Effects And Mechanism Of PS-NPs Exposure On Leydig Cells

Objective:Plastic particle pollution has posed a significant threat to ecological environment as well as human health.Plastic substances degrade in the environment into microplastics and nanoplastics,which can enter the human body via dietary,respiratory and skin exposure.In addition to gastrointestinal toxicity,neurotoxicity,hepatotoxicity and developmental toxicity,micro/nanoplastics can also cause reproductive toxicity.Studies have found that oral exposure to polystyrene particles can lead to destruction of blood-testis barrier,decrease of sperm count,increase of sperm deformity rate and reduction of testosterone content in mice.However,the mechanism of toxicity of polystyrene particles remains unclear.In this study,the mechanism of testicular reproductive toxicity induced by nanoplastics was explored by detecting the effects of exposure to polystyrene nanoparticles(PS-NPs)in vitro on mitochondria and cell membrane of TM3 mouse Leydig cells.Methods:TM3 cells were cultured for 24 h and then exposed to different concentrations of PS-NPs(0,50,100,150 μg/m L)for 24 h.Cell viability was measured by CCK8 method.Cell density and morphology were observed under an inverted microscope.The levels of reactive oxygen species(ROS),superoxide dismutase(SOD),glutathione(GSH)and malondialdehyde(MDA)in TM3 cells were detected by oxidative stress detection kit.JC-1 probe was used to detect mitochondrial membrane potential.The morphological structure of mitochondria was observed by transmission electron microscope.Mitochondrial DNA(mt DNA)copy number was detected by PCR.The apoptosis rate of TM3 cells was detected by Annexin V/PI double staining.The expression levels of apoptosis-related proteins BAX and BCL-2 were detected by Western blot.The activity of caspase-3 was detected by colorimetry.Real-time PCR was used to detect the expression of steroid synthesis-related genes St AR,P450 scc and 17β-HSD.Testosterone levels were detected by ELISA.Targeted metabolomics was performed to analyze of cell metabolites.The leakage rate of lactate dehydrogenase(LDH)was detected by colorimetry.Whole cell patch clamp was used to detect the change of cell membrane potential.Results:(1)CCK8 assay showed that cell viability decreased with the increase of PS-NPs concentration(P < 0.05).Inverted microscopy showed that PS-NPs reduced cell density and caused morphological changes.(2)After TM3 cells were treated with red fluorescent labeled PS-NPs for 24 h,the intracellular fluorescence intensity significantly increased in a dose-dependent manner(P < 0.05),indicating that PS-NPs were internalized by TM3 cells.(3)Oxidative stress test showed that PS-NPs increased ROS production in TM3 cells in a dose-dependent manner(P < 0.05).After exposure to medium and high doses of PS-NPs,MDA production increased,while antioxidant SOD activity and GSH content decreased(P < 0.05).(4)Annexin V-FITC/PI staining flow cytometry showed that PS-NPs caused a significant increase in apoptosis rate(P < 0.05).After exposure to 100 and 150μg/m L PS-NPs for 24 h,the expression of anti-apoptotic protein BCL-2 was downregulated,while the expression of pro-apoptotic protein BAX was up-regulated in TM3 cells(P < 0.05).Furthermore,PS-NPs treatment significantly increased caspase-3 activity.(5)Transmission electron microscopy showed that the cells in the PS-NPs treatment group displayed mitochondrial swelling and crista structure vanishment.JC-1 fluorescence probe found that PS-NPs caused a decrease in mitochondrial membrane potential in a dose-dependent manner(P < 0.05),indicating that PS-NPs disrupted mitochondrial function in TM3 cells.(6)Targeted metabolomics showed that 14 metabolites were significantly changed after medium-dose PS-NPs treatment(P < 0.05),of which pyruvate was upregulated,while 5’-adenosine triphosphate,cyclic adenosine monophosphate,thiamine pyrophosphate,adenosine diphosphate,5’-guanosine diphosphate,lactic acid,succinic acid,dihydroxyacetone phosphate,β-D-fructose 6-phosphate,citric acid,Dfructose 1,6-diphosphate,isocitric acid and cis-aconitic acid were down-regulated,indicating that PS-NPs affected the energy metabolism of TM3 cells.(7)Real-time PCR showed that PS-NPs reduced the expression of steroid synthesis-related genes St AR,P450 scc and 17β-HSD in TM3 cells.In addition,the testosterone levels significantly decreased in the middle and high dose treatment groups(P < 0.05),indicating that PS-NPs affected the synthesis of testosterone in TM3 cells.(8)In 100 and 150 μg/m L PS-NPs treatment groups,LDH leakage rate significantly increased(P < 0.05).In addition,whole-cell patch clamp results showed that medium and high doses of PS-NPs exposure led to cell membrane depolarization(P < 0.05),indicating that PS-NPs caused damage to TM3 cell membrane.Conclusion:(1)PS-NPs can be internalized by TM3 cells and reduce cell activity;(2)PS-NPs can induce oxidative stress and cause mitochondrial damage and apoptosis in TM3 cells;(3)PS-NPs can result in abnormal glucose metabolism and steroid synthesis in TM3 cells;(4)PS-NPs can damage membrane integrity of TM3 cells.