Effects of uteroplacental insufficiency on growth-restricted rats with altered lung development: A metabolomic analysis

Abstract

Background: Intrauterine growth restriction (IUGR) is among the most challenging problems in antenatal care. Several factors implicated in the pathophysiology of IUGR have been identified. We aimed to investigate the effect of UPI on lung development by identifying metabolic changes during the first seven days of postnatal life.

Materials and methods: On gestation day 17, four time-dated pregnant Sprague Dawley rats were randomized to a IUGR group or a control group, which underwent an IUGR protocol comprising bilateral uterine vessel ligation and sham surgery, respectively. On gestation day 22, 39 control and 26 IUGR pups were naturally delivered. The rat pups were randomly selected from the control and IUGR group on postnatal day 7. The pups' lungs were excised for histological, Western blot, and metabolomic analyses. Liquid chromatography mass spectrometry was performed for metabolomic analyses.

Results: UPI induced IUGR, as evidenced by the IUGR rat pups having a significantly lower average body weight than the control rat pups on postnatal day 7. The control rats exhibited healthy endothelial cell healthy and vascular development, and the IUGR rats had a significantly lower average radial alveolar count than the control rats. The mean birth weight of the 26 IUGR rats (5.89 ± 0.74 g) was significantly lower than that of the 39 control rats (6.36 ± 0.55 g; p < 0.01). UPI decreased the levels of platelet-derived growth factor-A (PDGF-A) and PDGF-B in the IUGR newborn rats. One-way analysis of variance revealed 345 features in the pathway, 14 of which were significant. Regarding major differential metabolites, 10 of the 65 metabolites examined differed significantly between the groups (p < 0.05). Metabolite pathway enrichment analysis revealed significant between-group differences in the metabolism of glutathione, arginine-proline, thiamine, taurine-hypotaurine, pantothenate, alanine-aspartate-glutamate, cysteine-methionine, glycine-serine-threonine, glycerophospholipid, and purine as well as in the biosynthesis of aminoacyl-tRNA, pantothenate, and CoA.

Conclusions: UPI alters lung development and metabolomics in growth-restricted newborn rats. Our findings may elucidate new metabolic mechanisms underlying IUGR-induced altered lung development and serve as a reference for the development of prevention and treatment strategies for IUGR-induced altered lung development.

Keywords: intrauterine growth restriction; lung development; metabolomics; radial alveolar count; uteroplacental insufficiency.

Figure 1

Metabolomic data analysis workflow. The workflow included a discrete combination of quadrupole time-of-flight mass spectrometry, multivariate statistics, multivariate machine learning, filtering of differentially expressed metabolites, compound identification, and pathway enrichment analysis.

Figure 2

(A) Representative lung sections stained with hematoxylin and eosin and radial alveolar count and mean septal thickness (B) representative Western blots of PDGF-A and PDGF-B in the lung tissue samples of the control and intrauterine growth restriction (IUGR) groups on postnatal day 7. ^*Denotes a respiratory bronchiole. Compared with the control rats, the IUGR rats had a significantly higher radial alveolar count, significantly lower average PDGF-A levels, and comparable mean septal thickness and PDGF-B levels. Data are presented as means ± SDs. ^*p < 0.05, ^**p < 0.01.

Figure 3

(A) Principal component analysis (PCA) score plot of IUGR rat lung tissue samples harvested on postnatal day 7 (in green) relative to the control samples (in orange). (B) Partial least squares discriminant analysis (PLS-DA) score plot of the IUGR rat lung tissue samples relative to the control samples. (C) Three-dimensional PCA score chart of IUGR rat lung tissue samples harvested on postnatal day 7 (in green) relative to the control samples (in orange). (D) Three-dimensional PLS-DA score chart of the IUGR rat lung tissue samples relative to the control samples (n = 5).

Figure 4

(A) Hierarchical clustering analysis and heat map of control and IUGR rat lung tissue samples harvested on postnatal day 7. Color scale represents the scaled abundance of each variable, with red indicating high abundance and blue indicating low abundance. Compounds represented in the heat map are numbered according to their peak numbers (n = 5). (B) Volcano plots of ultraperformance liquid chromatography–mass spectrometry (MS)/MS datasets. The y-axis represents p value converted to –log [p value] and the x-axis represents log₂ [fold change]. Significant metabolites (fold change <1.2, p value < 0.05) were highlighted in blue and red. Gray points represent non-significant metabolites.

Figure 5

(A) Bar chart of the results of the enrichment analyses highlighting the altered metabolic pathways in the control and IUGR rat lung tissue samples harvested on postnatal day 7. (B) Bubble plot of the results of pathway enrichment of the control and IUGR rat lung tissue samples harvested on postnatal day 7 (n = 5).

Figure 6

Relative ion intensities of significantly differential metabolites in the control and IUGR rat lung tissue samples harvested on postnatal day 7 (n = 5). Data are presented as means ± SDs. ns, not significant. ^*p < 0.05; ^**p < 0.01; ^***p < 0.001.