| Objective:Environmental heavy metal pollutions have become public health problems seriously affecting people’s health,in which heavy metals with serious harm and strong biological effect mainly include lead(Pb),cadmium(Cd)and mercury(Hg).These heavy metals often coexist in human living environment and are extremely difficult to degrade.Therefore,the health hazards caused by long-term low-dose exposure to these heavy metal mixtures,especially the nervous system damage,are worthy of attention.Our previous study found that low-dose mixed exposure of heavy metals under general population load and related levels can cause learning and memory as well as neuronal dendritic spine damage,but the mechanism is still unclear.LIM kinase 1(LIMK1)/ slingshot phosphatase 1(SSH1)-Cofilin is an important signal pathway regulating the structure and function of the cytoskeleton.which is involved in the remodeling of neuronal dendritic spines by changing the actin balance of dendritic spines.In this study,we established a model of primary hippocampal neurons exposed to low-dose heavy metal mixtures to regulate the expression of LIMK1/SSH1-Cofilin pathway,and to explore the dendritic spinous actin balance disorders in hippocampal neurons and their mechanism after low-dose lead,cadmium and mercury exposure,so as to provide a new target for the prevention and treatment of nerve injury caused by heavy metal combined pollution.Methods:(1)Establish a hippocampal neuron model which exposed to low dose lead,cadmium and mercury under general population load and its related concentration gradients,and to detect the effects of low dose heavy metal exposure on dendritic spinous cytoskeleton F-actin/G-actin balance and dendritic complexity.The expression of Cofilin,which is the disaggregation factor of actin dynamic balance in dendritic spine skeleton,and its effect on actin filament binding ability and actin filament growth regulation ability were detected.(2)After the primary cultured hippocampal neurons were exposed to low dose heavy metal mixture,the expression of Cofilin upstream regulatory proteins LIMK1 and p-LIMK1 were detected.The expression of p-LIMK1 was increased by lentivirus HBLV-p LIMK1 transfection.The Cofilin expression,F-actin/G-actin balance and the complexity of neuronal dendrites were observed.In addition,the inhibitor of LIMK1(BMS-5)was used to detect the effects of BMS-5 on the expression of LIMK1 and Cofilin as well as the dynamic balance of F-actin/G-actin induced by low dose heavy metal exposure.(3)After the primary cultured hippocampal neurons were exposed to low dose heavy metal mixture,the expression of Cofilin upstream regulatory proteins SSH1 and p-SSH1 were detected.The effects of sh RNA-SSH1 plasmid transfection on the Cofilin expression and F-actin/G-actin dynamic balance were observed after knocking down the expression level of SSH1 in the neurons.In addition,the interaction between LIMK1 and SSH1 in their simultaneous regulation of Cofilin and actin filament was further analyzed.Results:(1)The effects of low dose heavy metal exposure on the balance of cytoskeletal actin in dendritic spines of neurons and its depolymerizing factor Cofilin.Low dose of lead,cadmium and mercury mixed exposure caused a concentration-dependent decrease in F-actin expression and F-actin/G-actin ratio,as well as an increase in G-actin expression(P<0.05).which accompanied by the concentration-dependent increase of Cofilin and the decrease of p-Cofilin(P<0.05),as well as the degree of phosphorylation of Cofilin(p-Cofilin/Cofilin)also decreased(P<0.05).In addition,with the increase of mixed exposure dose of heavy metals,the ratio of Cofilin/F-actin increased(P<0.05)in a concentration-dependent manner,indicating that the Cofilin on actin filaments increased and the shearing ability enhanced.The ratio of ARP/Cofilin decreased(P<0.05)in a concentration-dependent manner,suggesting that Cofilin may reduce the growth ability of dendritic actin filaments after heavy metal exposure.(2)The role of Cofilin pathway regulated by LIMK1 in the disorder of F-actin/G-actin balance in neuronal dendritic spines induced by low dose heavy metal exposure.The level of p-LIMK1 and p-LIMK1/LIMK1 in primary hippocampal neurons decreased in a concentration-dependent manner after exposure to low dose of lead,cadmium and mercury.HBLV-p LIMK1 lentivirus transfection effectively increased the ratio of p-LIMK1,LIMK1 and p-LIMK1/LIMK1 in neurons.Further detection showed that HBLV-p LIMK1 transfection also improved the decrease of neuronal p-Cofilin/Cofilin,F-actin/G-actin ratio and dendritic complexity induced by low dose of heavy metal mixture(P<0.05).BMS-5,an inhibitor of LIMK1,aggravated the decreases of p-LIMK1/LIMK1,p-Cofilin/Cofilin and F-actin/G-actin ratio in neurons induced by low dose heavy metal mixture exposure.(3)The role of Cofilin pathway regulated by SSH1 in the disorder of F-actin/G-actin balance in dendritic spines induced by low dose heavy metal mixture exposure.Low dose of lead,cadmium and mercury mixture exposure increased the expression of SSH1 in a concentration-dependent manner,while the degree of phosphorylation of SSH1(p-SSH1/SSH1)decreased.Sh RNA-SSH1 plasmid transfection effectively decreased the levels of SSH1 and p-SSH1 and increased the ratio of p-SSH1/SSH1 in neurons.Further detection showed that sh RNA-SSH1 transfection also improved the decrease of neuronal p-Cofilin/Cofilin,F-actin/G-actin ratio induced by low dose heavy metal mixture exposure.(4)The interaction of SSH1 and LIMK1 in regulating the disturbance of actin balance induced by low dose heavy metal mixture exposure.HBLV-p LIMK1 lentivirus transfection effectively improved the damages of neuronal SSH1 and p-SSH1/SSH1 ratio induced by low dose of heavy metal mixture.Similarly,sh RNA-SSH1 plasmid transfection also increased the decrease of p-LIMK1,p-LIMK1/LIMK1 ratio.These results suggest that there is an interaction between LIMK1 and SSH1 regulating the disturbance of F-actin/G-actin impaired by low doses of heavy metal mixture.In addition,when the changes of p-LIMK1 and SSH1 expression induced by low dose heavy metal mixture were interfered to the normal level,the changes of F-actin/G-actin and p-Cofilin/Cofilin levels induced by heavy metals returned to the normal level after p-LIMK1 lentivirus transfection,however,these changes induced by heavy metals did not return to the normal level after the intervention of SSH1 plasmid.To a certain extent,it is suggested that the regulatory contribution of LIMK1 is more significant than that of SSH1 in this process.Conclusions:The disordered cytoskeletal actin balance of dendritic spines in neurons induced by low-dose heavy metal lead,cadmium and mercury mixtures might be caused by the Cofilin pathway regulated by LIMK1/SSH1.It is suggested that this may be a new target for the prevention and treatment of neuronal dendritic spinous cytoskeletal actin disorder and nerve injury induced by low dose heavy metal mixture exposure. |
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