电针激活AKT/GSK-3β/CREB通路促进脊髓损伤小鼠内源性神经干细胞增殖和分化的研究

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

目的探讨电针(EA)治疗对脊髓损伤(SCI)小鼠内源性神经干细胞(eNSCs)增殖、分化的影响及其与蛋白激酶B(AKT)/糖原合成酶激酶3β(GSK-3β)/环磷酸腺苷反应元件结合蛋白(CREB)信号通路的关系。方法48只C57BL/6小鼠采用完全随机方法分为假手术组(Sham组)、SCI模型组(SCI组)、SCI+EA处理组(EA组)、SCI+EA+AKT抑制剂MK-2206处理组(EA+MK-2206组),每组12只。Sham组仅行椎板切除术,其余3组均建立T8~9脊髓全横断模型。各组于SCI后第7、14天取损伤处脊髓组织,其中SCI后第7天采用免疫荧光染色方法检测巢蛋白(Nestin)和Ki-67的表达水平,以评估eNSCs的增殖情况,并采用蛋白质免疫印迹(WB)法检测Nestin蛋白的表达水平;SCI后第14天采用免疫荧光染色检测神经生长相关蛋白(GAP-43)和Ki-67的表达水平,以评估eNSCs向神经元的分化情况,检测胶质纤维酸性蛋白(GFAP)以评估损伤处星形胶质细胞的活化情况;采用WB法检测GAP-43和AKT/GSK-3β/CREB信号通路蛋白的表达水平。结果SCI小鼠的建模成功率为94.7%(36/38)。免疫荧光染色结果显示,与Sham组相比,SCI组脊髓损伤部位Nestin与Ki-67共标阳性细胞数量显著增多(P<0.05);与SCI组相比,EA组脊髓损伤部位Nestin与Ki-67共标、GAP-43与Ki-67共标阳性细胞数量均明显增多,而GFAP阳性细胞数量减少(均P<0.05)。WB结果显示,EA组Nestin、GAP-43蛋白的表达量相较于SCI组均明显升高(均P<0.05);与Sham组相比,SCI组GSK-3β磷酸化水平减低(P<0.05),而AKT和CREB磷酸化水平与Sham组的差异均无统计学意义(均P>0.05);与SCI组相比,EA组AKT、GSK-3β以及CREB的磷酸化水平均显著升高(均P<0.05);EA+MK-2206组的AKT、GSK-3β和CREB的磷酸化水平均低于EA组(均P<0.05),而与SCI组相比AKT、GSK-3β和CREB磷酸化蛋白表达的差异均无统计学意义(均P>0.05)。结论EA治疗可促进小鼠SCI后eNSCs的增殖和向神经元的分化,且通过促进AKT、GSK-3β、CREB的磷酸化激活AKT/GSK-3β/CREB通路,提示EA治疗SCI的机制可能与AKT/GSK-3β/CREB通路有关。

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-3β/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 T8-9 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-3βin SCI group was decreased(P<0.05),and the phosphorylation level of AKT and CREB was not significantly different(both P>0.05).Compared with SCI group,the phosphorylation levels of AKT,GSK-3βand CREB in EA group were significantly increased(all P<0.05).The phosphorylation levels of AKT,GSK-3βand 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.