Objective: To observe the effects of acupuncture on the expression of type Ⅲ phosphatidylinositol 3-hydroxykinase (PI3K) and Beclin-1 in hippocampus of rats with cerebral ischemia/reperfusion injury (CI/RI), so as to explore the mechanism of acupuncture in regulating type Ⅲ PI3K pathway to activate autophagy in the hippocampal neurons of CI/RI rats.
Methods: SD rats were randomly divided into sham operation group (n=11) and operation group. Then after successful modeling, rats in the operation group were randomly divided into model, acupuncture, model+3-MA and acupuncture+3-MA groups, with 11 rats in each group. The model of CI/RI was established by occlusion of the middle cerebral artery. Rats in the model+3-MA and acupuncture+3-MA groups were injected with 3-MA (400 nmol/ 5 μL) 5 μL into the lateral ventricle 30 min before reperfusion. Rats in the acupuncture and acupuncture+3-MA groups were punctured with filiform needles at "Dazhui" (GV14), "Shuigou" (GV26) and "Baihui" (GV20) and stimulated manually once every 15 min. The acupuncture intervention was conducted for 30 min each time, once every 12 h for a total of 7 times. The degree of neurological impairment was evaluated 2 h after reperfusion and after intervention by Garcia score. After intervention, the percentage of cerebral ischemic area was observed by TTC staining, the protein expression levels of type Ⅲ PI3K, Beclin-1, microtubule-associated protein 1 light chain 3B (LC3B), lysosome associated membrane protein 2 (Lamp2) and P62 in ischemic hippocampal tissue were detected by Western blot, the ultrastructure of neurons in ischemic hippocampus was observed by transmission electron microscopy (TEM).
Results: Compared with the sham operation group, the Garcia score was decreased (P<0.01), the percentage of cerebral ischemic area was increased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were decreased (P<0.01), and the expression level of P62 protein was increased (P<0.01) in ischemic hippocampal tissue in the model group. Compared with the model group, the Garcia score was increased (P<0.01), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ, Lamp2 proteins were increased (P<0.01, P<0.05) and the expression level of P62 was decreased (P<0.01) in ischemic hippocampal tissue in the acupuncture group； the percentage of cerebral ischemic area was increased (P<0.05), the expressions of type Ⅲ PI3K and Beclin-1 were decreased (P<0.01) and the expression level of P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the mo-del+3-MA group. Compared with the model +3-MA group, the Garcia score was increased (P<0.05), the percentage of cerebral ischemic area was decreased (P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, LC3B-Ⅱ/Ⅰ in ischemic hippo-campal tissue were increased (P<0.01, P<0.05) in the acupuncture+3-MA group. Compared with the acupuncture group, the Garcia score was decreased, the percentage of cerebral ischemic area was increased (P<0.05, P<0.01), the expression levels of type Ⅲ PI3K, Beclin-1, Lamp2 proteins were decreased (P<0.01, P<0.05) and P62 protein was increased (P<0.05) in ischemic hippocampal tissue in the acupuncture+3-MA group. The results of TEM showed that the edema of neurons was heavier, and few hypolysosomes existed in the model group; there was no obvious damage to neuronal structure, intracellular matrix was abundant, and a few lysosomes existed in the acupuncture group; the neuronal cells had mild edema and primary lysosomes were present in the acupuncture +3-MA group.
Conclusion: Acupuncture can improve the symptoms of neurological impairment and reduce the percentage of cerebral ischemic area in rats with CI/RI. The mechanism may be related to regulating type Ⅲ PI3K/Beclin-1 pathway, up-regulating the expressions of autophagy related factors LC3B-Ⅱ and Lamp2, and down-regulating the expression of P62.
目的：观察针刺对脑缺血再灌注损伤大鼠缺血侧海马组织Ⅲ型磷脂酰肌醇-3-羟激酶(PI3K)及Beclin-1等自噬相关指标的影响，探讨针刺调控Ⅲ型PI3K/Beclin-1通路适度激活脑缺血再灌注损伤大鼠缺血侧海马神经细胞自噬的作用机制。方法：SD大鼠按照随机数字表法分为假手术组、模型组、针刺组、模型+3-甲基腺嘌呤(3-MA)组、针刺+3-MA组，每组11只。使用改良线栓法制备大鼠大脑中动脉闭塞再灌注模型。模型+3-MA组、针刺+3-MA组于再灌注前30 min予侧脑室注射3-MA 5 μL(400 nmol/5 μL)。针刺组、针刺+3-MA组针刺“大椎”“水沟”“百会”，每15 min捻转1次，留针30 min，每12 h治疗1次，共7次。于再灌注后2 h、干预后进行Garcia神经功能评分，干预后通过TTC染色法观察大鼠脑组织缺血面积百分比，Western blot法检测缺血侧海马组织Ⅲ型PI3K、Beclin-1、自噬标志物微管相关蛋白1轻链3B（LC3B）、溶酶体相关膜蛋白 2(Lamp2)、P62的蛋白表达水平，透射电镜法观察缺血侧海马组织神经细胞超微结构。结果：再灌注后与假手术组比较，其余各组大鼠神经功能评分降低(P<0.01)。与假手术组比较，模型组大鼠神经功能评分显著降低、脑梗死面积百分比升高(P<0.01)，缺血侧海马组织Ⅲ型PI3K、Beclin-1、LC3B-Ⅱ/Ⅰ、Lamp2表达显著降低(P<0.01)，P62表达显著升高(P<0.01)，神经细胞水肿较重。与模型组比较，干预后针刺组神经功能评分升高、脑梗死面积百分比降低(P<0.01)，缺血侧海马组织Ⅲ型PI3K、Beclin-1、LC3B-Ⅱ/Ⅰ、Lamp2表达升高（P<0.01, P<0.05），P62表达降低(P<0.01)，神经元结构未见明显损伤，溶酶体增多；模型+3-MA组脑梗死面积百分比升高(P<0.05)，Ⅲ型PI3K、Beclin-1表达降低(P<0.01)，P62表达水平升高(P<0.05)，神经细胞中重度水肿，初级溶酶体存在。与模型+3-MA组比较，针刺+3-MA组神经功能评分显著升高、脑梗死面积百分比显著降低(P<0.05，P<0.01)，缺血侧海马组织Ⅲ型PI3K、Beclin-1、LC3B-Ⅱ/Ⅰ表达升高(P<0.01，P<0.05)，神经细胞轻度水肿，初级溶酶体存在。与针刺组比较，针刺+3-MA组神经功能评分显著降低、脑梗死面积百分比升高（P<0.05，P<0.01），缺血侧海马组织Ⅲ型PI3K、Beclin-1、Lamp2表达水平降低（P<0.01，P<0.05），P62表达水平升高（P<0.05）。结论：针刺可改善脑缺血再灌注损伤大鼠神经功能缺损症状，减少脑梗死面积，其机制可能与调控Ⅲ型PI3K/Beclin-1通路，上调海马区自噬相关因子 LC3B-Ⅱ/Ⅰ、Lamp2表达，下调P62表达有关。.
Keywords: Acupuncture; Autophagy; Cerebral ischemia/reperfusion injury; Type Ⅲ phosphatidylinositol 3-hydrorykinase/Beclin-1 signaling.