Comparative proteomics analysis of placenta from pregnant women with intrahepatic cholestasis of pregnancy

Abstract

Introduction: Intrahepatic cholestasis of pregnancy (ICP) usually occurs in the third trimester and associated with increased risks in fetal complications. Currently, the exact cause of this disease is unknown. In this study we aim to investigate the potential proteins in placenta, which may participate in the molecular mechanisms of ICP-related fetal complications using iTRAQ-based proteomics approach.

Methods: The iTRAQ analysis combined with liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to separate differentially expressed placental proteins from 4 pregnant women with ICP and 4 healthy pregnant women. Bioinformatics analysis was used to find the relative processes that these differentially expressed proteins were involved in. Three apoptosis related proteins ERp29, PRDX6 and MPO that resulted from iTRAQ-based proteomics were further verified in placenta by Western blotting and immunohistochemistry. Placental apoptosis was also detected by TUNEL assay.

Results: Proteomics results showed there were 38 differentially expressed proteins from pregnant women with ICP and healthy pregnant women, 29 were upregulated and 9 were downregulated in placenta from pregnant women with ICP. Bioinformatics analysis showed most of the identified proteins was functionally related to specific cell processes, including apoptosis, oxidative stress, lipid metabolism. The expression levels of ERp29, PRDX6 and MPO were consistent with the proteomics data. The apoptosis index in placenta from ICP patients was significantly increased.

Conclusion: This preliminary work provides a better understanding of the proteomic alterations of placenta from pregnant women with ICP and may provide us some new insights into the pathophysiology and potential novel treatment targets for ICP.

Figure 1. Cluster analysis of differentially expressed proteins in the placental tissue from pregnant women with ICP and healthy pregnant women.

Hierarchical cluster analysis for the 38 differentially expressed proteins displaying significantly altered expression levels in the placental tissue from pregnant women with ICP and healthy pregnant women. “N” represents healthy pregnant women and “P” represents pregnant women with ICP. The protein expression levels are shown as colored boxes; red indicates a high expression level and green indicates a low expression level.

Figure 2. Bioinformatic analysis of differentially expressed proteins in the placental tissue from pregnant women with ICP and healthy pregnant women predicted by PathwayStudio™.

Analysis of the cellular pathways affected by the differentially expressed proteins was performed using the PathwayStudio software. Proteins are shown as red ovals, regulated processes are represented by yellow squares. Regulation events are displayed with arrows and documented by literature citations.

Figure 3. Western blot analysis of PRDX6, ERp29 and MPO in the placental tissue from pregnant women with ICP and healthy pregnant women.

The expression levels of PRDX6 and ERp29 were significantly upregulated, whereas MPO was significantly downregulated in placenta from pregnant women with ICP compared to that in placenta from healthy pregnant women (P = 0.002, P = 0.018 and P = 0.009, respectively); β-tubulin was used as an internal control. (*P<0.05; **P<0.01).

Figure 4. Immunohistochemical staining for PRDX6, ERp29 and MPO in the placental tissue from pregnant women with ICP and healthy pregnant women (×400).

Immunohistochemstry images demonstrated higher expression of PRDX6 (B) and ERp29 (D), and lower expression of MPO (F) in cytoplasm and/or nucleus of trophoblasts in the placenta from pregnant women with ICP than those in placenta from healthy pregnant women (A, C, E).

Figure 5. Comparison of the incidence of apoptosis in placenta from pregnant women with ICP compared to that in placenta from healthy pregnant women (×400).

TUNEL assay demonstrated the incidence of apoptosis in placenta from pregnant women with ICP was higher than that in placenta from healthy pregnant women (A and B). Semi-quantitative analysis indicated there was significantly higher incidence of apoptosis in placenta from pregnant women with ICP (P = 0.007) (C).