Donepezil Promotes Stroke-Induced Neurogenesis Within Subventricular Zone Via Src Signaling

Background and ObjectiveCerebral infarction is the most common type of stroke,and currently effective treatments are limited.Studies have shown that neural stem cells(NSCs)in subventricular zone(SVZ)are in a relatively static state under physiological conditions.After cerebral infarction,neurogenesis is activated,and a large number of NSCs proliferate and migrate around the cerebral infarction area and differentiate into mature neurons to repair damaged brain tissue.So SVZ neurogenesis may be a key target for the treatment of cerebral infarction.There are a group of neurons expressing choline acetyltransferase(ChAT)in SVZ,which can regulate SVZ neurogenesis after cerebral infarction.ChAT~+neurons play a role mainly by acetylcholine receptor(AChRs),but it is unclear how to promote NSCs proliferation.Src is the upstream signal of epidermal growth factor receptor(EGFR).The EGFR signaling pathway is a classic pathway of NSCs proliferation.In this study,the choline level in the brain of mice was upregulated by donepezil(cholinesterase inhibitor),and the Src signaling pathway was blocked by KX-01(Src inhibitor)to explore the role of Src between AChRs and EGFR signaling pathways,and to analyse the effect of Src in the process of ChAT~+neurons promoting the neurogenesis in SVZ after cerebral infarction.MethodsAdult male Kunming mice were divided into Sham+vehicle group,MCAO+vehicle group,MCAO+donepezil group and MCAO+donepezil+KX-01group at random.Transient middle cerebral artery occlusion(tMCAO)model was established.Donepezil at a dose of 5mg/kg and KX-01 at a dose of 1mg/kg were administered once daily for 7 days after surgery.We evaluate the neurological recovery in tMCAO mice by modified neurological severity score(mNSS).The proliferated cells were labeled by Ki67 and neuroblasts were labeled by Double Cortin(DCX)to observe the number of Ki67~+/DCX~+cells in SVZ of mice in each group by immunofluorescence.Western blot was used to measure the level of Ki67,p-EGFR,p-raf,Src and p-Akt in SVZ of mice in each group.Repeated measurement and Multi-way ANOVA were used to analyze the mNSS score of each group.The expression of neonatal neuroblasts and each protein was analyzed by One-way ANOVA.LSD test was used for inter-group comparison.P<0.05 was considered statistically significant.Results1.The mNSS score of the MCAO+donepezil group was significantly lower than that of the MCAO+donepezil+KX-01 group and MCAO+vehicle group.2.Immunofluorescence results showed the number of Ki67~+/DCX~+cells in the MCAO+vehicle group was significantly higher than that of the Sham+vehicle group.The number of Ki67~+/DCX~+cells in the MCAO+donepezil group was significantly higher than that of the MCAO+vehicle group and MCAO+donepezil+KX-01 group.3.The expression of Ki67,Src,p-EGFR,p-Raf and p-Akt in the MCAO+vehicle group was significantly higher than that of the Sham+vehicle group.The protein expression in MCAO+donepezil group was higher significantly than that of MCAO+vehicle group and MCAO+donepezil+KX-01 group.Conclusions1.The neurological function recovery after cerebral infarction can be promoted by enhanced cholinergic signal.2.Enhanced cholinergic signal can promote SVZ neurogenesis after cerebral infarction.3.Donepezil promotes stroke-induced neurogenesis via Src/EGFR signaling.