“通督调神”电针预处理大鼠缺血半暗带区miR-124和皮质区蛋白的表达

Expression of microRNA-124 and cortical proteins in ischemic penumbra of rats pretreated with“Tongdu Tiaoshen”electroacupuncture

背景:“通督调神”电针预处理法是结合传统针刺和现代电刺激的新兴物理治疗方法,既往文献报道其对缺血性脑卒中具有显著治疗作用,但其具体机制尚不明确。目的:探讨“通督调神”电针预处理作用于细胞凋亡,对大鼠缺血半暗带区miR-124和皮质区Notch-1、p-JNK和半胱氨酸天冬氨酸特异性蛋白酶3蛋白相对表达量的影响及其可能的作用机制。方法:将75只雄性SD大鼠随机分为模型组、假手术组、电针预处理组、miR-124抑制剂预处理组和电针+miR-124激动剂预处理组,每组15只。7 d干预期内,电针预处理组取“风府”“百会”“大椎”穴行电针,1次/d,20 min/次;miR-124抑制剂预处理组第7天注射miR-124 antagomir至大鼠侧脑室内;电针+miR-124激动剂预处理组第7天在电针基础上注射miR-124 agomir;模型组及假手术组不予治疗。干预结束后造模,假手术组只做鼠板上固定和钝性分离血管,其余组均采用大脑中动脉闭塞方法并参照Zea Longa线栓法建立大鼠右侧脑缺血再灌注损伤模型。造模完成后24 h应用改良神经损伤程度评分定损,评分结束后使用TUNEL染色法测细胞凋亡,电镜观察大鼠皮质脑区超微结构,Western Blot法检测皮质区蛋白相对表达量,实时荧光定量PCR检测mRNA相对表达量。结果与结论:①与假手术组比较,其余各组大鼠改良神经损伤程度评分均升高(均P<0.01);与模型组比较,miR-124抑制剂预处理组和电针预处理组改良神经损伤程度评分显著降低(均P<0.05);②与假手术组比较,模型组大鼠凋亡细胞数量显著升高(P<0.01);与模型组比较,各预处理组大鼠凋亡细胞数量显著降低(均P<0.01);③模型组细胞结构及内容物缺损较重;电针预处理组细胞结构及内容物轻度缺损;miR-124抑制剂预处理组细胞较完整;电针+miR-124激动剂预处理组细胞结构及内容物存在损伤;④与模型组比较,miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区半胱氨酸天冬氨酸特异性蛋白酶3、Notch-1和p-JNK蛋白相对表达量显著降低(均P<0.01);⑤与模型组比较,各预处理组大鼠皮质脑区miR-124-3p和Notch-1 mRNA相对表达量降低(均P<0.01),miR-124抑制剂预处理组和电针预处理组大鼠皮质脑区半胱氨酸天冬氨酸特异性蛋白酶3 mRNA和p-JNK mRNA相对表达量降低(均P<0.01),电针+miR-124激动剂预处理组大鼠皮质脑区p-JNK mRNA相对表达量也降低(P<0.05);⑥结果表明,“通督调神”电针预处理可通过干预脑缺血再灌注损伤大鼠脑组织内miR-124表达水平,抑制脑缺血再灌注损伤后神经元细胞凋亡,发挥减轻脑缺血再灌注损伤神经功能缺损的作用,其作用机制可能与miR-124对Notch通路的正向调节机制有关。

BACKG ROUND:"Tongdu Tiooshen" electroacupuncture pretreatment is an eme rging physiotherapy combining traditional acupuncture with modern electrical stimulation,which has been reported to have significant therapeutic effects on ischemic stroke,but its specific mechanism is still unclear.OBJECTIVE:To investigate the effects of Tongdu Tiaoshen" electroacupuncture pretreatment on cell apoptosis,the relative expression of microRNA-124(miR-124) in the ischemic penumbra and Notch-1,p-JNK and cysteinyl-aspartate specific protease-3(caspase-3) proteins in the co rtex of rats and their possible mechanisms of action.METHODS:Seventy-five male Sprague-Dawley rats were randomly divided into five groups(n=15 per group):model group,sham-operated group,electroacupuncture pretreatment group,miR-124 antagomir pretreatment group and electroacupuncture+miR-124 agomir pretreatment group.In the electroacupuncture pretreatment group,20-minute electroacupuncture at Fengchi,Baihui,and Dazhui was performed once a day,for 7 continuous days.In the miR-124 antagomir pretreatment group,miR-124 antagomir was injected into the lateral ventricle of rats on day 7.In the electroacupuncture+miR-124 agomir pretreatment group,miR-124 agomir was injected based on electroacupuncture pretreatment.Model and sham-operated groups were not treated.After the intervention,animal models were created.In the sham-operated group,the rats were only fixed on the operating board and the vessels were blunt-se pa rated.In the other groups,the right cerebral ischemia-repe rfusion injury model was established by the middle cerebral artery occlusion method and the Zea Longa suture method.Twenty-four hours after the modeling,the modified neurological severity scoring was performed to determine the damage.TUN EL staining method was then used to measure cell apoptosis in the rat cerebral cortex.Ultrastructure of the rat cerebral co rtex was observed under electron microscopy.RESULTS AND CONCLUSION:Compared with the sham-operated group,the modified neurological severity sco res we re significantly elevated in the other groups(all P <0.01).Compared with the model group,the miR-124 antagomir pretreatment group and electroacupuncture pretreatment group had significantly reduced modified neurological seve rity scores(both P <0.05).Compared with the sham-operated group,the number of apoptotic cells was elevated in the model group(P <0.01).Compared with the model group,the number of apo ptotic cells was reduced in the pretreatment groups(all P <0.01).The model group had more severe defects in cell structure and contents.The electroacupuncture pretreatment group had mild defects in cell structure and contents.Cells in the miR-124 antagomir pretreatment group were more intact.There was damage to cell structure and contents in the electroacupuncture+miR-124agomir pretreatment group.The relative expressions of caspase-3,Notch-1 and p-JNK proteins in the cortex of rats were significantly lower in the miR-124antagomir pretreatment and electroacupuncture pretreatment group compared with the model group(all P <0.01).The relative expression of miR-124-3p and Notch-1 mRNA in the cortex of the pretreated rats was significantly reduced compared with that of the model group(all P <0.01).The relative expression of caspase-3 mRNA and p-JNK mRNA in the cortex of rats was significantly reduced in the miR-124 antagomir pretreatment and electroacupuncture pretreatment groups compared with the model group(all P <0.01).The relative expression of p-JNK mRNA in the cortex of rats in the electroacupuncture+miR-124 agomir pretreatment group was also reduced compared with the model group(P <0.05).To conclude,"Tongdu Tiaoshen" electroacupuncture pretreatment can inhibit the apoptosis of neuronal cells after cerebral ischemia-repe rfusion injury by interfering with the expression level of miR-124 in the brain tissue of rats,there by reducing neurological deficits after cerebral ischemia-reperfusion injury.The mechanism of this action may be related to the positive regulation mechanism of miR-124 on the Notch pathway.