目的 探究丁苯酞（NBP）是否通过EphB2/ephrinB2信号通路调节突触相关蛋白的表达，改善β淀粉样蛋白(Aβ_1-42)诱导的阿尔茨海默病（AD）模型大鼠认知功能障碍。方法 将SD雄性大鼠随机分为7组：对照组、假手术组、模型组、空载组、溶媒组、NBP (100 mg·kg^-1)组、NBP (100 mg·kg^-1)+shEphB2（EphB2敲低）组，每组8只。所有大鼠适应性喂养1周后，空载组、NBP+shEphB2组大鼠分别双侧海马立体定位注射空病毒和EphB2敲低病毒。等待4周病毒起效后，除对照组、假手术组以外，在大鼠双侧海马立体定位注射Aβ_1-42寡聚体(6.67μg·μL^-1)3.5μL；假手术组注射同等体积0.9%氯化钠溶液3.5μL。NBP组、NBP+shEphB2组大鼠于注射Aβ_1-42寡聚体前2周开始ig NBP，每天1次，共计28 d。各组大鼠在注射Aβ_1-42后第7天开始进行水迷宫实验；苏木精-伊红染色观察海马区神经元的病理变化；透射电镜下观察海马神经元及突触结构的变化；Western blotting检测海马组织中EphB2、突触后致密区蛋白95（PSD95）、突触素（Syn）、ephrinB2、淀粉样前体蛋白（APP）蛋白表达水平；ELISA法检测海马组织中Aβ_1-40、Aβ_1-42蛋白的水平；免疫荧光观察大鼠海马组织中EphB2蛋白的表达。结果 与模型组相比，NBP组水迷宫逃避潜伏期有所缩短（P<0.05），穿过平台的次数增加（P<0.05）;NBP+shEphB2组逃避潜伏期较NBP组延长（P<0.05），穿越平台的次数较减少（P<0.05）。在苏木精-伊红染色实验中，对照组和假手术组大鼠海马CA1/CA3区神经元排列整齐，细胞结构完整，核仁清晰，胞核与胞质界限分明；模型组、空载组、溶媒组及NBP+shEphB2组大鼠海马区神经元排列紊乱，细胞结构异常；NBP组大鼠海马组织病理损伤程度较上述各组有所减轻，但仍可见部分神经元坏死。透射电镜观察到，模型组、空病毒组、溶媒组及NBP+shEphB2组海马神经元呈现典型凋亡特征，突触结构模糊，三层结构不清晰；NBP组神经元及突触病理改变较AD模型组减轻：细胞核形态基本正常，少数突触结构模糊。与对照组相比，AD模型组大鼠海马组织中EphB2、PSD95、Syn、ephrinB2蛋白的表达量下降（P<0.05、0.001）,APP蛋白表达量升高（P<0.001），模型组与空载组、溶媒组和NBP+shEphB2组之间无明显差异；与模型组相比，NBP组大鼠海马组织中EphB2、PSD95、Syn、ephrinB2蛋白的表达量有所升高（P<0.05、0.01、0.001）,APP蛋白表达显著降低（P<0.01）。通过ELISA实验发现，与对照组相比，模型组、空载组及溶媒组Aβ_1-40和Aβ_1-42含量显著升高（P<0.001）;NBP组Aβ_1-40和Aβ_1-42含量较模型组显著降低（P<0.001），而NBP+shEphB2组Aβ_1-40和Aβ_1-42含量水平较NBP组明显升高（P<0.001）。在免疫荧光实验中，EphB2在海马组织中均有分布，主要表达在各组大鼠海马神经细胞的胞质中，并与PSD95存在共定位；模型组EphB2水平较对照组下降；NBP组EphB2水平较模型组升高；NBP+sh Eph B2组Eph B2水平较NBP组下降。结论 NBP对Aβ_1-42诱导的AD模型大鼠的学习记忆障碍具有改善作用，其作用机制可能与调控Eph B2/ephrin B2信号通路有关，通过该通路调节突触相关蛋白的表达，进而改善AD模型大鼠的认知功能障碍。

Objective To investigate whether butylphthalide regulates the expression of synaptic-related proteins through the EphB2/ephrinB2 signaling pathway and thereby improves cognitive dysfunction in a rat model of Alzheimer’s disease（AD） induced by Aβ_1-42. Methods Male SD rats were randomly divided into seven groups: control group, sham operation group, model group, empty vector group, solvent group, NBP(100 mg·kg^-1) group, and NBP(100 mg·kg^-1) + shEphB2（EphB2 knockdown） group, with 8 rats in each group. After one week of adaptive feeding, rats in the empty vector group and NBP + shEphB2 group were bilaterally injected with empty virus and EphB2 knockdown virus into the hippocampus, respectively. After waiting for four weeks for the virus to take effect, except for the control group and sham operation group, Aβ_1-42 oligomers(6.67 μg·μL^-1) 3.5 μL were bilaterally injected into the hippocampus of rats in the model group, empty vector group, solvent group, and NBP + shEphB2 group; the sham operation group was injected with the same volume of 0.9% sodium chloride solution 3.5 μL. Rats in the NBP group and NBP + shEphB2 group began to receive ig administration of NBP for two weeks before the injection of Aβ_1-42 oligomers, once a day for a total of 28 days. Water maze experiments were conducted on the 7 th day after the injection of Aβ_1-42 in all groups; hematoxylin-eosin staining was used to observe the pathological changes of neurons in the hippocampus; transmission electron microscopy was used to observe the changes in the structure of hippocampal neurons and synapses; Western blotting was used to detect the expression levels of EphB2, PSD95, Syn, ephrinB2, and APP proteins in the hippocampus; ELISA was used to detect the levels of Aβ_1-40 and Aβ_1-42 proteins in the hippocampus; immunofluorescence was used to observe the expression of EphB2 protein in the hippocampus of rats. Results Compared with the model group, the escape latency in the water maze was shortened in the NBP group（P ＜ 0.05）, and the number of times crossing the platform increased（P ＜ 0.05）; the escape latency in the NBP + shEphB2 group was longer than that in the NBP group（P ＜ 0.05）, and the number of times crossing the platform was reduced（P ＜ 0.05）. In the hematoxylin-eosin staining experiment, the neurons in the CA1/CA3 regions of the hippocampus in the control group and sham operation group were neatly arranged, with complete cell structures, clear nucleoli, and distinct boundaries between the nucleus and cytoplasm; in the model group, empty vector group, solvent group, and NBP + shEphB2 group, the neurons in the hippocampus were disordered and the cell structures were abnormal; the pathological damage of the hippocampus in the NBP group was less severe than that in the above groups, but some neurons were still necrotic. Transmission electron microscopy showed that the neurons in the hippocampus of the model group, empty vector group, solvent group, and NBP + shEphB2 group presented typical apoptotic features, with blurred synaptic structures and unclear three-layer structures; the pathological changes of neurons and synapses in the NBP group were less severe than those in the AD model group: the nuclear morphology was basically normal, and a few synaptic structures were blurred. Compared with the control group, the expression levels of EphB2, PSD95, Syn, and ephrinB2 proteins in the hippocampus of the AD model group decreased（P ＜ 0.05, 0.001）, and the expression level of APP protein increased（P ＜ 0.001）; there was no significant difference between the model group and the empty vector group, solvent group, and NBP + shEphB2 group. Compared with the model group, the expression levels of EphB2, PSD95, Syn, and ephrinB2 proteins in the hippocampus of the NBP group increased（P ＜ 0.05, 0.01, 0.001）, and the expression of APP protein significantly decreased（P ＜ 0.01）. Through the ELISA experiment, it was found that the contents of Aβ_1-40 and Aβ_1-42 in the model group, empty vector group, and solvent group were significantly higher than those in the control group（P ＜ 0.001）; The content of Aβ_1-40 and Aβ_1-42 in the NBP group was significantly lower than that in the model group（P ＜ 0.001）, while the content of Aβ_1-40 and Aβ_1-42 in the NBP + shEphB2 group was significantly higher than that in the NBP group（P ＜ 0.001）. In the immunofluorescence experiment, EphB2 was distributed in the hippocampal tissue of all groups of rats, mainly expressed in the cytoplasm of hippocampal neurons, and co-localized with PSD95. The level of EphB2 in the model group was lower than that in the control group; the level of EphB2 in the NBP group was higher than that in the model group; the level of EphB2 in the NBP + shEphB2 group was lower than that in the NBP group. Conclusion NBP has an improvement effect on learning and memory impairment in AD model rats induced by Aβ_1-42, and its mechanism may be related to the regulation of the EphB2/ephrinB2 signaling pathway, which regulates the expression of synaptic-related proteins and thereby improves cognitive dysfunction in AD model rats.

R285.5

贵州省科技厅科技支撑计划项目(黔科合支撑[2020]4Y144号); 贵州省卫生和计划生育委员会科学技术资助项目(gzwjkj2018-1-001); 遵义医科大学“12345”未来人才培养计划“未来临床名医”项目(20211004)