The Role And Mechanism Of Neuropathy Target Esterase In The Pathogenesis Of Preeclampsia

Objective:Abnormal trophoblast invasion is an important factor of preeclampsia, its causeis unknown. Recent years, some researches had shown that neuropathy target esterase(NTE) was closely related to the development of placenta. Phosphatidyl choline,which was regulated by NTE and could induce model of preeclampsia when it wasinjected into pregnant mice, was closely associated with trophoblast invasion.Therefore, we hypothesized that NTE might be involved in the preeclampsia. Thisstudy aimed to investigate the expression of NTE in preeclampsia, and the effect ofNTE on migration and invasion of placental trophoblast cell, as well as itsmechanism.Methods:(1) Using real-time quantitative PCR and immunohistochemical techniques,NTE mRNA and protein expression were detected in normal pregnancy andpreeclampsia placenta;(2) By transfecting pEGFP-N3-NTE and pGenesil-1-shNTE plasmid, HTR-8/SVneo trophoblast cells were over expressed or silenced NTE;(3) Using scratch method and transwell experiment, it wa observed the effect ofNTE on migration and invasion of HTR-8/SVneo trophoblast cell, then Using MTS todetect the effect of over expressing or silencing of NTE on proliferation ofHTR-8/SVneo trophoblast cell;(4) Using gelatin zymography and ELISA, it was to detect MMP-2,9andTIMP-1,2of the cell supernatants;(5) Using Western blot, it was observed the effects of NTE on ERK1/2andAKT signaling pathway of HTR-8/SVneo trophoblast cell.Results:(1) Real-time quantitative PCR showed that NTE mRNA was significantlyinduced in preeclampsia placenta compaired to normal pregnancy placenta (p<0.01).(2) Immunohistochemistry showed: both in normal pregnancy and preeclampsia placenta, NTE protein expressed in cytotrophoblast cell and syncytiotrophoblast cell.Compaired to normal pregnancy placenta, NTE protein was significantly decreased inpreeclampsia placenta.(3) Real-time quantitative PCR and Western blot showed that there were nosignificant difference in the NTE mRNA and protein expression of pGenesil-1group,Control shRNA group and Blank group (p>0.05). Compaired to Control shRNAgroup, NTE shRNA group significantly decreased NTE mRNA (p<0.05) and protein(p<0.05). Compaired to pGenesil-1group, NTE-EGFP group significantly increasedboth NTE mRNA (p<0.01) and protein (p<0.001).(4) Scratch assays and Transwell experiment showed there were no significantdifference in the HTR-8/SVneo cell’s migration and invasion of pGenesil-1group,Control shRNA group and Blank group (p>0.05). Compaired to ControlshRNA group, NTE shRNA group significantly decreased migration (p<0.01) andinvasion (p<0.01) of HTR-8/SVneo cell. Compaired to pGenesil-1group, NTE-EGFPgroup significantly increased migration (p<0.05) and invasion (p<0.05) ofHTR-8/SVneo cell.(5) MTS analysis showed: at24h,36h,48h in vitro, compaired to the sametime point of othergroups,20%FBS positive control group significantly increased theproliferation of HTR8/SVneo cells (p<0.01), at1h,24h,36h,48h and72h,there was no significant difference in Blank group, pGenesil-1group, NTE-EGFPgroup, Control shRNA group and NTE shRNA group (p>0.05).(6) Gelatin zymography analysis showed:24h and48h post-transfection,compaired to Blank group, there were no significant different in the MMP-9ofpGenesil-1group and Control shRNA group (p>0.05). Compaired to Control shRNAgroup, NTE shRNA group significantly decreased MMP-9secretion of HTR-8/SVneocells (p<0.01). Compaired to pGenesil-1group, NTE-EGFP significantly increasedMMP-9secreion of HTR-8/SVneo (p<0.05). However, there were no significantchanges in the MMP-2secreion among all the groups.(7) ELISA analysis showed: at24h and48h post-transfection, there were nosignificant differences in the TIMP-1, TIMP-2of HTR-8/SVneo cells of Blank group,pGenesil-1group, NTE-EGFP group, Control shRNA group and NTE shRNA group(p>0.05).(8) Western blot analysis showed:48h post-transfection, there were nosignificant difference in the ERK1/2and AKT phosphorylation of pGenesil-1group,Control shRNA group and Blank group (p>0.05). Compaired to the Control shRNAgroup, NTE shRNA could significantly inhibit the ERK1/2phosphorylation (p<0.01)and AKT phosphorylation (p<0.05) of HTR-8/SVneo cell. Compaired to pGenesil-1group, NTE-EGFP significantly promoted ERK1/2phosphorylation (p<0.01) andAKT phosphorylation (p<0.001) of HTR-8/SVneo cell.Conclusion:(1) Compaired to the normal pregnancy, NTE mRNA and protein were bothsignificantly decreased in the placenta of preeclampsia.(2) NTE regulated the migration and invasion of HTR-8/SVneo trophoblast cellby MMP-9.(3) ERK1/2and AKT signaling pathways were involved in the regulation ofNTE on HTR-8/SVneo trophoblast.