电针调节糖原合成酶激酶3β/β-catenin信号通路对脊髓损伤大鼠室管膜区细胞CD133蛋白表达的影响

Effect of electroacupuncture modulation of glycogen synthase kinase 3 beta/beta-catenin signaling pathway on CD133 protein expression in rat ventricular zone cells after spinal cord injury

背景:前期研究表明,电针可改善脊髓损伤后功能障碍,并促进脊髓损伤后大鼠内源性神经干细胞增殖,然而其具体作用机制不明确。目的:观察电针对脊髓损伤大鼠糖原合成酶激酶3β/β-catenin信号通路相关因子表达和室管膜区细胞CD133蛋白表达的影响,探讨电针促进内源性干细胞增殖的作用机制。方法:将45只SD雌性大鼠随机分为假手术组、模型组、电针组,每组15只,后两组以精密打击器构建脊髓损伤大鼠模型。在造模后24 h,电针组接受电针治疗,选取督脉“百会”“脊中”穴及损伤脊髓上下节段夹脊穴,予疏密波,频率1 Hz/20 Hz,强度1.0-1.2 mA,30 min/次,1次/d,持续14 d。应用BBB运动功能评分评估大鼠运动功能,Nissl染色观察脊髓组织病理形态学改变及神经元数量,免疫荧光检测脊髓组织中室管膜区细胞CD133、脊髓灰质和白质中Nestin荧光强度,实时荧光定量PCR检测脊髓组织中Nestin、糖原合成酶激酶3β、β-catenin mRNA表达,Western blot检测脊髓组织中Nestin、糖原合成酶激酶3β、磷酸化糖原合成酶激酶3β、β-catenin、p-β-catenin蛋白表达。结果与结论:(1)与模型组比较,电针组BBB运动功能评分术后1,3,5 d时无显著差异(P>0.05),术后7,14 d时显著提高(P<0.05、P<0.01);(2)病理形态上,电针组脊髓组织部分神经细胞形态较完整,尼氏小体溶解较轻;与模型组比较,电针组脊髓组织神经元数量增加(P<0.05);(3)与模型组比较,电针组脊髓组织中室管膜区CD133荧光强度升高(P<0.001),灰质中Nestin荧光强度均明显升高(P<0.01),白质中Nestin荧光强度显著升高(P<0.001);(4)与模型组比较,电针组Nestin mRNA表达水平显著升高(P<0.001),糖原合成酶激酶3βmRNA表达水平明显降低(P<0.01),β-catenin mRNA表达水平显著降低(P<0.001);(5)与模型组比较,电针组Nestin、磷酸化糖原合成酶激酶3β蛋白表达水平显著升高(P<0.001),p-β-catenin蛋白表达水平显著降低(P<0.001);(6)结果表明,电针能够促进脊髓损伤后大鼠室管膜区CD133蛋白表达,其机制可能与调节糖原合成酶激酶3β/β-catenin信号通路有关。

BACKGROUND:Preliminary studies have shown that electroacupuncture can improve functional impairment a?ter spinal cord injury and promote the proliferation of endogenous neural stem cells in rats a?ter spinal cord injury.However,its specifc mechanism of action is unclear.OBJECTIVE:To investigate the effect of electroacupuncture on the expression of glycogen synthase kinase 3β(GSK-3β)/β-catenin signaling pathway-related factors and CD133 protein in ventricular canal area cells in rats with spinal cord injury,and to explore the mechanism by which electroacupuncture promotes endogenous stem cell proliferation.METHODS:A total of 45 female Sprague-Dawley rats were randomly divided into sham-operated group,model group,and electroacupuncture group,with 15 rats in each group.Animal models of spinal cord injury were constructed in the latier two groups using a precision percussion device.The electroacupuncture group received electroacupuncture treatment at Baihui and Jizhong acupoints of the Governor’s Vessel and at Jiaji acupoints of the upper and lower segments of the injured spinal cord,with sparse and dense waves at a frequency of 1 Hz/20 Hz and an intensity of 1.0-1.2 mA,once for 30 minuntes,once a day,for 14 continuous days.The motor function of the rats was assessed using the Basso,Beatie and Bresnahan motor function score and Nissl staining was used to observe the histomorphological changes and the number of neurons in the spinal cord.Immunofuorescence was used to detect the fuorescence intensity of CD133 in ependymal cells in spinal cord tissue and Nestin in spinal cord gray matier and white matier.Real-time fuorescence quantitative PCR was used to detect Nestin,GSK-3β,andβ-catenin mRNA in spinal cord tissue.Western blot assay was used to detect Nestin,GSK-3β,p-GSK-3β,β-catenin,and p-β-catenin protein expression in spinal cord tissue.RESULTS AND CONCLUSION:(1)Compared with the model group,the Basso,Beatie and Bresnahan scores showed no signifcant changes in the electroacupuncture group at 1,3,and 5 days a?ter surgery(P>0.05)but were signifcantly increased at 7 and 14 days a?ter surgery(P<0.05,P<0.01).(2)The morphology of some nerve cells in the spinal cord tissue was relatively intact in the electroacupuncture group and Nissl bodies were slightly dissolved.Compared with the model group,the number of neurons in the spinal cord tissue was signifcantly increased in the electroacupuncture group(P<0.05).(3)Compared with the model group,in the electroacupuncture group,the fuorescence intensity of CD133 in the ventricular zone of spinal cord tissue was increased(P<0.001),and the fuorescence intensity of Nestin was signifcantly increased in the gray matier(P<0.01)and white matier(P<0.001).(4)Compared with the model group,in the electroacupuncture group,the expression of Nestin mRNA was signifcantly higher(P<0.001),GSK-3βmRNA expression was signifcantly lower(P<0.01),andβ-catenin mRNA expression was also signifcantly lower(P<0.001).(5)Compared with the model group,Nestin and p-GSK-3βprotein expression was signifcantly higher(P<0.001)and p-β-catenin protein expression was signifcantly lower(P<0.001)in the electroacupuncture group.(6)All these fndings indicate that electroacupuncture can promote CD133 protein expression in the ventricular canal area of rats a?ter spinal cord injury,and the mechanism may be related to the regulation of GSK-3β/β-catenin signaling pathway.