| Objective:Microplastics are widely distributed in the ocean,land and air,and can be ingested into the human body through various routes such as food chain contamination and air inhalation in daily life,and distributed to multiple tissues and organs,accumulating in the brain,liver and other tissues.GABA is a natural non-protein amino acid that is widely distributed in plants,animals and microorganisms,and is an important inhibitory neurotransmitter in mammals.GABA acts through the activation of GABA_A receptors on the cell membrane of neurons to open chloride channels,leading to an intracellular flow of chloride ions and generating inhibitory postsynaptic potentials to inhibit neuronal excitation.Gamma-aminobutyric acid receptor-associated protein(GABARAP),a GABA_A receptor transporter protein,is responsible for transporting GABA_A receptors from the Golgi apparatus to the cell membrane surface.It was shown that autophagy-associated protein P62 and GABARAP can interact and form a complex,which may be a pathway for the interaction between autophagy and GABAergic system.The current study found that microplastics may affect learning memory function in mice by inducing oxidative stress pathways,but whether autophagy and GABAergic systems play a role in this and their possible mechanisms have not been reported.In this study,based on the previous work of the group,we established a subchronic polystyrene exposure experimental animal model,and used neurobehavioral,histopathological,biochemical and molecular biological techniques to investigate the effects of polystyrene exposure on learning and memory functions and emotional disorders in mice;the effects on mitochondrial injury-related characteristics including mitochondrial ROS content,mitochondrial membrane potential level,mitochondrial autophagy-related protein expression;the effects on the interaction between P62 and GABARAP protein,and the membrane surface protein expression of GABA_A receptor.We also intervened with the antioxidant N-acetyl-L-cysteine(NAC)and the mitochondrial division inhibitor Mdivi-1 to observe the changes of the above indicators and to explore the possible mechanisms of the interaction between mitochondrial autophagy and GABAergic synaptic transmission in subchronic polystyrene exposure-induced neurotoxicity,in order to elucidate the mechanisms of cognitive and emotional impairment caused by polystyrene exposure and to explore possible interventions.This study will provide important data and clues to elucidate the mechanisms of cognitive and emotional impairment caused by polystyrene exposure and explore possible interventions.Methods:A subchronic polystyrene exposure mouse model was established by gavage of toxic polystyrene for 5 w.The experimental animals were divided into control,1μm,100 nm and 1μm+100 nm groups,and the mice were tested for learning and memory ability in the T-maze test and depression in the open field test.The protein levels of PINK1,Parkin,LC3,P62and GABARAP in the hippocampus were measured by Western Blot.An in vitro polystyrene-exposed cell model was established and SH-SY5Y cells were used for the assay.The cell viability was determined by CCK-8 method,and the doses of polystyrene and the intervention agents NAC and Mdivi-1 were determined.The morphological changes of the cells were observed under a microscope,the mitochondrial ROS content was detected by Mito SOXTM Red fluorescent probe,the mitochondrial membrane potential level was detected by TMRM method,the cell membrane surface expression of GABA_A receptor was detected by immunofluorescence,and the P62 and GABARAP protein-protein interaction was detected by fluorescence co-localization and immunoprecipitation.The antioxidant NAC and the mitochondrial division inhibitor Mdivi-1 were also used to detect the role of mitochondrial autophagy and altered GABAergic system in polystyrene neurotoxicity.Results:1.In vivo experiments.The T-maze experiment showed that the mice in the smaller particle size(100 nm)polystyrene group had longer time to explore the new iso-arm,increased the number of times to enter the new iso-arm,and decreased the learning memory ability,and the decrease in the mixed particle size group(1μm+100 nm)was more significant than that in the separate particle size group.HE staining showed that the neuronal structure of the hippocampal CA1 area was loosened and the number of neurons was reduced in the 100 nm PS group and the mixed particle size group.Elisa assay of hippocampal GABA content showed that the 100 nm PS group was elevated compared to the control group,and the most significant increase was seen in the mixed particle size group.2.In vitro experiments.The results of cell viability assay showed that the survival rate of SH-SY5Y cells decreased gradually with the increase of polystyrene concentration,and NAC and Mdivi-1 intervention could reverse the decreasing trend;polystyrene exposure increased the mitochondrial ROS content,decreased the mitochondrial membrane potential,increased the expression level of mitochondrial autophagy-related PINK1 and Parkin protein,increased the LC3 II/I ratio increased,and the increase was more significant in the mixed particle size group than in the 1μm and 100 nm groups;polystyrene exposure caused a decrease in P62 protein expression and an increase in GABARAP protein expression in SH-SY5Y cells,and the interaction between P62 and GABARAP protein was weakened,and the expression of GABA_A receptor on cell membrane surface increased;the in vitro experimental damage was all reduced to different degrees after NAC or Mdivi-1 pretreatment.Conclusion:Subchronic polystyrene exposure can cause impaired learning and memory capacity and emotional impairment in mice,and is associated with histopathological changes,oxidative mitochondrial damage,activation of the PINK1/Parkin pathway mediating mitochondrial autophagy,attenuated P62-GABARAP interaction,increased GABA_A receptor cell membrane translocation,and enhanced GABAergic neurotransmitter transduction;the damage was significantly reversed by NAC or Mdivi-1 treatment intervention. |
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