3.3 Changes in adult adrenal GC-IGF1 axis and steroidogenic function induced by PDE and its possible mechanisms
To confirm that the PDE-induced inhibition of fetal adrenal steroidogenesis could continue after birth and explore its epigenetic programming mechanism, we detected the changes in adrenal steroidogenesis in PW 12 and PW28. The results showed that compared with the respective controls in PW12 and PW28, the serum corticosterone levels and the mRNA expression of the adrenal steroidogenic genes (StAR and P450scc) and IGF1 signaling pathway were significantly reduced in the PDE group (P <0.05, P <0.01, Fig 3A, B and D), and immunohistochemical examination confirmed that the protein expression of StAR was also significantly reduced (P <0.05, P <0.01, Fig 3C). Meanwhile, PDE decreased the expression of adrenal GRα and miR-370-3p, increased Sirt3 expression, and decreased the H3K27ac level of IGF1 promoter region (P <0.05, P <0.01, Fig 3E-H), while H3K9ac and H3K14ac levels of IGF1 did not change significantly (Fig 3H). This suggested that the adrenal steroidogenic function was continually reduced in the PDE offspring after birth, and GRα/miR-370-3p/Sirt3/IGF1 signaling pathway might mediate the low adrenal steroidogenesis in the PDE adult offspring rats through histone acetylation modification.
3.4 Changes in positive programming of adrenal GC-IGF1 axis in PDE adult offspring with chronic stress and its related mechanisms
Furthermore, we confirmed the changes in positive programming of the adrenal GC-IGF1 axis in PDE adult rats through chronic stress (2 weeks of ice-water swimming). We found that serum corticosterone levels increased in both the control group and the PDE group with chronic stress, but the increase was more significant in the PDE group (P <0.05, P <0.01, Fig 4A). Next, compared with the groups without chronic stress, the expressions of adrenal IGF1 and steroidogenesis-related genes (StAR and P450scc) were significantly decreased in the control group with chronic stress, while significantly increased in the PDE group with chronic stress (P <0.05, P <0.01, Fig 4B, C). These results indicated that the adrenal GC-IGF1 axis of the normal offspring rats showed the negative changes, while that of the PDE offspring rats showed positive changes.
We also detected the changes of relevant indicators of the GC-IGF1 axis positive programming mechanism in the PDE offspring rats. Compared with the respective groups without chronic stress, the expression of adrenal GRα was increased, the expression of miR-370-3p was decreased, the expression of Sirt3 was increased, and the H3K27ac level of IGF1 promoter region was decreased in the control group with chronic stress (P <0.05, P <0.01, Fig 4D-G). However, these indicators showed consistent opposite changes in the PDE group with chronic stress (P <0.05, P <0.01, Fig 4D-G). These data indicated that the expression changes of the GRα/miR-370-3p/Sirt3 pathway and the H3K27ac level of the IGF1 promoter region were presented in a glucocorticoid-dependent manner in the adult PDE offspring rats.
3.5 Inhibition of dexamethasone on IGF1 andsteroidogenic function inNCI-H295R cells
Dexamethasone can easily enter the fetal blood through placentas. This study sought to determine whether dexamethasone in utero affected fetal adrenal steroidogenic function and exhibited an intrauterine programming effect. First, we detected the effects of dexamethasone with different concentrations (0, 20, 100, 500 nM) on the mRNA and protein expression of IGF1 and StAR, and the level of intracellular cortisol in the human adrenocortical cell line (NCI-H295R). The results showed that compared with the control, dexamethasone concentration-dependently decreased the above indicators (P< 0.05, P< 0.01, Fig 5A-D). Compared with the 500 nM dexamethasone group, exogenous IGF1 treatment reversed the inhibitory effect of dexamethasone on StAR expression (P< 0.05, P< 0.01, Fig 5E). Meanwhile, we observed the effects of dexamethasone on histone acetylation of the IGF1 promoter region, and found that the H3K9ac level of the IGF1 promoter region was significantly reduced (P< 0.05, Fig 5F), while there were no significant changes in the H3K14ac and H3K27ac levels (Fig 5F). This suggested that dexamethasone reduced steroidogenic function by inhibiting the H3K9ac and expression levels of IGF1 in the human adrenocortical cells.
3.6 Inhibition of GC-IGF1 axis andsteroidogenic function by low physiological concentrations of cortisol in NCI-H295R cells
As mentioned above, we had confirmed a low level of maternal glucocorticoid in fetal rats induced by PDE. Here, we further explored the effect of low glucocorticoid levels on adrenal steroidogenic function in NCI-H295R cells. Studies have reported that physiological cortisol concentration fluctuation range of normal neonatal is 11.67 +/- 4.68 μg/dl (303.5 +/- 129.1 nM), while neonates treated with dexamethasone during pregnancy have a range of 8.45 +/- 6.31 μg/dl (233.1 +/- 174.1 nM) (Srivastava et al. , 1994). Thus, we treated NCI-H295R cells with different concentrations (300, 150, and 75 nM) of cortisol for 24 h. Compared with the physiological control (300 nM) (Ruyet al. , 2017), the StAR expression and intracellular cortisol level were decreased in a cortisol concentration-dependent manner (P <0.05, P <0.01, Fig 6A,B). Meanwhile, the expression of GRα and miR-370-3p were decreased, the expression of Sirt3 was increased, and the H3K27ac and expression levels of IGF1 promoter region were decreased (P <0.05,P <0.01, Fig 6C-G), while the H3K9ac and H3K14ac levels of IGF1 promoter region did not change significantly (Fig 6F). Moreover, the protein expressions of StAR, Sirt3, and IGF1 were consistent with their mRNA expression (Fig 6H). These data indicated that low physiological concentrations of cortisol might inhibit steroidogenic function by altering GRα/miR-370-3p/Sirt3/IGF1 expression in the human adrenocortical cells.