Study on electroacupuncture promoting the proliferation and neuronal differentiation of endogenous neural stem cells after spinal cord injury via AKT/GSK-3β/CREB pathway in mice
Objective To investigate the effect of electroacupuncture(EA)on the proliferation and differentiation of endogenous neural stem cells(eNSCs)and its relationship with protein kinase B/glycogen synthase kinase-3 β/camp response element binding protein(AKT/GSK-3β/CREB)signaling pathway after spinal cord injury(SCI).Methods A total of 48 C57BL/6 mice were randomly divided into sham group,SCI group,SCI+EA treatment group and SCI+EA+AKT inhibitor MK2206 treatment group,with 12 mice in each group.The sham group only underwent laminectomy,and the Tg_9 spinal cord transection model was established in the other three groups.On the 7th day after SCI,immunofluorescence staining was used to detect Ki-67 and Nestin to evaluate the proliferation of eNSCs,and the Western blot(WB)was used to detect the expression of Nestin protein.On the 14th day after SCI,nerve growth-associated protein(GAP-43)and Ki-67 were detected by immunofluorescence staining to evaluate the differentiation of eNSCs into neurons,and glial fibrillary acidic protein(GFAP)was detected to evaluate the activation of astrocytes at the injury site.WB was used to measure the protein expression levels of GAP-43 and AKT/GSK-3β/CREB signaling pathway.Results The success rate of model building was 94.7％(36/38).Compared with the Sham group,the SCI group had a significant increase in the number of Nestin/Ki-67 positive double cells(P＜0.05).Compared with the SCI group,the number of Nestin/Ki-67 double positive cells and GAP-43/Ki-67 double positive cells increased significantly in the EA group,and the number of GFAP positive cells decreased(all P＜0.05).WB results showed that the expression of Nestin and GAP-43 in the EA group was significantly higher than that in the SCI group(both P＜0.05).Compared with Sham group,the phosphorylation level of GSK-3β in SCI group was decreased(P＜0.05),and the phosphorylation level of A KT and CREB was not significantly different(both P＞0.05).Compared with SCI group,the phosphorylation levels of AKT,GSK-3β and CREB in EA group were significantly increased(all P＜0.05).The phosphorylation levels of AKT,GSK-3β and CREB in the EA+MK-2206 group were significantly lower compared with those in the EA group(all P＜0.05),and there was no significant difference between the EA+MK-2206 group and the SCI group(all P＞0.05).Conclusions EA treatment can promote the proliferation and neuronal differentiation of eNSCs after SCI,and inhibition of AKT activation can inhibit that after SCI.It is suggested that the mechanism of EA treatment may be related to the AKT/GSK-3β/CREB pathway.
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