3.1 The phenomenon of low maternally derived glucocorticoid exposure in fetal blood induced by PDE and its occurrence mechanism
The level of basic glucocorticoid (cortisol in humans and corticosterone in rodents) during the intrauterine period is a key factor in regulating the maturity and function of fetal tissue. To explore the effects of PDE on fetal serum glucocorticoid levels and its occurrence mechanism, we first examined the corticosterone levels in maternal and fetal serum. Compared with the maternal control rats, the serum corticosterone levels were significantly reduced in the PDE group (P <0.05, Fig 1A), while the serum ACTH levels were not markedly changed (Fig 1B). Meanwhile, qRT-PCR analysis showed that the mRNA expressions of adrenal steroidogenic genes, including StAR, P450scc, P450c21, and P450c11, were significantly reduced in the PDE maternal rats (P <0.05,P <0.01, Fig 1C). Compared with the fetal control rats, the serum corticosterone levels were also significantly reduced in the PDE group (P <0.01, Fig 1D). The placental glucocorticoid barrier is a crucial factor controlling the transfer of maternal glucocorticoid to the fetus. Hence, we examined the expression of the placental glucocorticoid barrier-related transport protein (P-gp) and glucocorticoid metabolic enzymes (11β-HSDs). The results showed that compared with the control group, the mRNA expression of P-gp encoding genes — Mdr1a and Mdr1b in placental were significantly reduced in the PDE group, and the mRNA expression of placental 11β-HSD1 was increased (P <0.01, Fig 1E) while that of 11β-HSD2 was not significantly changed, resulting in the markedly increased ratio of 11β-HSD1/11β-HSD2 mRNA expression (P <0.01, Fig 1F). Meanwhile, western blotting analysis also suggested similar conclusions at the protein expression level (P <0.05,P <0.01, Fig 1G-I). Furthermore, we analyzed the correlation between maternal serum corticosterone levels and fetal serum corticosterone levels, and found a significant positive correlation between them (P <0.01, Fig 1J). These results indicated that the serum corticosterone levels were reduced in the PDE fetal rats, and its occurrence was mainly related to the decrease of maternal corticosterone levels and the open placental glucocorticoid barrier.
3.2 Changes in fetal adrenal GC-IGF1 axis andsteroidogenic function induced by PDE and its possible mechanisms
Compared with the control group, the mRNA expressions of fetal adrenal steroidogenic genes, including StAR, P450scc, P450c21, and P450c11, were reduced in the PDE group (P <0.01, Fig 2A), and StAR protein expression was also significantly reduced (P <0.05, Fig 2B, C). We also observed the expression of fetal adrenal IGF1 and its downstream signaling pathway, and found that the mRNA expression levels of IGF1, IGF1R, and Akt1 were markedly decreased in the PDE group (P <0.01, Fig 2D). Further, we explored their occurrence mechanism. The results showed that compared with the control, adrenal glucocorticoid receptor α (GRα) mRNA expression was significantly increased in the PDE fetal rats (P <0.01, Fig 2E). Fetal adrenal microarray analysis indicated that the expression of miR-370-3p was reduced in the PDE group (Fig 2F), and Sirt3 might be the target gene of miR-370-3p through bioinformatics prediction. We confirmed that adrenal miR-370-3p expression was decreased and Sirt3 expression increased in the PDE fetal rats using qRT-PCR (P <0.01, Fig 2G, H). Moreover, screening revealed that the H3K9ac and H3K27ac levels of the IGF1 promoter region were reduced in the PDE fetal adrenal glands (P <0.01, Fig 2I) while the H3K14ac level was not significantly changed (Fig 2I). These data indicated that the GRα/miR-370-3p/Sirt3/IGF1 signaling pathway might mediate the adrenal low steroidogenic function in the PDE fetal rats through histone acetylation modification.