The Role Of Neuronal Mitochondrial Axon Transport Disorders Caused By Nonylphenol Exposure In Axon Development

Objective: Nonylphenol is a persistent organic pollutant,which can enter the human body through the accumulation of food chain,and eventually cause a serious impact on various systems of the body.In recent years,there are more and more reports about the neurotoxicity of nonylphenol.Now there is clear evidence that nonylphenol has an effect on the axon development of hippocampal neurons,and there is also evidence that nonylphenol has an effect on the activation of AKT / GSK-3β signal pathway of hippocampal neurons.However,there are few reports about whether nonylphenol can cause the mitochondrial transport disorder of hippocampal neuron axons.Mitochondria are transported from the neuron body to the synapse through the axon,providing energy for the normal activities of the synapse.The obstacle of mitochondrial transport is bound to lead to abnormal axon growth.Research shows that the AKT / GSK-3β signal pathway regulates the movement of mitochondria in the axon by regulating the proteins related to the transport of mitochondria.In this study,we established a model of exposure to nonylphenol during pregnancy and lactation to explore the effects and possible mechanisms of nonylphenol on the axonal mitochondrial transport and axonal development of neurons;On this basis,a rat pheochromocytoma cell(PC12)exposure model of nonylphenol was established to explore the role of AKT / GSK-3β signal pathway in neuronal axonal mitochondrial transport disorder and abnormal axonal development induced by nonylphenol exposure during pregnancy and lactation via GSK-3β antagonist1-Azakenpaullone.Methods: 1.Establishment of infant rat model of exposure to nonylphenol during pregnancy and lactation: SD rats during pregnancy and lactation were administered with different doses of nonylphenol by gavage every day,with the concentration of 2、10 and50 mg/kg respectively.The control group was directly administered with corn oil without adding nonylphenol by gavage.On the 21 th day after birth,they were killed and taken.2.Detection of relevant indicators of axonal development in the hippocampus of offspring:collect samples of the hippocampus of offspring,observe the fluorescence expression of hippocampal neurons of offspring with immunofluorescence,and detect the expression level of axonal NFL protein by Western blot.3.Detection of mitochondrial axonal transport complex related proteins in the hippocampus of offspring: The expression level of mitochondrial axonal transport complex related proteins KHC,KLC,Miro1,Trak2 was detected by Western blot.4.Detection of relevant indicators of AKT / GSK-3β signal pathway in hippocampus of offspring: Expression level of AKT / GSK-3β signal pathway was detected by Western blot.5.Detection of PC12 cell viability after 24 hours’ NP exposure: Cell viability was detected by CCK-8.PC12 cell samples were collected at 5μM、8 μM、10 μM dose group;Western blot was used to detect the relevant indicators of axon mitochondrial transport and axon development,and the most significant dose group(10 μM)was conducted in follow-up inhibitor study.6.Detection of related indicators of PC12 cell neurite development: Collect PC12 cell samples,observe the fluorescence expression of PC12 cells with immunofluorescence,and detect the NFL protein expression level by Western blot.7.Detection of mitochondrial transport complex related proteins in PC12 cells: PC12 cell samples were collected to detect the expression level of mitochondrial transport complex related proteins KHC,KLC,Miro1,Trak2 by Western blot.8.Detection of relevant indicators of PC12 cell AKT / GSK-3β signal pathway:Collect PC12 cell samples and detect expression level of AKT / GSK-3β signal pathway by Western blot.9.Detection of PC12 cell viability after 24 hours’ 1-A exposure: Cell viability was detected by CCK-8 dose,and the 5 μM dose group of the antagonist was conducted in follow-up inhibitor study.10.Detection of related indicators of neurite development of PC12 cells under the application of GSK-3β antagonist 1-A: Select 10 μM NP dose group,add 1-A,observe PC12 cell fluorescence expression of NFL,and use Western blot to detect NFL protein expression level.11.Detection of mitochondrial transport complex related proteins in PC12 cells under the application of GSK-3βantagonist 1-A: Select 10 μM NP dose group,add 1-A,and detect the expression level of mitochondrial transport complex related proteins KHC,KLC,Miro1,Trak2 by Western blot.Results: 1.Nonylphenol exposure resulted in abnormal axonal development of hippocampal neurons: The immunofluorescence results showed that the NFL fluorescence morphology of neurons in the CA1 and DG areas of hippocampus in the nonylphenol exposure group during pregnancy and lactation changed.Western blot showed that the content of NFL in the 10 mg/kg dose group and 5 mg/kg dose group increased significantly(P < 0.05).2.Nonylphenol exposure resulted in mitochondrial transport disorder of hippocampal neurons: Western blot results showed that the expression of KHC protein in the hippocampus of offspring in the 10 mg/kg dose group and 50 mg/kg dose group was lower(P < 0.05);The expression of KLC protein in hippocampus of offspring in 2 mg/kg dose group,10 mg/kg dose group and 50 mg/kg dose group was lower(P < 0.01);The expression of Miro1 protein in hippocampus of offspring in 2 mg/kg dose group,10 mg/kg dose group and 50 mg/kg dose group was lower(P < 0.05);The expression of Trak2 protein in the hippocampus of offspring in the 50 mg/kg dose group was lower(P < 0.05).3.Nonylphenol exposure activated AKT / GSK-3β signal pathway in hippocampus: Western blot results showed that the expression of p-GSK-3β decreased significantly in the 10mg/kg dose group and 50 mg/kg dose group;The total GSK-3β protein level remained stable(P < 0.05).The expression of p-AKT in the 10mg/kg dose group and 50mg/kg dose group decreased significantly;The total AKT protein level remained stable(P < 0.05).4.Nonylphenol exposure resulted in abnormal neurite of PC12 cells: The immunofluorescence results showed that the NFL fluorescence morphology of PC12 cells in the nonylphenol exposure group changed.Western blot showed that the expression of NFL in the 5、8、10 μM dose group decreased significantly(P < 0.05).5.Nonylphenol exposure caused mitochondrial transport disorder in PC12 cells: Western blot results showed that the expression level of KHC protein in PC12 cells of 8 μM dose group and 10μM dose group was lower(P < 0.01);The expression level of KLC protein in PC12 cells of 8 μM dose group and 10 μM dose group was lower(P < 0.01);The expression level of Miro1 protein in PC12 cells of 8 μM dose group and 10 μM dose group was lower(P <0.01);The expression level of Trak2 protein in PC12 cells in the 10 μM dose group was lower(P < 0.05).6.Nonylphenol activated AKT / GSK-3β signal pathway of PC12 cells:Western blot results showed that the expression of p-GSK-3β in the 8 μM dose group and10 μM dose group decreased significantly;The total GSK-3β protein level remained stable(P < 0.01).The expression of p-AKT in the 8 μM dose group and 10 μM dose group decreased significantly;The total AKT protein level remained stable(P < 0.05).7.The antagonist of GSK-3β reduced the neurite abnormalities of PC12 cells induced by nonylphenol exposure: The immunofluorescence results showed that the NFL fluorescence morphology of PC12 cells in NP exposure group changed,while the morphology of NP+1-A combined treatment group improved compared with NP exposure group.Western blot showed that the expression of NFL in NP exposure group decreased significantly,while the expression of NFL in NP+1-A combined treatment group increased significantly compared with NP exposure group(P < 0.05).The expression of NFL in 1-A alone treatment group had no significant difference.8.The antagonism of GSK-3β can alleviate the mitochondrial transport disorder of PC12 cells caused by nonylphenol exposure:Western blot results showed that the expression of KHC in the NP exposure group decreased significantly,while the expression of KHC in the NP+1-A combined treatment group increased significantly compared with the NP exposure group(P < 0.05),and the expression of KHC in the 1-A alone treatment group had no significant difference.The expression of KLC in the NP exposure group decreased significantly,while the expression of KLC in the NP+1-A combined treatment group increased significantly compared with the NP exposure group(P < 0.05).The expression of KLC in the 1-A single treatment group was not significant.The expression of Miro1 in NP exposure group decreased significantly,while the expression of Miro1 in NP+1-A combined treatment group increased significantly compared with NP exposure group(P < 0.05).There was no significant difference in the expression of Miro1 between 1-A alone treatment group and the control group.The expression of Trak2 in the NP exposure group decreased significantly,while the expression of Trak2 in the NP+1-A combined treatment group increased significantly compared with the NP exposure group(P < 0.05).The expression of Trak2 in the 1-A single treatment group had no significant difference.Conclusion: 1.Nonylphenol exposure results in abnormal axonal development of hippocampal neurons.2.Nonylphenol exposure results in the impairment of mitochondrial transport in axons of hippocampal neurons.3.The AKT / GSK-3β signal pathway may be involved in the mitochondrial transport disorder of neuronal axons caused by nonylphenol exposure,which leads to the abnormal development of neuronal axons caused by nonylphenol exposure.