| Objective To investigate the effect of low-dose Botulinum toxin A on the proliferation of neural stem cells and its mechanism.Methods Neural stem cells(NSCs)were isolated from the subventricular zone of the brain of Sprague-Dawley rat fetus(E15),then the same concentration of 0.001,0.01,0.1,1 and 10U/ml of botulinum toxin A(BoNT/A)and the same amount of saline(Control Group)a total of six groups.After 24 h,48h and 72 h,the absorbance of the six groups was measured by Methods CCK-8 to obtain the optimal concentration of BoNT/A to promote the proliferation of NSCS.NSCS were isolated and purified by the methods mentioned above,then the optimal concentration of BoNT/A was added as Experimental Group,and the same amount of saline was added as Control Group,the expression of Value-associated protein(cyclin D1,PCNA)and Wnt Pathway-associated protein(Rac1,?-catenin)was detected by Western blotting.The appropriate amount of Rac1 inhibitor(NSC23766)was added in the process of cell culture.The expression changes of Value-associated protein(cyclin D1,PCNA)and Wnt Pathway-associated protein(Rac1,?-catenin)was separately detected by Western blotting,in order to make the further conformation of the role of Rac1 in the process of NSCs proliferation and its upstream and downstream relationship with ?-catenin.Results 0.01U/ml BoNT/A can significantly increase the proliferation of NSCs by Methods CCK-8;0.01U/ml BoNT/A significantly increased the expression of cyclin D1,PCNA,Rac1,?-catenin by Western blotting.After adding the Rac1 inhibitor(NSC23766),the expression of cyclin D1?PCNA?Rac1 and ?-catenin decreased to some extent,which indicated that the Rac1 inhibitor was in the upstream of these four proteins.Conclusion:Low-dose BoNT/A could significantly promot the proliferation of NSCs;and the Rac1/?-catenin branch of Wnt pathway played an important role in this cellular activity of BoNT/A in promoting the proliferation of NSCs. |
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