Objective: To observe the effect of acupuncture on the expression of miR-34c-5p, autophagy-related proteins and apoptosis rate in hippocampus of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to explore its mechanism in regulating autophagy in hippocampal neurons in CI/RI rats.
Methods: SD rats were randomly divided into sham operation, model, medication and acupuncture groups, with 20 rats in each group. The rat model of CI/RI was established by occlusion of the middle cerebral artery. In the acupuncture group, "Dazhui" (GV14), "Baihui" (GV20) and "Shuigou" (GV26) were punctured with filiform needles and stimulated manually once every 15 min, for 30 min. The rats of the medication group were intraperito-neally injected with edaravone (5 mg/kg). The treatment was conducted once every 12 h for a total of 7 times. The neurological de-ficit score of all the rats were evaluated according to Garcia's methods, and TTC staining was employed to assess the cerebral ischemic area (percentage of cerebral infarct area, CIA). Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of hippocampal neurons. The expression of hippocampal miR-34c-5p was measured by real-time PCR, and the protein expressions of hippocampal LC3B, Beclin1 and p62 were measured by Western blot. The apoptosis rate of ischemic brain tissue was observed by TUNEL staining.
Results: Compared with the sham operation group, the neurological deficit score, the expressions of miR-34c-5p, Beclin1, p62 and LC3B-Ⅱ/LC3B-Ⅰ were significantly decreased (P<0.01, P<0.05), and the CIA and the apoptosis rate significantly increased (P<0.01) in the model group. Compared with the model group, the neurological deficit scores, the expressions of miR-34c-5p, Beclin1, p62 and LC3B-Ⅱ/LC3B-Ⅰ were significantly increased (P<0.01, P<0.05), and the CIA and the apoptosis rate significantly decreased (P<0.01) in the medication and acupuncture groups. Compared with the medication group, the expression of miR-34c-5p was significantly increased (P<0.01). The results of electron microscope showed that the neurons in the acupuncture and medication groups were less damaged than those in the model group, the cells showed mild edema, and the structures were relatively complete. Some normal organelles could be seen, and autophagy bodies, autophagy lysosomes and their encapsulated organelles could still be observed.
Conclusion: Acupuncture can improve the neurological deficit and reduce the area of cerebral infarction in CI/RI rats, which is closely with its effect in promoting hippocampal neuronal autophagy and anti-apoptosis via up-regulating the expression of miR-34c-5p.
目的：观察针刺对脑缺血再灌注损伤(CI/RI)大鼠缺血侧海马组织miR-34c-5p、自噬相关蛋白表达及凋亡率的影响, 探讨针刺通过miR-34c-5p调控CI/RI大鼠海马神经细胞自噬的机制。方法：SD大鼠随机分为假手术组、模型组、药物组及针刺组, 每组20只。采用线栓法制作大脑中动脉阻塞再灌注大鼠模型。假手术组只剥离血管, 不插入线栓。针刺组针刺“大椎”“百会”“水沟”, 每15 min行针1次, 留针30 min；药物组腹腔注射依达拉奉(5 mg/kg)；均1次/12 h, 共7次。用改良Garcia评分法评估神经功能损伤程度, TTC法测定脑梗死面积百分比, 透射电镜观察缺血侧海马区神经细胞超微结构, 实时荧光定量PCR法检测海马miR-34c-5p的表达量, Western blot法检测海马Beclin-1、LC3B、p62的表达, TUNEL法检测缺血侧脑组织细胞凋亡率。结果：与假手术组比较, 模型组Garcia评分明显降低(P<0.01), 脑梗死面积百分比、细胞凋亡率明显升高(P<0.01), 海马miR-34c-5p、Beclin-1、p62表达及LC3B-Ⅱ/LC3B-Ⅰ均降低(P<0.05, P<0.01)。与模型组比较, 针刺组、药物组Garcia评分明显升高(P<0.01), 脑梗死面积百分比、细胞凋亡率明显降低(P<0.01), 海马miR-34c-5p、Beclin-1、p62表达及LC3B-Ⅱ/LC3B-Ⅰ均升高(P<0.01, P<0.05)。与药物组比较, 针刺组海马miR-34c-5p表达水平明显升高(P<0.01)。电镜结果显示, 假手术组大鼠海马神经细胞结构正常；模型组大鼠神经细胞有明显病理变化；针刺组、药物组较模型组神经细胞损伤减轻, 细胞呈轻度水肿, 结构较完整, 可见部分正常细胞器, 仍可观察到自噬小体、自噬溶酶体及其包裹的细胞器。结论：针刺可改善CI/RI大鼠神经功能, 减少脑梗死面积, 可能与其上调miR-34c-5p进而调控细胞自噬抗凋亡有关。.
Keywords: Acupuncture; Autophagy; Cerebral ischemia/reperfusion injury; miR-34c-5p.