and suggests that leptin supplementation may be a promising treatment modality to enhance lung development in IUGR neonates.