Objectives: To observe the effect of electroacupuncture on miR-142-5p and ADAMTS1/PI3K/AKT pathway in rats with ischemic stroke, so as to explore the regulatory mechanism of electroacupuncture on angiogenesis after ischemic stroke.
Methods: This study was divided into two parts. The first part of the experiment：SD rats were randomly divided into sham operation group, model group and electroacupuncture group. There were 20 rats in each group. The middle cerebral artery occlusion (MCAO) rat model was prepared using a modified Longa's method. In the electroacupuncture group, "Shuigou" (GV26) was selected for electroacupuncture intervention (4 Hz/20 Hz) for 30 min each time. The rats in the electroacupuncture group were given electroacupuncture immediately after successful modeling, once a day for 4 times. Hunter score and TTC staining were used to observe the neurological deficits and infarct volumes respectively；HE staining was used to observe the cortical pathological changes；immunohistochemistry was used to determine the changes of cerebral microvascular density. Real-time quantitative PCR and Western blot were used to observe the miR-142-5p expression, mRNA and protein expression levels of ADAMTS1, VEGF, PI3K, AKT, eNOS in ischemic cortex. The second part of the experiment：The rats were randomly divided into electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group with 8 rats in each group. MCAO model was established after injection. Electroacupuncture+control group was given 0.9% sodium chloride solution injected into the right ventricle.The rats in the electroacupuncture+miR-142-5p Antagomir group were injected with miR-142-5p inhibitor into the right ventricle 30 min before modeling. Rats in electroacupuncture+control group and electroacupuncture+miR-142-5p Antagomir group were all given the same electroacupuncture treatment. Real-time fluorescence quantitative PCR was used to observe the effect of miR-142-5p Antagomir on the expression of miR-142-5p and ADAMTS1 mRNA. The effect of miR-142-5p Antagomir on ADAMTS1 protein was observed by Western blot.
Results: In the first part of the experiment, compared with the sham operation group, the Hunter score in the model group was significantly increased (P<0.01)；the volume of cerebral infarction in the model group was significantly increased (P<0.01)；the degree of brain edema and neuronal necrosis and the density of cerebral microvessels was increased；the cerebral microvascular density was significantly increased (P<0.01)；the expression levels of miR-142-5p and the mRNA expression levels of VEGF, AKT and eNOS were significantly decreased (P<0.01, P<0.05), and the protein expression levels of VEGF, p-AKT and eNOS were significantly down-regulated (P<0.01), while the mRNA expression levels of ADAMTS1 and PI3K, and the protein expression levels of ADAMTS1 and p-PI3K were all up-regulated (P<0.01, P<0.05) in the model group. Compared with the model group, after intervention, the Hunter score in the electroacupuncture group was decreased (P<0.01), the volume of cerebral infarction was significantly decreased (P<0.01)；the degree of brain edema and neuronal necrosis were alleviated；the cerebral microvascular density was significantly increased (P<0.01)；the expression of miR-142-5p and the mRNA expression of VEGF, PI3K, AKT and eNOS were increased (P<0.01), the protein expressions of VEGF, p-PI3K, p-AKT and eNOS were increased (P<0.01, P<0.05), while the mRNA and protein expression of ADAMTS1 were decreased (P<0.05, P<0.01). After injection of miR-142-5p inhibitor, compared with electroacupuncture+control group, the expression of miR-142-5p in electroacupuncture+miR-142-5p Antagomir group was decreased(P<0.05), while the mRNA and protein expression of ADAMTS1 were increased (P<0.01, P<0.05).
Conclusions: Electroacupuncture at GV26 can improve the neurological damage of ischemic stroke rats, reduce the volume of cerebral infarction and promote angiogenesis. The mechanism may be associated with the function of electroacupuncture in promoting the expression of miR-142-5p, so as to inhibit the expression of its target gene ADAMTS1, mediate the up-regulation of VEGF expression, activate PI3K/AKT pathway, promote the release of eNOS, and participate in promoting angiogenesis in ischemic stroke rats.
目的: 观察电针对缺血性脑卒中大鼠miR-142-5p及ADAMTS1/磷脂酰肌醇-3激酶（PI3K）/丝氨酸-苏氨酸蛋白激酶（AKT）通路的影响，探讨电针对缺血性脑卒中后血管新生的调控机制。方法: 本研究分为两部分实验。第一部分实验：将SD大鼠随机分为假手术组、模型组、电针组，每组20只。采用改良的Longa法制备大脑中动脉栓塞大鼠模型。电针组于“水沟”进行电针干预（4 Hz/20 Hz），30 min/次，造模成功后即时干预1次，以后每日1次，共4次。采用亨氏评分法评价造模后大鼠神经功能缺损程度；TTC染色法检测大鼠脑梗死体积；HE染色法观察大鼠大脑皮层的病理改变；免疫组织化学法测定脑微血管密度（MVD）；实时荧光定量PCR法和Western blot法检测大鼠大脑皮层缺血半暗带的miR-142-5p及ADAMTS1/PI3K/AKT通路中ADAMTS1、血管内皮生长因子（VEGF）、PI3K、AKT、内皮型一氧化氮合酶（eNOS）mRNA和蛋白表达水平。第二部分实验：将同批次SD大鼠随机分为电针+对照组、电针+miR-142-5p Antagomir（拮抗剂）组，每组8只。电针+对照组给予右侧侧脑室注射0.9%氯化钠溶液，电针+拮抗剂组大鼠造模前30 min给予右侧侧脑室注射miR-142-5p拮抗剂，大鼠注射后进行造模，电针+对照组、电针+拮抗剂组均给予上述同样的电针治疗。实时荧光定量PCR法和Western blot法检测两组大鼠大脑皮层miR-142-5p、ADAMTS1 mRNA及蛋白表达。结果: 与假手术组比较，模型组大鼠神经缺损体征评分升高（P<0.01），脑梗死体积增加（P<0.01），脑组织水肿及神经元坏死程度增加，MVD升高（P<0.01），miR-142-5p表达水平及VEGF、AKT、eNOS mRNA表达水平均降低（P<0.01，P<0.05），VEGF、磷酸化（p）-AKT、eNOS蛋白表达水平均降低（P<0.01），ADAMTS1、PI3K mRNA及ADAMTS1、p-PI3K蛋白表达水平均升高（P<0.01，P<0.05）。与模型组比较，电针组大鼠神经缺损体征评分降低（P<0.01），脑梗死体积缩小（P<0.01），脑组织水肿及神经元坏死程度减轻，MVD升高（P<0.01），miR-142-5p及VEGF、PI3K、AKT、eNOS mRNA的表达水平升高（P<0.01），VEGF、p-PI3K、p-AKT、eNOS蛋白表达水平均升高（P<0.01，P<0.05），ADAMTS1 mRNA及蛋白表达水平降低（P<0.05，P<0.01）。注射miR-142-5p拮抗剂后，与电针+对照组比较，电针+拮抗剂组miR-142-5p表达水平降低（P<0.05），ADAMTS1 mRNA及蛋白表达水平升高（P<0.01，P<0.05）。结论: 电针“水沟”可改善缺血性脑卒中大鼠的神经功能损伤，减少脑梗死体积，促进血管新生，其机制可能是通过促进miR-142-5p表达抑制其靶基因ADAMTS1的表达，上调VEGF表达，从而进一步激活PI3K/AKT通路，促进eNOS释放，促进缺血性脑卒中大鼠脑血管新生。.
Keywords: ADAMTS1/ PI3K/AKT pathway; Electroacupuncture; Ischemic stroke; Middle cerebral artery occlusion; miR-142-5p.