苯丙胺对PC12细胞AKT/GSK-3β/CRMP-2信号通路的影响
摘要
目的：
探讨苯丙胺对PC12细胞的神经毒性作用和机制。
方法：
将PC12细胞分为四组：空白对照组,苯丙胺组(2mM),苯丙胺加NGF(神经生长因子)组,苯丙胺加SB216763组。各组培养24h后,采用倒置显微光镜,免疫荧光技术及免疫印迹法观察苯丙胺对PC12细胞的神经毒性作用以及对AKT/GSK-3β/CRMP-2信号通路的影响,并且观察该信号通路上游激活剂NGF和下游GSK-3β的抑制剂SB216763能否减轻苯丙胺对PC12细胞的神经毒性作用。
结果：
1.倒置显微光镜形态学结果：空白对照组PC12细胞膜光滑,边界清晰,胞体饱满,透光,多呈椭圆形或梭形,有两条或以上的细长突起,许多细胞的突起交织在一起形成网；苯丙胺组与空白对照组相比,细胞体萎缩,呈圆形,突起变短、消失(P<0.05),突起形成的网状结构消失；苯丙胺加NGF组和苯丙胺加SB216763组与苯丙胺组相比,突起长度增长(P<0.05)。四组的细胞计数进行比较,差异无显著性(P>0.05)。多巴胺免疫荧光技术观察PC12细胞,各组间细胞长度的变化与上述的基本一致,各组细胞胞体及突起内都有多巴胺激发的荧光,其中空白对照组细胞及突起内多巴胺荧光强度比其他各组的更强,苯丙胺组与其他各组相比,多巴胺细胞的数量有所减少。
2.苯丙胺、NGF及SB216763对AKT/GSK-3β/CRMP-2信号传导通路中蛋白表达的影响：
1)TrkA和P-TrkA:苯丙胺组与空白对照组比较,TrkA和P-TrkA的表达差异均无统计学意义(P>0.05)。苯丙胺加NGF组和苯丙胺加SB216763组P-TrkA的表达与苯丙胺组相比均增多,差异有显著性(P<0.05),但TrkA的表达与苯丙胺组相比,差异均无统计学意义(P>0.05)。
2)AKT和P-AKT:苯丙胺组与空白对照组相比,苯丙胺组P-AKT的表达减少,差异有显著性(P<0.05)。苯丙胺加NGF组和苯丙胺加SB216763组与苯丙胺组相比,P-AKT的表达增加,差异有显著性(P<0.05)。而AKT的表达在各组间的差异无统计学意义(P>0.05)。
3)GSK-3β和P-GSK-3β:苯丙胺组与空白对照组相比,苯丙胺组P-GSK-3β表达减少,差异有显著性(P<0.05)；而苯丙胺组GSK-3β表达增加,差异有显著性(P<0.05)。苯丙胺加NGF组与苯丙胺组相比,苯丙胺加NGF组P-GSK-3β的表达增加有显著性(P<0.05), GSK-3β表达差异无显著性(P>0.05)。苯丙胺加SB216763组和苯丙胺组相比,苯丙胺加SB216763组P-GSK-3β的表达差异无显著性(P>0.05),苯丙胺加SB216763组GSK-3β表达减少,差异有显著性(P<0.05)。
4)CRMP-2和)P-CRMP-2:苯丙胺组与空白对照组相比,苯丙胺组P-CRMP-2的表达增多,差异有显著性(P<0.05),苯丙胺加NGF组和苯丙胺加SB216763组与苯丙胺组相比,P-CRMP-2的表达均减少,差异有显著性(P<0.05)。CRMP-2在各组间的表达差异无统计学差异(P>0.05)。
结论：
苯丙胺可以抑制PC12细胞突起的生长,其机制可能与抑制AKT/GSK-3β/CRMP-2信号通路有密切关系。2mM浓度的苯丙胺对PC12细胞数量无明显影响。AKT/GSK-3β/CRMP-2信号通路的上游激活剂NGF可以减轻苯丙胺对PC12细胞的神经毒性作用,且其下游GSK-3β的抑制剂SB216763也具有减轻苯丙胺对PC12细胞的神经毒性作用,这可能与激活AKT/GSK-3β/CRMP-2信号通路的分子有关。
Objective
To explore mechanism of amphetamine-induced neurotoxicity on PC 12 cells.
Methods
The 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.
Results
1.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).
Conclusion
Amphetamine 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.
探讨苯丙胺对PC12细胞的神经毒性作用和机制。
方法：
将PC12细胞分为四组：空白对照组,苯丙胺组(2mM),苯丙胺加NGF(神经生长因子)组,苯丙胺加SB216763组。各组培养24h后,采用倒置显微光镜,免疫荧光技术及免疫印迹法观察苯丙胺对PC12细胞的神经毒性作用以及对AKT/GSK-3β/CRMP-2信号通路的影响,并且观察该信号通路上游激活剂NGF和下游GSK-3β的抑制剂SB216763能否减轻苯丙胺对PC12细胞的神经毒性作用。
结果：
1.倒置显微光镜形态学结果：空白对照组PC12细胞膜光滑,边界清晰,胞体饱满,透光,多呈椭圆形或梭形,有两条或以上的细长突起,许多细胞的突起交织在一起形成网；苯丙胺组与空白对照组相比,细胞体萎缩,呈圆形,突起变短、消失(P<0.05),突起形成的网状结构消失；苯丙胺加NGF组和苯丙胺加SB216763组与苯丙胺组相比,突起长度增长(P<0.05)。四组的细胞计数进行比较,差异无显著性(P>0.05)。多巴胺免疫荧光技术观察PC12细胞,各组间细胞长度的变化与上述的基本一致,各组细胞胞体及突起内都有多巴胺激发的荧光,其中空白对照组细胞及突起内多巴胺荧光强度比其他各组的更强,苯丙胺组与其他各组相比,多巴胺细胞的数量有所减少。
2.苯丙胺、NGF及SB216763对AKT/GSK-3β/CRMP-2信号传导通路中蛋白表达的影响：
1)TrkA和P-TrkA:苯丙胺组与空白对照组比较,TrkA和P-TrkA的表达差异均无统计学意义(P>0.05)。苯丙胺加NGF组和苯丙胺加SB216763组P-TrkA的表达与苯丙胺组相比均增多,差异有显著性(P<0.05),但TrkA的表达与苯丙胺组相比,差异均无统计学意义(P>0.05)。
2)AKT和P-AKT:苯丙胺组与空白对照组相比,苯丙胺组P-AKT的表达减少,差异有显著性(P<0.05)。苯丙胺加NGF组和苯丙胺加SB216763组与苯丙胺组相比,P-AKT的表达增加,差异有显著性(P<0.05)。而AKT的表达在各组间的差异无统计学意义(P>0.05)。
3)GSK-3β和P-GSK-3β:苯丙胺组与空白对照组相比,苯丙胺组P-GSK-3β表达减少,差异有显著性(P<0.05)；而苯丙胺组GSK-3β表达增加,差异有显著性(P<0.05)。苯丙胺加NGF组与苯丙胺组相比,苯丙胺加NGF组P-GSK-3β的表达增加有显著性(P<0.05), GSK-3β表达差异无显著性(P>0.05)。苯丙胺加SB216763组和苯丙胺组相比,苯丙胺加SB216763组P-GSK-3β的表达差异无显著性(P>0.05),苯丙胺加SB216763组GSK-3β表达减少,差异有显著性(P<0.05)。
4)CRMP-2和)P-CRMP-2:苯丙胺组与空白对照组相比,苯丙胺组P-CRMP-2的表达增多,差异有显著性(P<0.05),苯丙胺加NGF组和苯丙胺加SB216763组与苯丙胺组相比,P-CRMP-2的表达均减少,差异有显著性(P<0.05)。CRMP-2在各组间的表达差异无统计学差异(P>0.05)。
结论：
苯丙胺可以抑制PC12细胞突起的生长,其机制可能与抑制AKT/GSK-3β/CRMP-2信号通路有密切关系。2mM浓度的苯丙胺对PC12细胞数量无明显影响。AKT/GSK-3β/CRMP-2信号通路的上游激活剂NGF可以减轻苯丙胺对PC12细胞的神经毒性作用,且其下游GSK-3β的抑制剂SB216763也具有减轻苯丙胺对PC12细胞的神经毒性作用,这可能与激活AKT/GSK-3β/CRMP-2信号通路的分子有关。
Objective
To explore mechanism of amphetamine-induced neurotoxicity on PC 12 cells.
Methods
The 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.
Results
1.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).
Conclusion
Amphetamine 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.
引文
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