Effect Of MEHP Treatment Of Placental Trophoblast Cells On The Differentiation Of Co-cultured Neural Stem Cells

Background Di-(2-ethylhexyl)phthalate [di-(2-ethylhexyl)phthalate,DEHP] is a widely exposed endocrine disruptor that can damage the thyroid hormone regulation system of pregnant women and fetuses,and can interfere with placental nourishment The transport of thyroid hormones(THs)in layered cells may be toxic mainly through phthalate(2-ethylhexyl)monoester [mono-2-ethylhexyl phthalate,MEHP] after entering the human body.Thyroid hormones needed for embryonic development before birth are mainly supplied by the mother’s transport through the placenta.This study intends to observe the effect of MEHP on the differentiation of embryonic neural stem cells.Objective To explore the effect of MEHP-treated placental trophoblast cells on the differentiation of embryonic neural stem cells.Methods Human placental trophoblast cells HTR-8 were treated with MEHP(0μM,50μM,100μM,180μM)for 24 hours,and then embedded in a transwell co-culture system and human embryonic neural stem cells Re Ncell-CX(cell confluence about70%)were co-cultured for 72 hours.Before embedding,collect MEHP-treated HTR-8cell culture medium for 0 and 24 hours,use high performance liquid chromatography-tandem mass spectrometry(LC-MS)to detect MEHP content;use cell proliferation-toxicity detection kit(CCK8)to detect MEHP treatment Cell viability of HTR-8 cells for 24 hours;enzyme-linked immunosorbent assay(ELISA)was used to detect the 0 and 24 hours culture medium and intracellular triiodothyronine(T3)concentration of HTR-8 cells treated with MEHP,q RT-PCR and Western blotting(WB)detect the m RNA and protein levels of thyroid hormone metabolism enzymes(DIO2,DIO3)and receptors(TRα,TRβ).After embedding,use 1% activated charcoal adsorption treatment serum(think not containing thyroid hormone)stem cell differentiation culture medium(referred to as differentiation I liquid),after 72 hours of co-cultivation,ELISA test the content of T3 in the medium,immunofluorescence cytochemistry(IFC)and WB detect Re Ncell-CX cell-specific antibody SOX2,IFC labeled astrocyte marker protein GFAP,WB detects neuronal cell marker β-TubulinⅢ.In addition,in the neural stem cell differentiation experiment,a non-co-cultivation positive control group of differentiation medium(differentiation II for short)with 1%serum without activated carbon adsorption treatment was set up,and a non-co-culture negative control cultured in differentiation I medium.Results(1)The LC-MS test results showed that the MEHP content in the culture medium was consistent with the designed dose.(2)The proliferation rate of HTR-8cells in the highest dose MEHP(180μM)treatment group was 88%,indicating that the designed dose did not significantly inhibit cell proliferation and did not produce significant cytotoxicity.(3)MEHP treatment reduced the consumption of T3 in HTR-8cell culture medium(P<0.01).(4)MEHP treatment caused a decrease in the m RNA expression and protein level of thyroid hormone metabolizing enzyme DIO2 in HTR-8cells,while the m RNA expression and protein level of DIO3 increased(P<0.05).(5)MEHP treatment caused a down-regulation of m RNA expression and protein levels of thyroid hormone receptors(TRα,TRβ)in HTR-8 cells(P<0.05).(6)After co-cultivation,the differentiation of neural stem cells toward neurons decreased(P<0.05),and the differentiation toward astrocytes increased(P<0.05).Conclusions MEHP can affect the expression of HTR-8 thyroid hormone deiodinase and thyroid hormone receptor in human placental trophoblast cells.The treated HTR-8cells will reduce the differentiation of co-cultured human neural stem cells Re Ncell-CX towards neurons.The differentiation of astrocytes increased.