督脉电针通过调控糖原合酶激酶3β及其磷酸化水平减少脊髓损伤大鼠神经细胞凋亡

Electroacupuncture reduces neuronal apoptosis in rats with spinal cord injury by regulating GSK-3β expression and its phosphorylation

目的:观察督脉电针对脊髓损伤(SCI)大鼠糖原合酶激酶3β(GSK-3β)蛋白及其磷酸化水平及神经元凋亡的影响,探讨督脉电针治疗SCI的部分作用机制。方法:SD大鼠随机分为假手术组、模型组、电针组,每组15只。采用改良式Allen’s打击装置制备SCI大鼠模型。电针组电针“大椎”“命门”,每次20 min,每天治疗1次,治疗28 d。分别于第1、7、14、21、28天进行BBB行为学评分评价大鼠后肢运动功能;HE染色法观察大鼠脊髓组织病理形态改变;免疫组织化学法、Western blot法检测大鼠脊髓组织GSK-3β及其Ser9位点磷酸化表达水平;TUNEL染色法观察脊髓组织神经元凋亡指数(AI)。结果:与假手术组比较,术后各时点模型组大鼠BBB评分下降(P<0.01);造模后7~28 d,与模型组比较,电针组BBB评分升高(P<0.05,P<0.01)。模型组大鼠SCI区域脊髓组织结构紊乱,炎性浸润严重;电针组大鼠脊髓组织结构较完整,组织空腔及炎性浸润减少。与假手术组比较,模型组大鼠脊髓组织中GSK-3β蛋白的表达量增高(P<0.01),p-GSK-3β蛋白的表达降低(P<0.01),AI上升(P<0.01);与模型组相比,电针组大鼠脊髓组织中GSK-3β蛋白的表达降低(P<0.05),p-GSK-3β蛋白的表达升高(P<0.01),AI降低(P<0.01)。结论:督脉电针可改善SCI大鼠神经功能损伤症状,减少脊髓组织神经元的凋亡,可能是通过调控GSK-3β蛋白及其磷酸化水平实现的。

Objective To observe the effect of electroacupuncture(EA)stimulation of“Dazhui”(GV14)and“Mingmen”(GV4)of the Governor Vessel(GV)on the apoptosis of neurons in rats with spinal cord injury(SCI)by regulating the expression of glycogen synthase kinase-3β(GSK-3β)protein and its phosphorylation level,so as to reveal partial mechanism for the treatment of SCI with EA of GV.Methods A total of 45 male SD rats were randomly divided into sham-operation,model and EA groups,15 rats in each group.The SCI model was established by using the modified Allen’s percussion device.EA stimulation(2 Hz,1 mA)was applied to GV14 and GV4 for 20 min,once daily for 28 days.Basso-Beattie-Bresnahan(BBB)locomotor rating scale was used to assess the rat’s hind limb motor function at the 1st,7th,14th,21th and 28th day after modeling.H.E.staining was used to observe the histopathological changes of the injured spinal cord tissue.The expressions of GSK-3βand its phosphorylation at Ser9 site in the spinal cord were detected by immunohistochemistry and Western blot,respectively.The TUNEL staining was used to observe the apoptosis of neuron in the spinal cord.Results In comparison with the control group,the BBB scores from day 1 to day 28,and the expression and immunoactivity of p-GSK-3β were significantly decreased(P<0.01),while the expression and immunoactivity of GSK-3βand the apoptosis index were significantly increased(P<0.01)in the model group.In contrast to the model group,the EA group had a remarkable increase in the BBB score from day 7 to day 28,and the expression and immunoactivity of p-GSK-3β(P<0.05,P<0.01),and an obvious decrease(P<0.05,P<0.01)in the expression and immunoactivity of GSK-3βand the apoptosis index.H.E.staining showed that in the model group,the spinal cord structure of the SCI region was disorganized,with tissue cavity,blurred gray matter boundary,severe inflammatory infiltration,reduction of normal neurons,loss of cytoplasm,and nuclear contraction,which was relatively milder in the EA group.Conclusion EA at GV14 and GV4 can improve the symptoms of neurological function injury and reduce the apoptosis of neurons in rats with SCI,which may be achieved by regulating GSK-3βprotein and its phosphorylation level.