ITRAQ Based Proteomics Reveals the Potential Mechanism of Placental Injury Induced by Prenatal Stress

Abstract

Maternal stress experienced during prenatal development is recognized as a significant risk factor for neurodevelopmental and neuropsychiatric disorders across the offspring's lifespan. The placental barrier serves a crucial function in safeguarding the fetus from detrimental exposures during gestation. However, previous investigations have not yet comprehensively elucidated the extensive connections between prenatal stress and the expression of placental proteins. In this study, we used iTRAQ-based quantitative proteomics to elucidate the placental adaptive mechanisms of pregnant rats in response to fear-induced stress. Our results showed that during pregnancy, exposure to fear-induced stress led to a pathological hypercoagulable state in the mother's body. Placental circulation was also disrupted, significantly reducing placental efficiency and blood oxygen saturation in newborn rats. Proteomic analyses showed that most of the DEPs were annotated to the PI3K-Akt and ECM-receptor interaction signaling pathway. In addition, the expressions of CDC37, HSP90β, AKT, p-AKT and p-mTOR were down-regulated significantly in the placenta. Our results demonstrated that prenatal fear-induced stress led to inhibition of the cellular signal transduction of placental PI3K/AKT/mTOR, which affected biological processes such as rRNA processing, translation, protein folding, protein stability, and oxygen transport in the placenta. These abnormalities in biological functions could potentially damage the barrier function of the placenta and thereby result in abnormal development in the offspring.

Keywords: PI3K/AKT/mTOR pathway; placental damage; prenatal stress; proteomics analyses.

Figure 1

Behavioral assessment of pregnant rats and their pups. The results of (A) the Sucrose Preference Test and the immobility time of pregnant rats are presented. (B) The results of Open-Field Test of pregnant rats. (C) The concentrations of serum stress hormones in pregnant rats. (D) The physical and reflex development of offspring. (E) The sucrose consumption and the immobility times in the Tail suspension test of pups. (F) The escape latency during the 4 training days in the MWM and (G) the swimming speeds, the retention time that rats spent in the target quadrant, and the number of times that the pups crossed the original platform during the probe test in the MWM. (H) The frequency of ambulation, rearing, and self-grooming in the open field-tests of the pups. (I) The result of the average litter size of pregnant rats. NC group, normal control group; FSM group, fear stress model group; SP, Sucrose Preference; IT, immobility time; AF, Ambulation frequency: number of floor units entered with all four feet; RF, Rearing frequency, number of instances of standing on the hindlimbs without touching the wall; SF, Self-grooming frequency: number of self-grooming actions performed; ACTH, Adrenocorticotropic hormone; CORT, Corticosterone; OFSM, offspring from the FSM group; ONC, offspring from the normal control group; EYO, eye opening; EAO, ear opening; IE, incisor eruption; ASR, auditory startle reflex; SRR, surface righting reflex; MWM, Morris water maze. Values are expressed as means ± standard deviations.* p < 0.05, ** p < 0.01 versus NC group or ONC group.

Figure 2

Effects of prenatal fear stress on placental efficiency, coagulatory biomarkers, and placental histopathology in pregnant rats. (A) the morphological features of placental tissues; (B) the diameters and (C) the thicknesses of placentas; (D) the weights of the fetus and placentas, and the placental efficiency; (E) the blood oxygen saturations of pregnant rats and their pups; (F) quantification of red blood cells in placental blood vessels using imageJ; (G,H) the coagulatory biomarkers of pregnant rats using the detection kits; (I) Quantitative analysis of the vascular network distribution in placental tissue using imageJ; (J) the H&E staining and Masson staining of the placentas were performed to evaluate the pathological changes of the placenta. The arrow indicates the presence of red blood cells within the blood vessels. NC group, normal control group; FSM group, fear stress model group; OFSM, offspring from the FSM group; ONC, offspring from the normal control group; PND 21, postnatal days of 21, PT, prothrombin time; APTT, activated partial thromboplastin time; TT, thrombin time; FIB, fibrinogen; ATIII, antithrombin III. Values are expressed as means ± standard deviations.* p < 0.05, ** p < 0.01 versus NC group or ONC group.

Figure 3

The volcano map of the identified proteins and GO classification of DEPs were shown. (A) The volcano map of the identified proteins was plotted based on its logarithmic fold change and p-value. x-axis represents the log2-fold change of protein expressions in FSM group compared to the NC group; The y-axis corresponds to the p-value of this fold change. Red dots: significant difference proteins; blue dots: proteins with fold changes > 0.83 or <1.2, and p-value < 0.05; grey and green dots: proteins with no significant difference in expression. (B–D) the GO classification of DEPs; (B) distributions of the DEPs for cellular components; (C) distributions of the DEPs for molecular functions; (D) distributions of the DEPs for biological processes. Only the top 20 Gene Ontology (GO) terms with significant differences (p-value < 0.05) are shown.

Figure 4

PPI network construction and hub genes extraction of DEPs. (A) The PPI network of the DEPs is shown. Network nodes represent proteins; edges represent protein–protein associations. Network analysis was set at high confidence (STRING score = 0.4) and high FDR stringency (0.01). Red indicates significantly increased; green indicates significantly decreased; width represents the degree of the interactions. (B) The top 10 hub genes were extracted with cytoHubba plug-in of Cytoscape software, and the eight algorithms are shown. Network nodes represent proteins; edges represent protein–protein associations. Color represents the score. MCC, Matthews Correlation Coefficient; DMNC, Neighborhood Component Centrality; MNC, Maximum Neighbor Connectivity; EPC, Edge Percolated Component.

Figure 5

The results of KEGG enrichment of DEPs. (A) The significantly enriched pathways (p-value < 0.05). (B) The significant interactions of the pathways (p-value < 0.05). Circle-nodes represent DEPs. Arrow-shaped nodes represent the pathways. Edges represent connections between the nodes. Node color represents the pathways to which they belong. Yellow coloring represents the multiple pathways to which the proteins belong.

Figure 6

The expressions of PI3K/AKT/mTOR pathway in the placenta of rats detected by western blotting assay. (A) Representative western blotting images of PI3K, AKT, and p-AKT; (B) Representative western blotting images of mTOR and p-mTOR. The protein expressions of PI3K (C), AKT (D), and p-AKT (E) determined by image J software (1.53n); (F) The p-AKT/AKT ratio calculated. The protein expressions of mTOR (G) and p-mTOR (H) determined by image J software (1.53n); (I) The p-mTOR/mTOR ratio calculated. (J) Representative western blotting images of CDC37 and HSP90β. The protein expressions of CDC37 (K), and HSP90β (L) determined by image J software (1.53n). Results presented as means ± SD (n = 6). ** p < 0.01 and * p < 0.05 versus ONC group.

Figure 7

Schematic diagrams for prenatal stress-induced placental damage. Prenatal fear stress induces down-regulation of PI3K/AKT/mTOR pathway in placenta. These abnormalities in protein expression might damage the barrier function of the placenta and thereby cause abnormal development in the offspring.

Figure 8

Fear stress schema illustration.

Figure 9

Schematic diagrams for the experimental protocol.