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.