| Objective:The influence of environmental heavy metal pollution on human health has become a global public health problem,among which lead(Pb),cadmium(Cd)and mercury(Hg)are the main harmful factors.They usually coexist in the human living environment,through a variety of ways into the human body,and in the general population of the blood can be detected at the same time of these three heavy metals.In previous studies,our team found that the blood load of the general population and its related levels of mixed exposure to low doses of heavy metals Pb,Cd and Hg can cause learning and memory impairment and synaptic plasticity impairment in rats,but the mechanism is still unclear.Arp2/3 complex and Cofilin both play important roles in dendritic branch morphogenesis.Recent studies have shown that Cofilin can accelerate the dissociation rate of Arp2/3 from actin filaments and promote the dissociation of actin filaments branches,resulting in disturbance of actin balance and thus damage of dendrite morphological structure.Therefore,in this study,the primary hippocampal neuron model of mixed exposure to low doses of heavy metals was established to regulate the expression of Cofilin-Arp2/3 pathway,and to explore the morphological abnormalities of hippocampal neuron dendrite and its mechanism after mixed exposure to low doses of lead,cadmium and mercury,providing a new target for the prevention and treatment of nerve damage caused by heavy metal combined pollution.Methods:(1)To establish the primary hippocampal neuron model of mixed exposure of low dose heavy metals Pb,Cd and Hg to the blood load level of the general population.F-actin was stained with phalloidin to detect the effect of mixed exposure to low doses of heavy metals on the morphological complexity of hippocampal neuronal dendrites.The F-actin/G-actin balance of dendritic skeleton was determined by Western Blot,and the expression of Arp2 and Arp3 subunits,the key proteins for dendritic spinous branch formation,were detected.Binding ability of Arp3 and actin filaments co-stained by immunofluorescence.Lentivirus HBLV-Arp3 was used to infect high-expression neurons with Arp3 expression levels,and the effects of mixed exposure to low doses of heavy metals on the binding ability of Arp3 and F-actin,actin balance and dendrite complexity were observed.(2)The expression level of Cofilin and the binding ability of Cofilin and F-actin were detected after the primary hippocampal neurons were exposed to mixed heavy metals with a concentration gradient.By interfering si RNA-Cofilin knocking down Cofilin expression in hippocampal neurons,the effects of Cofilin binding with actin filaments,actin balance and dendrite complexity in hippocampal neurons were observed.(3)The co-expression of Cofilin and Arp3 in hippocampal neurons was detected by immunofluorescence co-staining after the primary hippocampal neurons were exposed to low-dose heavy metals.After knocking down Cofilin expression level in hippocampal neurons by si RNA-Cofilin,the effect of Arp3 expression induced by low dose of heavy metals on its binding ability to actin filaments was observed.At the same time,after Arp3 expression level of lentivirus HBLV-Arp3 high expression neurons,the influence of low dose heavy metal induced abnormal Cofilin expression was observed.(4)In vitro reaction system,Cofilin was purified by Escherichia coli expression system,and EMR mass spectrometry was used to analyze in vitro the binding effect between heavy metal ions and mixed exposure and Cofilin proteomic molecules.The actin kinetics of actin during actin polymerization and depolymerization was detected by multifunctional enzyme labeled instrument.On this basis,Cofilin and heavy metal were added into the reaction system to investigate the effect of Cofilin on actin shearing and low dose heavy metal mixed exposure.Results:(1)The effect of Arp2-3 on the morphological abnormalities of neuronal dendrites after mixed exposure to low doses of heavy metals.The dendrite complexity decreased in concentration-dependent way due to low doses of Pb,CD and Hg combined exposure,which showed decreased average dendrite length and number of branch ends(P<0.05).In addition,the balance of skeletal actin was disturbed,the expression level of filamentous actin decreased while that of G-actin increased,and the ratio of F-actin to G-actin decreased(P < 0.05).The expression level of Arp3,a key protein for dendritic branching,and its binding ability to actin filaments decreased concentration-dependent with the increase of low-dose heavy metal exposure(P<0.05).Lentivirus HBLV-Arp3 infection effectively increased the expression level of Arp3 in neurons.Further tests showed that HBLV-Arp3 infection also improved the binding capacity of Arp3 and F-actin,actin balance and dendrite complexity damage caused by mixed exposure to low doses of heavy metals(P <0.05).(2)The effect of Cofilin on the morphological abnormalities of neuronal dendrites induced by mixed exposure to low-dose heavy metals.The expression level of dendrite shear related protein Cofilin was increased concentration-dependent by low dose exposure of heavy metals such as lead,cadmium and mercury,and the binding capacity of Cofilin and actin filaments was increased,and the shear capacity was enhanced(P<0.05).Small interference fragments si RNA-Cofilin transfection effectively knocked down the expression level of Cofilin in neurons.Further tests showed that si RNA-Cofilin transfection also improved Cofilin and F-actin binding capacity,actin balance,and dendrite complexity damage caused by low dose heavy metal mixed exposure(P<0.05).(3)Cofilin regulates Arp2/3 in the abnormal balance of dendritic skeleton actin induced by low dose mixed heavy metal exposure.Cofilin and Arp3 expression in hippocampal neurons increased concentration-dependent after exposure to low doses of lead,cadmium and mercury(P<0.01).si RNA-Cofilin knockdown of Cofilin expression in hippocampal neurons improved the expression of Arp3 and its binding ability to actin filaments induced by mixed exposure to low-dose heavy metals(P<0.05).However,high expression of HBLV-Arp3 had no effect on abnormal Cofilin expression caused by mixed exposure to low dose heavy metals(P>0.05).It was revealed that Cofilin,as an upstream molecule,plays an important molecular role in regulating Arp3 in the abnormal morphology of neuronal dendrites induced by mixed exposure to low doses of heavy metals.(4)The direct binding effect of low dose heavy metal ions on Cofilin protein molecules and its effect on the inhibition of actin polymerization by Cofilin.Mass spectrometry showed that Hg ions at low doses could combine with Cofilin in vitro,and Pb ions and Cd ions could promote Cofilin protein molecules to combine with Hg ions.In addition,through the actin polymerity-depolymerization system,it was found that mixed exposure to low doses of heavy metals in vitro did not directly affect the inhibition of actin polymerization by Cofilin.Conclusions:The abnormal dendrite morphology of hippocampal neurons induced by low dose of heavy metals Pb,Cd and Hg may be caused by Cofilin-regulated Arp2/3.And low dose heavy metal ions and their mixed exposure can directly bind to Cofilin protein molecules.These results suggest that it may be a new target for prevention and treatment of nerve damage caused by abnormal dendrite morphology caused by mixed exposure to low dose heavy metals. |
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