Proteomics Analysis And Functional Study Of Placental Tissue In Patients With Intrahepatic Cholestasis Of Pregnancy

Intrahepatic cholestasis of pregnancy (ICP) is a pregnancy-specific disease,which is characterized by pruritus and elevated liver enzymes and/or serum bile acids.ICP usually occurs during the third trimester and the symptoms of the disease and theliver dysfunction resolve quickly after delivery. However, it can have severeconsequences for the fetus and is associated with an increased risk of spontaneouspre-term labour, fetal distress and sudden intrauterine death. Up to date, the exactcause of this disease is unknown, although genetic and hormonal, and probablyenvironmental factors are involved in the development of ICP. To explore thepathogenesis of ICP and fetal complications, we performed an isobaric tags forrelative and absolute quantification (iTRAQ) based proteomics approach andidentified2120proteins (7399peptides) in the placental tissues of all samplesincluding ICP patients and normal controls, and a total of38differentially expressedproteins were separated, of which29were upregulated and9were downregulated inICP. Cluster analysis was performed to characterize the specific and uniqueexpression patterns of the38differentially expressed proteins, it displayed thesignificantly altered expression levels of the proteins in ICP patients and normalcontrols. Bioinformatic analysis was performed to analyze the role of these proteinsin cellular processes and the results showed that most of the identified proteins werefunctionally related to specific cell processes, including apoptosis, oxidative stress,lipid metabolism, cell cycle, immune response, cell proliferation and cell growth.Three differentially expressed proteins, namely endoplasmic reticulum protein29(ERp29), myeloperoxidase (MPO) and peroxiredoxin6(PRDX6) were chosen forverification in4cases of ICP patients and4controls using Western blotting andimmunohistochemical analysis. The results showed that PRDX6and ERp29wereupregulated, whereas MPO was downregulated in ICP patients versus controls (P < 0.05). The expression patterns of the three proteins were consistent with theproteomic and Western blotting analysis. Based on previous studies and ourproteomic results, which suggested the appearance of apoptosis in the placental tissueof ICP patients, we also quantified the apoptotic index in the placenta. We found thatthere were few TUNEL-positive cells in the placental tissue of the normal controls,whereas, the apoptotic index was significantly higher in the placental tissue of ICPpatients. Recent studies have shown that ERp29is an endoplasmic reticulum protein,and high expression of ERp29inversely correlates with tumor progression due toinduction of apoptosis and inhibition of the cell cycle in a variety of cancer cells,including breast cancer cells. In this study, we found that ERp29was upregulated inthe placental tissue of ICP patients. So, we speculated that ERp29may play a key rolein the molecular pathogenesis of apoptosis in the placenta and ICP-related fetalcomplications. In order to verify this hypothesis, we quantified the expression ofERp29and apoptotic index in HTR-8/SVneo cells after treatment with differentconcentrations of taurocholic acid (TCA) for6h. We demonstrated that theexpression of ERp29and apoptotic index were significantly higher in the100μMTCA group than the10μM TCA groups and negative control group (P <0.05),respectively. Cell cycle analysis showed that the proportions of cells in the G1phase,S phase and G2/M phase at different time points (24h,48h and72h) were notsignificantly different among different concentrations of TCA groups, respectively. Tofurther explore the mechanism of ERp29overexpression on apoptosis in the placentaof ICP patients, we investigated the expression of p38and phosphorylated (p)-p38inHTR-8/SVneo cells after treatment with TCA. Western blotting result indicated thatthe levels of p-p38significantly increased in the100μM TCA group, compared withthe10μM TCA group and negative control group (P <0.01). Caspase-3activity alsosignificantly increased in the100μM TCA group, compared with the10μM TCAgroup and negative control group (P <0.01). This quantitative proteomics mayprovide us new insights into the pathophysiology and novel treatment targets for ICP.Among the38differentially expressed proteins, we discovered that ERp29may playa key role in placental apoptosis by inducing p38phosphorylation and activating caspase-3, which suggests that ERp29may represent a novel target for the treatmentof ICP.