Role Of ROS-Mediated Activation Of Endoplasmic Reticulum PERK/ATF4 Signaling In Impaired Estradiol Synthesis By Bisphenol A And Bisphenol S Exposure In JEG-3 Cells

Background This study investigates the role and mechanism of ROS-mediated activation of endoplasmic reticulum stress PERK/ATF4 signaling in the impairment of estradiol(E2)synthesis in human placental choriocarcinoma cells(JEG-3)caused by Bisphenol A(BPA)and its analogue Bisphenol S(BPS)exposure,and provides a scientific basis for elucidating the mechanism of BPA/ BPS exposure leads to the reduction of E2.Methods In this study,we investigated the mechanism of impaired E2 synthesis in JEG-3 cells caused by BPA/BPS exposure through in vitro cellular experiments,which consisted of four parts.Experiment 1: JEG-3 was co-incubated with the reactive oxygen species inhibitor NAC and divided into control,NAC incubation,BPA/BPS exposure and BPA/BPS+NAC co-incubation groups for 12 h.JEG-3 was co-incubated with the PERKspecific inhibitor GSK2606414(GSK)and divided into control,GSK incubation,BPA/BPS exposure and BPA/BPS+GSK co-incubation groups for 12 h.The JEG-3 was treated with BPA/BPS+GSK co-incubation group for 12 h.The supernatants were collected after different times(0,2,4,6,12 and 24 h)of BPA/BPS exposure with 50 μM,and the E2 content was measured by ELISA.Experiment 2: Using reactive oxygen species inhibitor NAC co-incubation,JEG-3 was divided into control group,NAC incubation group,BPA/BPS exposure group and BPA/BPS+NAC co-incubation group,after corresponding treatment for 12 h;50 μM of BPA/BPS was given for different times(0,2,4,6 and 12 h),and after the end of staining,the cells were detected by DCFH-DA staining method ROS levels.Experiment 3: JEG-3 cells were treated with BPA(0,25,50,100 μM)or BPS(0,25,50,100 μM)for 12 h;50 μM BPA/BPS for 2 h,4 h,6 h,12 h,24 h,respectively;NAC/GSK pretreatment for 2 h and then treated with 50 μM BPA/BPS,and cells were collected at the end of staining and detected by RT-qPCR was used to detect the cellular CYP19A1 mRNA levels;Experiment 4: Using the reactive oxygen species inhibitor NAC co-incubation,JEG-3 was divided into control,NAC incubation,BPA/BPS exposure,and BPA/BPS+NAC co-incubation groups;using the PERK-specific inhibitor GSK co-incubation,JEG-3 was divided into control,GSK incubation,BPA/BPS-exposed group,BPA/BPS+GSK co-incubation group,respectively,and the cells were collected after 12 h of treatment to extract proteins,and the expression levels of p-PERK,PERK,p-eIF2α,eIF2α and ATF4 proteins in the cells were detected by Western Blotting.Results ELISA experiments showed that: BPA/BPS exposure inhibited E2 synthesis in JEG-3 cells;the reactive oxygen species inhibitor NAC reversed E2 synthesis in JEG-3cells downregulated by BPA/BPS exposure;and the PERK activity inhibitor GSK reversed E2 synthesis in JEG-3 cells resulting from BPA/BPS exposure.DCFH-DA experiments showed that: BPA/BPS exposure led to Western Blotting assays showed that BPA/BPS exposure led to endoplasmic reticulum PERK/ATF4 signaling activation in JEG-3 cells.The reactive oxygen species inhibitor NAC reversed the activation of endoplasmic reticulum PERK/ATF4 signaling caused by BPA/BPS exposure.GSK,an inhibitor of PERK activity,reversed the activation of endoplasmic reticulum stress PERK/ATF4 signaling caused by BPA/BPS exposure.The PERK activity inhibitor GSK reversed the reduction of CYPI9A1 mRNA gene expression in JEG-3 cells caused by BPA/BPS exposure;the reactive oxygen species inhibitor NAC antagonized PERK/eIF2α/ATF4 signaling activation and reversed the downregulation of CYPI9A1 mRNA gene expression in JEG-3 cells by BPA/BPS exposure.expression.Conclusion BPA/BPS exposure inhibits E2 synthesis through upregulation of ROSactivated PERK/eIF2α/ATF4 signaling,and the results of this study provide a basis for further exploration of the mechanism of BPA/BPS-induced placental estrogen synthesis.