| Objective:Missed abortion(MA)is a common adverse pregnancy that seriously affects the physical and mental health of women at childbearing age.Although the importance of maintaining oxidative stress balance in the growth and development of the placenta is well-known,the mechanism of oxidative stress imbalance in missed abortion is still poorly understood.APPL1 protein is one of the differential proteins identified by proteomics in the villous tissue of missed abortion.So far,the understanding of the association of APPL1 protein with oxidative stress in missed abortion is still blank.Therefore,this study aims to assess the potential impact of APPL1 on oxidative stress balance and the molecular mechanisms involved.Methods:1.Collect the villous tissues of 15 patients with missed abortion from January 2018 to May 2019 as the MA group,and collect the villous tissues of 15 patients with normal pregnancy requesting abortion as the Control group during the same period.Control group villous tissue was treated with different concentrations of antioxidants or oxidants to change intracellular reactive oxygen species(ROS)levels.DHE staining was used to detect the levels of ROS in villous trophoblasts.The activity of superoxide dismutase(SOD)in each group of samples was detected.The apoptosis of each group was detected by TUNEL staining.The expression pattern of APPL1 in villous tissue was observed by immunohistochemistry.The relative expression levels of APPL1,Nrf2,HO-1 protein and mRNA in villous trophoblasts were detected by Western Blot and qRT-PCR.2.Construction of multiple cell models by human chorionic trophoblast HTR-8/SVneo cell line to further verify the connection between APPL1 and Nrf2/HO-1 pathway.SiAPPL1 was transfected in HTR-8/Svneo cells to inhibit APPL1 expression,and a series of rescue experiments were performed,pretreatment of HTR-8/Svneo cells with antioxidants or oxidants was followed by transfection of siAPPL1.Use ROS detection kit and DCF staining to observe the production of ROS in each group of cells,use SOD activity kit to detect the antioxidant capacity of each group of cells,use TUNEL staining,CCK8 kit and flow cytometry to detect the apoptosis of each group of cells.The expression levels of APPL1,Nrf2/HO-1 signaling pathway-related genes were detected by Western Blot and qRT-PCR.Results:1.In the MA group and the Control group treated with oxidants,ROS accumulation in villous trophoblasts increased,apoptosis increased,SOD activity decreased,and APPL1,Nrf2 and HO-1 expression levels were down-regulated.Antioxidant treatment inhibited ROS production and apoptosis in villous trophoblasts,induced SOD activation,and promoted the expression of APPL1,Nrf2 and HO-1.2.The results of immunohistochemistry showed that in the samples of the MA group and the Control group treated with the oxidant,the positive signal of APPL1 immunostaining in syncytiotrophoblasts disappeared,and no significant changes in syncytiotrophoblasts and villous interstitial cells.3.After APPL1 gene was silenced in HTR-8/SVneo cells,intracellular ROS accumulation and apoptosis increased,and the antioxidant related factors SOD and Nrf2/HO-1 signaling pathways were down-regulated.4.After pretreatment of HTR-8/SVneo cells with antioxidants to reduce the basic level of intracellular ROS,the adverse effects of siAPPL1 on the cells can be rescued by antioxidants in a concentration-dependent manner.However,pretreatment of cells with oxidant aggravates ROS accumulation,apoptosis,and down-regulation of antioxidant pathways produced by siAPPL1.Conclusions:1.Outbreak of oxidative stress in the placenta severely affects the proliferation and differentiation of trophoblasts,and eventually leads to the occurrence of missed abortion.2.APPL1 protein has anti-oxidant properties,and can function as an upstream signaling molecule to positively regulate the Nrf2/HO-1 pathway,and plays an important role in oxidative stress balance and cell survival in trophoblasts.3.The application of antioxidants can effectively restore the oxidative stress damage caused by excessive ROS,so antioxidant drugs can participate in the clinical treatment of missed abortions. |
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