| Objective: 2,2’,4,4’-Tetrabromodiphenyl ether(2,2’,4,4’-tetrabromodiphenyl ether,BDE-47)is one of the most exposed polybrominated diphenyl ether congeners in humans,and studies have confirmed its toxic effects involve reproductive,nervous,endocrine and other systems.Epidemiological studies have shown that BDE-47 can lead to adverse pregnancy outcomes such as premature birth and low birth weight.Animal experiments have shown that BDE-47 can accumulate in the placenta and cause adverse pregnancy outcomes in mice by damaging the placenta,but the specific mechanism is still being explored.The exchange of substances between mother and fetus during pregnancy depends on the placenta,which is responsible for providing all the nutrients needed for fetal growth throughout pregnancy and is essential for maintaining fetal growth during pregnancy.There is increasing evidence that abnormal placental structure and function contribute to many adverse pregnancy outcomes.Substantial evidence suggests that many placental abnormalities and dysfunctions result from impaired angiogenesis,a process that relies on the proper invasion of trophoblasts,and the changes of trophoblast proliferation,apoptosis and autophagy are also closely related to the normal development of the placenta.However,the effect of BDE-47 on placental structure and function is not yet clear,and further research is urgently needed.p38 MAPK(Mitogen-activated protein kinase,MAPK)is an indispensable serine and threonine kinase that exists in the body,and it controls various cellular responses in the body,which is very important for maintaining cell survival.The migration,invasion,proliferation,apoptosis and autophagy of extravillous trophoblasts also require the participation of the p38 MAPK signaling pathway.However,the role of p38 MAPK in placental injury by BDE-47 is unclear and requires further study.Therefore,this study established a mouse model of BDE-47 exposure during pregnancy to study the effect of BDE-47 on the pregnancy outcome of mice,and by observing the changes in placental tissue structure,to explore the role of the placenta in the adverse pregnancy outcomes of mice exposed to BDE-47,and to reveal the relevant mechanism of BDE-47 induced reproductive and developmental toxicity at the body level.At the same time,the human extravillous trophoblast cell line HTR-8/SVneo was used as an in vitro research model to investigate the effect of BDE-47 on the migration,invasion,proliferation,apoptosis and autophagy,and to study the role of p38 MAPK signaling pathway in regulating these processes;This will clarify part of the reasons for the structural damage and dysfunction of placenta caused by BDE-47 exposure,and provide a scientific basis for the prevention and control of the adverse effects of BDE-47 on pregnancy.Methods: 1.The pregnant ICR mice were randomly assigned to 4 experimental groups according to body weight: control group(corn oil),25 mg/kg/day BDE-47 exposure group,and 50 mg/kg/day BDE-47 exposure group,100 mg/kg/day BDE-47 exposure group.From the 0.5th day to the 16.5th day of gestation,the mice were exposed to BDE-47 by gavage,and the mice were sacrificed on the 17.5th day of gestation to observe the effect of BDE-47 on the body weight,food consumption,water consumption and pregnancy outcome of the mice.HE staining was used to detect the changes of placental tissue structure.The expression levels of placental development related genes Esx1,Ascl2,Hand1,Eomes and Fosl1 were detected by Real-Time fluorescence quantitative PCR.2.Based on the BDE-47 exposured mouse model,immunohistochemical staining of CD34 in placental tissue was performed,and the expression levels of CD34 and VEGF-A were detected by Western blot to determine the effect of BDE-47 exposure on placental angiogenesis in mice.ELISA assay was used to detect the expression levels of angiogenesis factor VEGF and placental growth factor Pl GF in the serum of pregnant mice;Western blot was used to detect the protein expression levels of invasion related factors MMP9,MMP2,TIMP2,IGF1,IGFBP3 and the activation of p38 MAPK signaling pathway;Real-Time quantitative PCR was used to detect the m RNA expression levels of MMP9,MMP2 and TIMP2.HTR-8/SVneo cells were treated with multiple concentrations of BDE-47 for 24 hours,and the cell viability was detected by CCK-8 assay to determine the dose of BDE-47.Next,the cell experiments were divided into four groups: control group(DMSO),5 μM BDE-47 treated group,10 μM BDE-47 treated group,and 20 μM BDE-47 treated group.The cell wound healing assay was used to detect the change of cell migration ability;Transwell assay was used to detect the change of cell invasion ability;Western blot was used to detect the protein expression levels of VEGF-A and invasion-related factors and the activation of p38 MAPK signaling pathway.After using the inhibitor of p38 MAPK signaling pathway SB203580 to inhibit the activation of p38 MAPK signaling pathway,the changes of cell migration and invasion ability and the protein expression levels of invasion-related factors were detected.3.Based on the BDE-47 exposured mouse model,immunohistochemistry assay was used to detect the expression levels of proliferation related factors Ki67 and PCNA in placental tissue;Western blot was used to detect the expression levels of PCNA and CCND in placental tissue;The apoptosis of placental cells was observed by TUNEL fluorescence staining assay;The expression levels of apoptosis-related proteins Bcl2,Bax,Caspase9,cleaved-Caspase9,Caspase3,cleaved-Caspase3 and Cyt c in placental tissue were detected by Western blot assay;The m RNA expression levels of Bcl2,Bax,Caspase9 and Caspase3 were detected by Real-Time quantitative PCR;The expression levels of autophagy marker proteins p62,Beclin1 and LC3 were detected by Western blot and immunohistochemistry assay.Based on BDE-47 exposured HTR-8/SVneo cell model,flow cytometry was used to detect changes in cell cycle and apoptosis;Other detected indicators of proliferation and apoptosis were the same as those in animal experiments;Western blot and immunofluorescence staining were used to detect the expression levels of autophagy marker proteins in HTR-8/SVneo cells.After using SB203580 to inhibit the activation of p38 MAPK signaling pathway,the changes of cell proliferation,apoptosis and autophagy related indexes were detected.Results: 1.During the exposure period,compared with the control group,the body weight and the food consumption of pregnant mice in all BDE-47-treated groups were significantly reduced,and the water consumption of pregnant mice in the 25 and 50mg/kg/day BDE-47-treated groups was significantly reduced,and the 50 and 100mg/kg/day BDE-47 exposure can significantly increase the organ coefficient of the liver of pregnant mice,while the organ coefficient of the kidney has no significant change.The numbers of embryos and viable embryos in the 100 mg/kg/day BDE-47 treatment group were significantly reduced,and the number of absorbed embryos in the 50 mg/kg/day BDE-47 treatment group was significantly increased.The total uterine and fetal weight in the 100 mg/kg/day BDE-47 treatment group was significantly reduced,and the placental weight of the pregnant mice in the 25 and 50mg/kg/day BDE-47 treatment groups decreased.The placenta area,the placental labyrinth and spongiotrophoblast area were significantly reduced in all the BDE-47 treatment groups,and the ratio of placental spongiotrophoblast area to labyrinth area decreased in the 50 mg/kg/day BDE-47 treatment group.In addition,BDE-47 exposure significantly decreased the m RNA expression levels of placental development-related genes such as Esx1,Ascl2,Hand1,Eomes and Fosl1.2.Compared with the control group,the number of placental blood vessels in the BDE-47 treatment group was significantly reduced,and the levels of VEGF and Pl GF in the serum of pregnant mice were significantly reduced.The protein expression level of VEGF-A and invasion-related factors MMP9,MMP2,TIMP2,IGF1 and IGFBP3 in placental tissue were significantly decreased,and the m RNA level of MMP9 in the 100 mg/kg/day BDE-47 treatment group was significantly decreased,and the m RNA levels of MMP2 were significantly decreased in the three BDE-47-treated groups,while the m RNA levels of TIMP2 were not significantly changed.The viability of HTR-8/SVneo cells decreased gradually with the increase of BDE-47 dose after treatment with BDE-47.BDE-47 treatment can inhibit the expression level of VEGF-A in HTR-8/SVneo cells,and reduce the migration and invasion ability of the cells.The protein expression levels of MMP9,MMP2,TIMP2,IGF1 and IGFBP3 were significantly decreased.In mouse placental tissue and HTR-8/SVneo cells,the protein expression level of p-p38 in the BDE-47 treatment group was significantly increased,but the protein expression level of p38 did not change significantly.SB203580 could inhibit the increase of intracellular p-p38 protein induced by BDE-47 treatment.After inhibiting the p38 MAPK signaling pathway,the decrease in the expression level of VEGF-A caused by BDE-47 rebounded,and the decline in cell migration and invasion ability was restored,and the expression levels of MMP9,MMP2,TIMP2,IGF1 and IGFBP3 increased.3.Compared with the control group,BDE-47 treatment could significantly reduce the expression levels of Ki67,PCNA and CCND in placental tissue,and the number of apoptotic cells in the BDE-47 treatment group was significantly increased,and the expression levels of apoptosis-related factors Bcl2,Caspase9 and Caspase3 were significantly decreased,while the expression levels of Bax,cleaved-Caspase9,cleaved-Caspase3 and Cyt c were significantly increased.The m RNA levels of Bcl2 in all BDE-47 treatment groups were significantly decreased,and the m RNA level of Bax in the 100 mg/kg/day BDE-47 treatment group was significantly increased,while the m RNA levels of Caspase9 and Caspase3 did not change significantly.The protein expression level of p62 in the BDE-47 treatment group was significantly decreased,and the protein expression levels of Beclin1 and LC3-II were significantly increased.In HTR-8/SVneo cells,the protein expression levels of PCNA and CCND in the BDE-47 treatment group were significantly decreased,and the proportion of cells in S phase in the 20 μM BDE-47 treatment group was significantly decreased,and the apoptosis rate of 10 μM and 20 μM BDE-47 treatment groups increased significantly.The expression levels of anti-apoptotic protein Bcl2,Caspase9 and Caspase3 were significantly decreased,while the expression levels of pro-apoptotic proteins Bax,cleaved-Caspase9,cleaved-Caspase3 and Cyt c were significantly increased.The protein expression level of p62 was significantly decreased in BDE-47 treatment group,and the protein expression levels of Beclin1 and LC3-II were significantly increased.After inhibiting the p38 MAPK signaling pathway,the G0/G1 phase arrest of HTR-8/SVneo cells induced by BDE-47 treatment was reversed,the protein expression levels of PCNA and CCND increased,and the reduction of cell proliferation ability caused by BDE-47 was recovered.And the inhibition of p38 MAPK signaling pathway reduced the increase of apoptosis rate caused by BDE-47.Compared with the 20 μM BDE-47 treatment group,after inhibiting the p38 MAPK signaling pathway,the protein expression level of Bcl2 was significantly increased,and the protein expression levels of Bax,cleaved-Caspase9,cleaved-Caspase3 and Cyt c were significantly decreased.After inhibiting the p38 MAPK signaling pathway,p62 protein expression level was increased compared with BDE-47 treatment group,Beclin1 and LC3-II protein expression levels decreased.Conclusion: 1.BDE-47 exposure can reduce the body weight of pregnant mice,affect the food and water consumption of pregnant mice,and cause adverse pregnancy outcomes in mice by affecting the structure of the placenta and reducing the expression levels of placental development-related genes.2.BDE-47 can impair placental angiogenesis in mice,and activate the p38 MAPK signaling pathway in mouse placental tissue and HTR-8/SVneo cells.The p38 MAPK signaling pathway participates in the process of BDE-47 inhibiting the migration and invasion ability of HTR-8/SVneo cells.3.BDE-47 can inhibit proliferation,induce increased apoptosis and overactivate autophagy in mouse placenta and HTR-8/Svneo cells,and p38 MAPK signaling pathway is involved in the proliferation inhibition,apoptosis increase and autophagy activation process of HTR-8/SVneo cells induced by BDE-47. |
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