Functional Effects And Mechanisms Of Nickel Nanoparticles Exposure On HTR-8/SVneo In Trophoblast Cells

Objective:Nickel nanoparticles are widely used in various fields due to their excellent physicochemical properties.It has been found that Ni NPs are reproductive and embryotoxic and can accumulate in reproductive organs,leading to reduced fertility.However,whether they affect the function of trophoblast migration and invasion during gestation and the possible mechanisms are not clear.Therefore,the aim of this paper is to investigate whether Ni NPs affect the migration and invasion of extravillous trophoblast cells HTR-8/SVneo,and the study of its related mechanisms.Methods:1.Ni NPs solutions were prepared with RPMI 1640 basal medium at concentrations of25 μg/m L and 50 μg/ m L and characterized using scanning electron microscopy and particle size analysis after ultrasonic dispersion for 30 min.2.Cells were exposed to different concentrations of Ni NPs(0,25,50,75,100,200 and400 μg/m L)for 24 h.Cell proliferation viability was assayed using the CCK8 assay.3.Cells were exposed to different concentrations of Ni NPs(0,25,50,100 and 200μg/m L)for 24 h.Cell precipitates were collected by centrifugation,resuspended using Annexin V-FITC conjugate 100 μL,and the proportion of apoptotic cells was detected and imaged using flow cytometry after the addition of staining solution.4.Cells were exposed to Ni NPs at concentrations of 0,25,50 and 100 μg/m L and incubated for 24 h.Neat marks were made with a 20 u L sterile pipette tip in a culture dish with approximately 80% cell volume,and the healing of the marks was observed after 0 h and 24 h,respectively.5.Cells were inoculated in Transwell chambers,and 200 μL of cell suspension was added to the upper chamber and complete medium containing 20% FBS was added to the lower chamber,and the cell migration ability was analyzed after 24 h of continued incubation.For invasion assay,50 μL Matrigel gel should be added in Transwell in advance.6.Cells were exposed to Ni NPs at a concentration of 100 μg/m L,10 μM 740 Y-P and combined exposure to both solutions for 24 h.The expression of genes related to migration invasion of PI3 K,p-PI3 K,AKT,p-AKT,and MMP2 cells by Ni NPs was detected using RT-PCR and Western blot.Results:1.The Ni NPs are in the size range of 20 nm to 150 nm,with regular spherical morphology and some agglomeration.The particle size range of Ni NPs in the solution dispersion system was distributed between 90 nm and 615 nm with some agglomeration.2.The CCK-8 results showed that the cell viability showed a decreasing trend with the increase of Ni NPs concentration.The proliferation viability of the cells decreased by nearly 50% at Ni NPs concentrations greater than 100 μg/m L compared with the control group.3.Flow cytometry results showed that no significant apoptosis was observed at Ni NPs concentrations up to 100 μg/ml.Since cells showed significant apoptosis at high concentrations greater than 100 μg/m L,subsequent experiments chose to use a concentration of 100 μg/m L of Ni NPs as the maximum concentration for the experiment.4.The results of the cell scratch healing assay showed that the difference in migration distance of cells did not change significantly with the increase of Ni NPs concentration,and the results of Transwell assay showed that the number of migration and invasion of cells decreased gradually with the increase of Ni NPs concentration.5.Western blot and RT-PCR results showed that exposure to Ni NPs caused a decrease in the expression levels of PI3 K,p-PI3 K,AKT,p-AKT,and MMP2.6.740 Y-P agonist rescued the decrease in expression of related genes caused by exposure to Ni NPs and partially restored the migration and invasion ability of cells.Conclusion: Exposure to Ni NPs significantly affected the migratory and invasive ability of extravillous trophoblast HTR-8/SVneo and decreased the expression of PI3 K,p-PI3 K,AKT,p-AKT,and MMP2 at the m RMA and protein levels,and PI3 K activator partially improved the expression of related genes in the pathway and partially restored the migratory and invasive ability of HTR-8/SVneo ability.Thus,Ni NPs inhibit the migration and invasion of HTR-8/SVneo cells by downregulating the PI3K/AKT signaling pathway,resulting in reduced MMP2 expression.