Reduction Of TET Enzyme Activity Caused By Mitochondrial Dysfunction Mediates The Inhibition Of Mesenchymal Like Behavior Of Placental Trophoblasts Induced By Arsenic Exposure During Pregnancy

Objective This study investigated the mechanism of arsenic-induced inhibition of mesenchymal like behavior of placental trophoblasts,focusing on the role of decreased TET enzyme activity induced by mitochondrial dysfunction in the process.Methods The experimental scheme is implemented by two means:in vivo and in vitro experiment.Firstly,an animal model was established to repeat our previous study in which we identified that arsenic（As）exposure led to intrauterine growth restriction（IUGR）in ICR mice.In the animal experiment,the mice in the control group were given ultrapure water,while the mice in the As group were given ultrapure water containing 15mg/L sodium arsenite（Na As O₂）from the day in which the mating got successful until the end of the collection of biological materials.In the animal intervention experiment,pregnant rats were givenα-Ketoglutarate（α-KG）2g/kg/day by intragastric administration to explore whetherα-KG supplementation has a protective effect on the inhibition of mesenchymal like behavior of placental trophoblast induced by As.In vivo experiments mainly focused on the birth outcome of fetal mice.In addition,fresh placental tissues of mice were collected.A complete placenta per dam was need for H＆E staining.The whole genome DNA hydroxymethylation level andα-KG content of placenta were detected by LC-MS/MS.The activity of placental TET enzyme was detected by luciferase method.Besides,the expression levels of genes related to placental mesenchymal and epithelioid like behavior including ccn1,mmp1,mmp2,mmp9,n-cadherin,vimentin were detected.And so did proteins with regard to placental mesenchymal like behavior such as MMP2 and MMP9.In the part of in vitro experiment,human placental trophoblast cell line HTR-8/Svneo cells were introduced to explore the mechanism in detail.In the cell experiment,the cells in As group were exposed to 2μM Na As O₂ according to previous data of laboratory,while the cells inαKG+As group were pretreated with 20m Mα-KG for 2 hours,and then exposed to Na As O₂.We tested the expression levels of genes and proteins to verify whether the mesenchymal like behavior or oxidative phosphorylation process of As-treated cells were limited.The migration ability of cells were detected using Wound healing test and Transwell migration experiment.And the invasion ability was measured by Transwell invasion experiment.The mitochondrial membrane potential and the level of mitochondrial reactive oxygen species（ROS）were detected by immunofluorescence staining.The DNA hydroxymethylation modification level of specific gene target region was screened by detecting the cell transcriptome and cell DNA hydroxymethylation level of gene target regions.Results The results of animal experiments showed that As exposure resulted in the decrease of birth weight and crown-rump length of fetal rats,placental weight and diameter.The results of H＆E staining showed that the migration and invasion process of placental trophoblasts in As group was significantly delayed.It was found that As exposure led to the decrease of DNA hydroxymethylation level andα-KG content in placenta after the determination completed by LC-MS/MS.Luciferase detection showed that TET enzyme activity decreased significantly in As group,but no significant difference was observed in the expression levels of TET genes.It was found that As exposure resulted in the down-regulation of the expression of placental mesenchymal like behavior related indicators and the up-regulation of the expression of epithelioid behavior related indicators.Supplementation withα-KG could significantly improve the decrease of placental TET enzyme activity,placental dysplasia and IUGR caused by As in mice.In vitro,the expression levels of indicators promoting HTR-8 cell mesenchymal like behavior such as MMP2,MMP9 and N-cadherin in As group were significantly decreased.Though,the expression levels of gene Occludin related to epithelioid behavior of cells from As group were significantly increased.Wound healing test and Transwell experiments showed that the ability related to mesenchymal like behavior of cells in As group were significantly limited.And transwell invasion experiment indicated that the invasion ability of cells treated with As were extremely limited.The results of LC-MS/MS indicated that theα-KG content of cells in As group decreased.And the testing using luciferase method showed that the activity of TET enzyme decreased significantly in cells treated with As.The detection results of cell transcriptome and DNA hydroxymethylation level in target region of genes showed that the DNA hydroxymethylation level of the second target region of gene CCN1,which promotes mesenchymal like behavior,decreased significantly in As group.At the site level,the DNA hydroxymethylation level of CCN1,LAMA5 and IDH2 also decreased significantly in the arsenic treated group.Immunofluorescence staining showed that mitochondrial membrane potential decreased significantly in As treated cells.On the contrary,mitochondrial ROS increased significantly in the same group.At the protein expression level,we found As inhibited the oxidative phosphorylation of mitochondria of HTR-8 cells.In vitro,α-KG supplementation could significantly improve the mitochondrial dysfunction,the decrease of TET enzyme activity and the inhibition of mesenchymal like behavior of HTR-8 cells caused by As exposure.Conclusion In conclusion,the decrease of TET enzyme activity evoked by mitochondrial dysfunction mediates the As-induced inhibition of mesenchymal like behavior of placental trophoblasts.This may lead to placental dysplasia and IUGR.Fortunately,α-KG supplementation can play an obvious protective role.