Objective: To explore the mechanism of electroacupuncture (EA) at "Fengchi" (GB 20) and "Sishencong" (EX-HN 1) on learning and memory impairment in vascular dementia (VD) rats by observing the influences on the N-methyl-D-aspartate receptor (NMDAR)/cyclic adenosine monophosphate response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signaling pathway and the excitotoxicity induced by hippocampal calcium overload.
Methods: Thirty-two male SD rats of SPF grade were selected and randomized into a normal group (6 rats), a sham-operation group (6 rats) and an operation group (20 rats). VD model was established with the modified Pulsinelli's four-vessel occlusion (4-VO) method. Twelve rats after successfully modeled were assigned randomly into a model group and an EA group, 6 rats in each one. In the EA group, EA was delivered at bilateral "Fengchi" (GB 20) and "Sishencong" (EX-HN 1), with the continuous wave, the frequency of 2 Hz and the electric current of 1 mA. Stimulation intensity was adjusted depending on the slightly trembling of rat head. EA was given once daily, 30 min each time; and EA intervention was delivered for 21 days continuously. Using Morris water maze test, the learning and memory function was assessed. The neuronal morphology in the hippocampal CA1 was observed with HE staining; the level of glutamate (GLU) in serum and hippocampal tissue, as well as the activity of calcium pump (Ca2+-ATP) in the hippocampus were detected using colorimetric method. The protein expression of NMDAR, calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), phosphorylated calmodulin-dependent protein kinase Ⅱ (p-CaMKⅡ), phosphorylated cyclic phosphoradenosine effector element binding proteins (p-CREB), CREB, and BDNF in the hippocampal CA1 was detected using immunohistochemistry. The protein expression of NMDAR, CREB, p-CREB and BDNF in the hippocampal tissue was detected using Western blot method.
Results: Compared to the sham-operation group, in the model group, the escape latency was prolonged and the platform crossing times of rats were reduced (P<0.01), the hippocampal neuron structure was damaged to different degrees, the structure in hippocampal CA1 was loosened, the arrangement disorganized, with clear grid-like structure; the neuronal morphology was irregular, pyknosis and even dissolution occurred, glial cells increased, blood capillary was dilated and the inflammatory cells were infiltrated and scattered. The level of GLU in the serum and hippocampal tissue and the protein expression of hippocampal NMDAR were elevated (P<0.01), the activity of Ca2+-ATP and the protein expression of CaMKⅡ, p-CaMKⅡ, CREB, p-CREB and BDNF were reduced (P<0.01, P<0.05); and the ratio of p-CaMKⅡ/CaMKⅡ and that of p-CREB/CREB were dropped (P<0.05). In comparison with the model group, in the EA group, the escape latency was shortened and the platform crossing times of rats rose (P<0.01), the arrangement was improved in the hippocampal CA1, the neuronal morphology was intact, the nucleoli were clear relatively and the pyknosis or dissolution were attenuated, the numbers of glial cells reduced relatively, the dilation of blood capillary was alleviated. The level of GLU in the serum and hippocampal tissue and the protein expression of NMDAR were reduced in the hippocampal tissue (P<0.01), the activity of Ca2+-ATP and the protein expression of CaMKⅡ, p-CaMKⅡ, CREB, p-CREB and BDNF were elevated (P<0.05, P<0.01); and the ratio of p-CaMKⅡ/CaMKⅡ and that of p-CREB/CREB increased (P<0.05).
Conclusion: EA at "Fengchi" (GB 20) and "Sishencong" (EX-HN 1) can attenuate learning and memory impairment in VD rats, which may be obtained by reducing GLU level in hippocampal tissue, inhibiting hippocampal excitotoxicity, mediating protein expression related to the NMDAR/CREB/BDNF signaling pathway, and maintaining neuronal survival and growth.
目的：观察电针“风池”“四神聪”对血管性痴呆（VD）大鼠海马N-甲基-D-天冬氨酸受体（NMDAR）/环磷腺苷效应元件结合蛋白（CREB）/脑源性神经营养因子（BDNF）信号通路及海马区钙超载所致兴奋性毒性的影响，探究电针“风池”“四神聪”改善大鼠学习记忆障碍的作用机制。方法：选用SPF级雄性SD大鼠32只，随机分为正常组（6只）、假手术组（6只）和手术组（20只）。手术组采用改良的Pulsinelli四血管阻断（4-VO）法制备VD模型，将造模成功的12只大鼠随机分为模型组和电针组，每组6只。电针组于双侧“风池”及左右“四神聪”进行电针干预，予连续波，频率2 Hz，电流1 mA，以大鼠头部微颤为宜。电针干预每日1次，每次30 min，持续21 d。Morris水迷宫实验检测大鼠学习记忆能力；HE染色观察大鼠海马CA1区神经元形态；比色法检测大鼠血清、海马组织谷氨酸（GLU）含量及海马组织Ca2+-三磷酸腺苷（ATP）酶活力；免疫组化法检测大鼠海马CA1区NMDAR、钙调蛋白依赖性蛋白激酶Ⅱ（CaMKⅡ）、磷酸化钙调蛋白依赖性蛋白激酶Ⅱ（p-CaMKⅡ）、磷酸化环磷腺苷效应元件结合蛋白（p-CREB）、CREB、BDNF蛋白表达；Western blot法检测大鼠海马组织NMDAR、CREB、p-CREB、BDNF蛋白表达。结果：与假手术组比较，模型组大鼠逃避潜伏期延长、穿越平台次数减少（P<0.01）；海马神经元结构不同程度受损，海马CA1区组织结构疏松，排列结构散乱，网格状结构明显，神经元形态不规则，核仁固缩甚至溶解，胶质细胞增多，毛细血管扩张，散见炎性细胞浸润；血清及海马组织GLU含量、海马组织NMDAR蛋白表达升高（P<0.01），海马组织Ca2+-ATP酶活力及CaMKⅡ、p-CaMKⅡ、CREB、p-CREB、BDNF蛋白表达水平降低（P<0.01，P<0.05），p-CaMKⅡ/CaMKⅡ、p-CREB/CREB值降低（P<0.05）。与模型组比较，电针组大鼠逃避潜伏期缩短、穿越平台次数增加（P<0.01）；大鼠海马CA1区组织排列散乱程度稍有好转，神经元形态相对完整，核仁形态相对明显，固缩或溶解现象减轻，胶质细胞数量相对减少，毛细血管扩张程度减轻；血清及海马组织GLU含量、海马组织NMDAR蛋白降低（P<0.01），海马组织Ca2+-ATP酶活力及CaMKⅡ、p-CaMKⅡ、CREB、p-CREB、BDNF蛋白表达升高（P<0.05，P<0.01），p-CaMKⅡ/CaMKⅡ、p-CREB/CREB值升高（P<0.05）。结论：电针“风池”“四神聪”可以改善VD大鼠学习记忆功能障碍，其机制可能通过降低海马组织GLU含量，抑制海马神经元兴奋性毒性，介导NMDAR/CREB/BDNF信号通路相关蛋白表达，维持神经细胞存活和生长有关。.
Keywords: N-methyl-D-aspartate receptor (NMDAR)/cyclic adenosine monophosphate response element-binding protein (CREB)/brain-derived neurotrophic factor (BDNF) signaling pathway; electroacupuncture; glutamic acid; learning and memory impairment; vascular dementia.