| ObjectiveTo explore mechanism of amphetamine-induced neurotoxicity on PC 12 cells.MethodsThe PC12 cells were divided into four groups:control group,amphetamine group(2.0mM), amphetamine plus NGF (nerve growth factor) group,and amphetamine plus SB216763 group. The cells were cultured for 24 hours after dosing, inverted microscope, immunofluorescence technique and western-blotting were used to observe amphetamine neurotoxicity on PC12 cells and AKT/GSK-3P/CRMP-2 signal pathway. Effects of NGF and SB216763 on PC12 cells and AKT/GSK-3β/CRMP-2 signal pathway were also explored.Results1.Morphology under light microscopy:PC12 cell membranes in control group were smooth and clear.Cell bodies were prominent,transparent and refractive with oval or spindle shapes.Each cell had two or more slender processes. Many cellular processes were connected with each other to form the neuronal network. Compared with the control group, PC12 cells in the amphetamine group were gradually shrunk in size,cell bodies became round,and cellular processes became sh-orter and broken(P<0.05),the cellular process network was disappeared.The cellular processes in the amphetamine plus NGF group and plus SB216763 group were longer than those in the amph-etamine group(P<0.05).There were no significant differences in cell number between four grou-ps(P>0.05).Immunofluorescence technique showed that PC 12 cells contain dopamine(DA).The changes of cellular processes observed by this technique are the same as above.Dopamine cells and processes in the control group had stronger fluorescence intensity than those in the other gr-oups, the cell numbers in the amphetamine group appeared to be decreased compared with the other groups.2.Changes of AKT/GSK-3β/CRMP-2 signaling pathway influenced by amphetamine,NGF and SB216763:1)TrkA and P-TrkA:The expressions of TrkA and P-TrkA had no significant differences be-tween the amphetamine and control groups(P>0.05). The expression of P-TrkA in amphetamine plus NGF group and plus SB216763 group was significantly increased compared with that of the amphtamine group(P<0.05).TrkA expression was not significantly different between plus group and amphetamine group(P>0.05).2)AKT and P-AKT:Compared with the control group,P-AKT expression was significantly reduced in amphetamine group (P<0.05).P-AKT expression in amphetamine plus NGF group and plus SB216763 group was significantly increased compared with that of the amphetamine groups(P<0.05).However,the expression of AKT had no any changes between all groups(P> 0.05).3)GSK-3βand P-GSK-3β:Compared with the control group,P-GSK-3βexpression was sig-nificantly decreased in amphetamine group(P<0.05),GSK-3βexpression was increased in amph-etamine group(P<0.05).Compared with the amphtamine group,P-GSK-3βexpression was signi-ficantly increased in amphetamine plus NGF group(P<0.05),however,GSK-3βexpression had no significantly changes in amphetamine plus NGF group(P>0.05).P-GSK-3βexpression in amphetamine plus SB216763 group was no significantly different compared with that of the amphetamine group(P>0.05),GSK-3p expression was significantly decreased in amphetamine plus SB216763 group(P<0.05).4)CRMP-2 andP-CRMP-2:P-CRMP-2 expression in amphetamine group was significantly increased than that of the control group(P<0.05).Compared with the amphtamine group,P-CRMP-2 expression was decreased in amphetamine plus NGF group and plus SB216763group(P< 0.05).CRMP-2 expression had no any changes between all groups(P>0.05).ConclusionAmphetamine inhibited the growth of PC 12 cell processes.Its mechanism may be closely rel-ated to inhibition of AKT/GSK-3β/CRMP-2 signaling pathway.2mM amphetamine had no effect on PC 12 cell number.NGF as an upstream signaling molecule of this pathway may reduce the amphetamine-induced neurotoxicity on PC12 cells.SB216763 as a downstream signaling mole-cule and GSK-3βinhibitor of this pathway also has a role in cell protection against amphetamine damage.They may activate AKT/GSK-3β/CRMP-2 signaling pathway in PC 12 cells. |
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