电针对脑缺血/再灌注损伤大鼠神经干细胞增殖的动态影响

The dynamic effect of electroacupuncture on increment of neural stem cells in rats with cerebral ischemia/reperfusion injury

目的:探讨电针对脑缺血/再灌注损伤大鼠海马区内源性神经干细胞NSCs含量的动态影响及可能机制.方法:取SPF级雄性Wistar大鼠96只按照随机原则分为:A组(假手术组)、B组(模型组)、C组(γ-分泌酶抑制剂,DAPT组)、D组(电针组)共4组,每组再根据处理时间不同(7、14、21 d)进一步分为3个亚组,共12个亚组.对于B、C、D组大鼠,参照Zea longa线栓法制备大脑中动脉阻塞/再灌注模型,电针组取"百会、大椎"穴,针后接韩氏电针治疗仪,选疏密波、2/15 Hz、电流1 m A.每次20 min,每天1次.应用Zea Longa 5分制进行各时间点大鼠神经功能缺损评分;采用Hematoxylin-eosin(HE)染色检测脑受损程度;运用免疫组织化学方法观察各时间点大鼠海马齿状回(DG)Brd U含量的变化;运用蛋白质印迹法(Western Blot)检测Notch信号通路相关蛋白表达水平的变化.结果:神经功能缺损评分结果显示各组缺损评分呈下降趋势,术后7、14、21 d,D组评分比B、C组低(P<0.05).同一时间点,与A组相比,其他各组大鼠海马齿状回Brd U阳性细胞数量均增加(P<0.01).D组的阳性细胞计数明显多于B和C组(P<0.01).各时间点,与B组比较,D组Notch1蛋白表达明显升高.结论:电针能改善模型大鼠神经缺损症状,并随着疗程延长而持续存在,其机制可能通过对Notch信号通路的调控维持高水平的NSCs数量,为NSCs向成熟神经元细胞分化提供充足的细胞来源.

Objective:To investigate the dynamic effect of electroacupuncture(EA)on the content of endogenous neural stem cells(NSCs)in the hippocampus of rats with cerebral ischemia/reperfusion injury and its possible mechanism.Methods:Ninety-six male Wistar rats of SPF grade were randomly divided into sham operation group(A),model group(B),γ-secretase inhibitor DAPT group(C)and electroacupuncture group(D).Each group was subdivided into 3 subgroups according to different treatment time(7,14 and 21 days),total 12 subgroups.In group B,C and D,rat models of middle cerebral artery occlusion/reperfusion was established according to method reported by Zea Longa et al.In group D,“Bai Hui(DU 20)”and“Da Zhui(DU 14)”was selected for acupuncture and then connected to Han electroacupuncture treatment instrument(density wave,2/15 Hz,1 mA),20 min per time,once a day.Zea Longa scale was used to score the neurological deficit in rats at different time points.The degree of brain damage was observed using Hematoxylin-eosin(HE)staining.The change of BrdU level in hippocampus dentate gyrus(DG)of rats at different time points was measured using immunohistochemical method.The expression of Notch signaling related protein was detected using Western blot(WB).Results:The neurological deficit score of each group showed a descending trend.7 d,14 d and 21 d after operation,the score of group D was lower than group B and C(P<0.05).Compared with group A,the BrdU labelled cells in hippocampus dentate gyrus obviously increased in the other groups(P<0.01)at same time point.The number of positive cells in group D was obviously more than that in group B and C(P<0.01).Compared with group B,the expression of Notch 1 protein in group D considerably increased at different time points.Conclusion:EA treatment improved the symptoms of neurological deficit in rats,which persisted with the prolongation of treatment course.The mechanism may be to maintain high level of NSCs through regulation of Notch signaling pathway,thereby providing enough cell source for differentiation of NSCs into mature neuron cells.