Cytoskeleton And Synaptic Toxicity Of Neuro-2a Exposed To Bisphenol A

Bisphenol A(BPA)is an important organic chemical raw material and one of the most widely used industrial compounds in the world.Nowadays,BPA has been widely used in dyes,food packaging materials,fungicides and the manufacture of medical devices and other products,and is one of the important raw materials for the production of baby bottles,drinking cups and food and beverage containers.Therefore,it is widely exposed in the environment,endangering the health of humans and animals.BPA has been shown to affect the development and function of neurons in vitro,but the mechanism of BPA's neurotoxicity is not yet clear.The research conducted has focused on neuroendocrine,inflammation,and oxidative stress,etc.There are relatively few related studies on synaptic damage.In order to further study the pathogenesis of neurotoxicity caused by BPA,this study was conducted.Microtubule-associated proteins-MAP2 and Tau proteins play an important role in the development of the nervous system and the maintenance of microtubule function.Synaptophysin(SYP)is an important marker protein for synaptic reconstruction and neural plasticity,which is used to detect synaptic density and distribution.Developmentally regulated brain protein(Drebrin,Dbn)is widely present in the cytoplasm of neurons,especially dendrites,and has the effect of regulating the shape of synapses.The above four neurocytoskeleton and synapse-related proteins play an important role in the development of the nervous system and the stability of kinetic energy,so they were selected as evaluation indexes.In this study,Neuro-2a cells were selected as cell model,and Neuro-2a cells were cultured to the third to fourth generation.BPA with 1 ‰ DMSO,0 ?M(MEM),50 ?M,100?M,150 ?M and 200 ?M was added to the cell model for 24 hours,and samples were collected.Taking advantage of(1)Observe the morphology and quantity of Neuro-2a by ordinary light microscope.(2)Observe the ultrastructure of Neuro-2a cells by transmission electron microscope.(3)CCK-8 method and LDH kit to detect the cell survival rate of BPA and cell membrane permeability.(4)Observe the distribution of cytoskeleton by phalloidin staining method.(5)Western blot technique to detect the expression of MAP2,tau,SYP andDbn protein.(6)Immunofluorescence analysis of MAP2 and tau.(7)Detection of MAP2,tau,Dbn and SYP mRNA expression by qRT PCR.A preliminary study on the dose-response relationship between cytoskeleton and synaptic toxicity induced by BPA in Neuro-2a cells.In real life,most BPA-exposed organisms are low-dose long-term exposure.In order to further study the time-effect relationship between cytoskeleton and synaptic toxicity induced by BPA exposure to Neuro-2a cells,this study selected 150 ?M BPA concentration to act on Neuro-2a cells.Samples were collected respectively at 12 h,24 h and 36 h.Taking advantage of(1)Observe the morphology and quantity of Neuro-2a by ordinary light microscope.(2)Observe the ultrastructure of Neuro-2a cells by transmission electron microscope.(3)CCK-8 method and LDH kit to detect the cell survival rate of BPA and cell membrane permeability.(4)Observe the distribution of cytoskeleton by phalloidin staining method.(5)Western blot technique to detect the expression of MAP2,tau,SYP and Dbn protein.The specific test results are as follows:1.When the BPA concentration was 50 ?M and 100 ?M,there was no significant change in the morphology and structure of Neuro-2a cells compared with the control group.With the increase of BPA concentration,the number of cells decreased,rounded,adherence weakened,cell synapses decreased,survival rate decreased,cell membrane permeability increased and cell mitochondria were swollen and vacuolated.When the BPA concentration was 150 ?M and 200 ?M,compared with the control group,the MAP2,Tau,Dbn protein expression and mRNA levels were reduced,and the SYP protein expression and mRNA levels were increased.MAP2 and Tau proteins were reduced or missing in dendrites and axons.2.When the concentration of BPA was 150 ?M,theNeuro-2a cells in the treatment group and the control group were arranged orderly and closely attached to the bottom.With the increase of the BPA action time,the cells were loosely arranged and the adherence of the cells decreased.The cells in the 36 h group were lysed and contained black substances in the cytoplasm.The mitochondria swelled and the nuclear membrane was damaged.The phalloidin results showed that the cell synapses retracted into the cell body.Western blot results showedthat the expression levels of MAP2,Tau,and Dbn protein decreased significantly,while the expression levels of SYP protein increased significantly.The results showed that as the concentration increased or the exposure time increased,BPA damaged the morphology and structure of Neuro-2a cells,disrupted the stability of the cytoskeleton and synaptic plasticity,and revealed that the effect of BPA onNeuro-2a cells was dose-and time-dependent.To provide a new perspective for further research on the toxic effects of BPA-induced nerve injury.