The Role And Its Mechanism Of GAP43 In Amphetamine-induced Injury Of PC12 Cells

Objective: To investigate the role and its mechanism of GAP43 in AMPH-induced injure of PC12 cells.Methods: The animal model was established with intraperitoneally injection of AMPH(2.5mg/kg/d),Sprague-Dawley rats were randomly divided into control and drug groups.The drug groups were subdivided into 1d,7d and 14 d groups according to the time of drug treatment.The control group was injected with equivalence saline.The changes of the expression of GAP43 in striatum were detected by Western blot.The model of DA neurons was established with PC12 cells.PC12 cells were exposed to AMPH(3m M)for difference times(1h,2h,6h,12 h,24h).The changes of GAP43 and p-GAP43 in PC12 cells were detected by Western blot,the expression of GAP43 m RNA were detected by q PCR.We compared the relationship between GAP43 level and the neurite length.After PC12 cells were treated with AMPH(3m M),PKC activator PMA(100u M),or PKC inhibitor Enzastuarin(10n M),the changes of PC12 cells were observed by immunofluorescence,Western blot were used to detect the changes of PKC,GAP43 proteins and apoptotic proteins,MTT assay was used to detect the activity of PC12 cells.Results: 1.The effects of AMPH on the level of GAP43 in rats striatum: GAP43 level was gradually decreased in the drug groups.But there was no significant difference between AMPH 1day and 7day groups.GAP43 level was significantly decreased at 14 d in the AMPH group than in the control group(P<0.01).2.The changes of GAP43 level and neurite lengths induced by AMPH in PC12 cells: After PC12 cells were treated with AMPH,the levels of GAP43 and p-GAP43 proteins were gradually decreased(P<0.05).The neurite lengths also gradually became shorter,and there was a positive correlation between the GAP43 levels and neurite lengths(R2=0.7241,P=0.000).The expression of GAP43 m RNA was gradually reduced(P<0.05).3.The effects of PKC activators and inhibitors on PC12 cells: 1)The changes of PKC,GAP43 levels and neurite lengths: Compared to the normal group,neurite lengths became shorter after PC12 cells were treated with AMPH and PKC inhibitors(P<0.001).AMPH and PKC inhibitors also reduced the levels of GAP43(P<0.001),p-GAP43(P<0.001)and PKC?1(P<0.001);After PC12 cells were treated with PMA,an PKC activator,the levels of GAP43(P<0.01)and PKC?1(P<0.001)were increased,but p-GAP43 level and neurite lengths had no changes.PKC activator plus AMPH group significantly increased the GAP43 and PKC?1 levels(P<0.05),and neurite lengths(P<0.05),compared to the AMPH group.But there were no significant changes in total PKC,PKC? and PKC? levels.2)The changes of apoptotic proteins and cell viability: 3m M AMPH decreased the level of anti-apoptotic Bcl-2 protein(P<0.001),and viability of PC12 cells(P<0.001),and increased the level of pre-apoptotic Bax protein(P<0.001);PMA had no effects on the levels of Bcl-2 and Bax.Compared with 3m M AMPH,PMA increased Bcl-2 level(P<0.01)and cell viability(P<0.05),decreased Bax level(P<0.001).Conclusions: 1.AMPH can inhibit neurite growth of PC12 cells,and its mechanism is closely related to reduction of GAP43.2.AMPH reduced GAP43 level through inhibition of PKC?1.Activation of PKC?1/GAP43 pathway can protect PC12 cells against AMPH-induced neurotoxicity.