Role Of Rat Primary Cultured Neuron And Astrocyte In PFOS-induced Neurotoxicity

Background:Perfluorooctane sulfonate(PFOS)is the end-degrade product of perfluorinated compounds(PFCs).Due to its hydrophobic,oleophobic and chemical stability,PFOS has been widely used in industrial and consuming products.Because of its accumulation,recalcitrance and carrying on the long-distance migration through the atmosphere,PFOS was classified as a new member of persistent organic pollutants(POPs).PFOS existed in atmosphere,water and soil widely,and had the ability to accumulate in biological tissues.Epidemiological investigation and animal studies both showed that PFOS had the potential effect of neurotoxicity,while the mechanism is unclear.It is generally considerated that abnormal apoptosis in neuron is one of the mechanism of PFOS-induced neurotoxicity.Whether autophagy was also involved in PFOS-induced neurotoxicity was unknown.Astrocytes(AS),a kind of glial cells,played important physiological functions in neurons formation,connectivity and activity.Glutamate(Glu)is one kind of an excitatory neurotransmitter,and plays an important role in neuron-glia interactions through Glu-glutamine(Gln)cycle mediated by neuron-AS coupling.Studies have shown that PFOS could induced decreased study ability in experimental animal,companion with increased glutamate content in the hippocampal organization.Objective:The model with the primary cultured neuron and astrocytes exposed to PFOS were built.Then,the molecules mechanism of PFOS-induced neurotoxicity was explored based on autophagy in neuron and glutamate transport system of astrocytes.Method:Neuron_:(1)The viability of cells was determined by the 3-(4,5-Dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide(MTT)assay and lactate dehydrogenase(LDH)release.(2)Oxidative stress:the level of intracellular ROS was quantified by Reactive Oxygen Species Assay Kit,the experiment for MDA was according to the kit instructions.(3)A fluorescent probe of JC-1 was applied to test mitochondrial membrane potential and Fluo-3AM was used to determine intracellular Ca2+ concentration of neuron.(4)The Hoechst 33342 and PI double-fluorescent staining assay kit were used for inspecting cellular apoptosis and Western Blot was applied to detect the expression of apoptosis related protein.(5)Lyso-Tracker Red used on examing cell autophagy and qRT-PCR was used to detect related mRNA expression.Primary rat astrocytes:(1)The viability of astrocytes was determined by MTT assay.(2)Oxidative stress:the level of intracellular ROS was quantified by Reactive Oxygen Species Assay Kit,and the experiments for CAT and SOD were according to the kit instructions.(3)Kit method were used to detect the contents of Glu,Gln and activity of GS.(4)qRT-PCR was used to detect related mRNA expression.Result:Rat primary hippocampal neurons:.(1)Compared with the controls,75?125 ?M PFOS could inhibit the neuron viability significantly(P<0.05),and 25?100?M PFOS induced an increasing in LDH release in neurons(P<0.05).(2)With the increased concentration of PFOS exposure remarkably increased ROS production and MDA content(P<0.05),25?100 PFOS decreased SOD activity(P<0.05),75?100?M PFOS reduced the concentration of CAT activity(P<0.05),50?100 ?M PFOS reduced concentration of GSH and GSSG in the neuron(P<0.05).(3)PFOS exposure decreased the mitochondrial membrane potential and induced increased calcium concentration in the primary cultured hippocamal neuron(P<0.05).(4)PFOS exposure increased neurons apoptosis and increased the activation of Caspase-3 significantly(P<0.05).(5)25?100?M PFOS exposure enhanced lysosome membrane permeability(LMP)significantly(P<0.05).100 ?M PFOS could disturb the expression of Atg 5,Atg 12 and Beclin-1 mRNA.Primary rat astrocytes:(1)Compared with the controls,PFOS at 75 and 100 ?M significantly decreased the viability of astrocytes(P<0.05),(2)15?75 PFOS induced increasing of ROS production in astrocytes(P<0.05),15?75 ?M PFOS significantly decreased the enzymatic activity of SOD in astrocytes(P<0.05)and 50?75?M PFOS significantly decreased the enzymatic activity of CAT(P<0.05).(3)15?75?M PFOS induced an increase in extracellular level of Glu(P<0.05)and decrease concentrations of extracellular Gln extracellular of astrocytes(P<0.05).15?75 ?M PFOS can lessen the GS activity(P<0.05).(4)50?75 ?M PFOS exposure to astrocytes significantly decreased the gene expression of GS,GLAST and GLT(P<0.05).Conclusion:(1)Apoptosis and autophagy were both involved in PFOS-induced rat primary hippocampal neurons death.(2)PFOS could disturb the glutamate transport system of primary rat astrocytes.In conclusion,the present study demonstrated that apoptosis and autophagy of neurons and disturbing the transport system maybe the mechanisms of PFOS-induced neurotoxicity.