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Role and mechanism of the PI3K / Akt signaling pathway in the embryonic neurodevelopment of mice
Li Shen, Guo Xiaolan, Guo Jin, et al
Journal of Developmental Medicine(Electronic Version)
2023, 11 (5):
321-330.
doi: 10.3969/j.issn.2095-5340.2023.05.001
【Abstract】 Objective To explore the role and mechanism of the phosphatidylinositol-3-kinase/
serine-threonine kinase (PI3K/Akt) signaling pathway in the embryonic neurodevelopment of mice,
for further clarifying the molecular mechanism of PI3K/Akt - related embryonic neurodevelopmental
abnormalities. Method Experimental animals were C57BL/6J mice and were randomly divided into
6 groups on embryonic day 7.5 (E7.5). In the experimental group, 12.5, 25, 50, 75 and 100 mg/kg of
LY294002 (the inhibitor of PI3K/Akt signaling pathway) were injected intraperitoneally, and the control
group mice were injected with normal saline solution. Pregnant mice were sacrificed and embryos
were examined under dissecting microscope on E13.5. After determining the optimal model dose,
embryonic nerve and spine samples were taken. Quantitative real-time polymerase chain reaction (RTqPCR) was used to detect the mRNA expressions of key genes of apoptosis and Shh (sonic hedgehog)
signaling pathway. The protein expression levels were determined by western blotting (WB). The mouse
neural stem cell NE-4C cell line was used for the study. The cell survival after treatment by different
concentrations of LY294002 was measured by methylthiazolyldiphenyl tetrazolium bromide (MTT).
The cell apoptosis was detected by TdT-mediated dUTP nick-end labeling (TUNEL), and cell cycle
was detected by flow cytometry. Statistical methods were performed by one-way analysis of Variance
and Dunnett's t test. Result The animal results showed that LY294002 affected embryonic neural
development, causing neural tube defects (NTDs). The optimal modeled dose was 50 mg/kg in which
the incidence of NTDs was 80.7% (46/57), all of which were myelomeningoeele and spina bifida. The
RT-qPCR results showed that, compared with the control group, the relative mRNA expressions of
tumor suppressor gene P53 and cysteinyl aspartate-specific proteinase 3 (Caspase3) were higher in nonmalformation, myelomeningoeele and spina bifida groups (P<0.01); and the relative mRNA expressions
of phosphatidylinositol-4,5-bisphosphate-3-kinase catalytic subunit alpha (PIK3CA) and Shh pathway
key genes [ smoothened (Smo), glioma-associated oncogene homolog 1 (Gli1), Gli2 ] were lower in the
three groups (P<0.01). The WB results showed that, compared with the control group, the relative protein
expressions of PI3K and Gli2 were lower in non-malformation, myelomeningoeele and spina bifida groups
(P<0.01); while the relative protein expressions of Smo and Gli1 were lower in myelomeningoeele and
spina bifida groups (P<0.01), but there was no significant difference in non-malformation group. The cell
experiment results showed that LY294002 inhibited cell proliferation, blocked the cell cycle in the G1 phase
and induced apoptosis. The MTT assay showed that the inhibition of cell survival was stronger with
increasing LY294002 concentration. The TUNEL staining showed that the ratios of positive cells to
total cells in the 20 and 25 μmol/L treated groups were higher than those in the control group (4.22±0.16,
8.56±0.24, 1.00±0.14, t=6.225 and 15.089, respectively, all P<0.001). The flow cytometry results showed
that the percentage of G1 cells in total cells was significantly increased [ (53.1±3.1) %, (66.4±2.1) %, and
(76.3±1.6) %, F=31.627, P<0.001] with the increase concentration of LY294002 (0, 20 and 25 μmol/L), and the proportion of cells in S and G2 phase were gradually decreased. Conclusion The inhibition of PI3K/Akt signaling pathway during the embryonic neurodevelopment of mice can inhibit Shh signaling pathway, promote cells apoptosis, and affect embryonic neural tube closure of mice, leading to the occurrence of NTDs.
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