海南医科大学学报

目的：探究血塞通软胶囊（Xuesaitong soft capsule,XST）对东莨菪碱所致阿尔茨海默病模型小鼠学习记忆能力的影响及突触可塑性的调节作用。方法：90只雄性昆明小鼠随机分为空白（Control）组，模型（Model）组，盐酸多奈哌齐（DPH）组及血塞通低、中、高剂量（XST-L、XST-M、XST-H）组。通过腹腔注射东莨菪碱制备阿尔茨海默病模型，使用Morris水迷宫检测小鼠空间认知记忆功能改变；H&E染色观测小鼠海马CA1区和皮质区细胞形态改变；透射电镜观察海马超微结构变化；ELISA测定脑组织中环磷酸腺苷（cAMP）含量变化，免疫组化染色检测突触素（SYN）、生长相关蛋白-43(GAP-43)、突触后致密物-95(PSD-95)蛋白水平表达；Western blot检测PSD-95、GAP-43、SYN、环磷酸腺苷反应元件结合蛋白（CREB）、蛋白激酶A(PKA)在海马和皮质区的表达。结果：Morris水迷宫结果显示，与Control组比较，Model组小鼠平均逃避潜伏时间明显延长，穿越平台次数出现减少（P<0.05），游泳轨迹冗长杂乱。与Model组相比，经XST治疗后小鼠平均逃避潜伏期明显缩短，穿越平台次数增加（P<0.05），探寻轨迹简短清晰。H&E染色结果显示，Model组细胞层变薄，神经元排列松散紊乱，部分细胞胞膜破裂，细胞核深染，染色质裂解，突起不同程度消失。与Model组比较，各用药组上述病理变化得到不同程度改善。透射电镜观察结果显示，Model组突触结构不完整，突触间隙模糊，突触小泡以及突触前致密物质减少，药物干预后突触结构相对完整，突触间隙清晰，突触小泡密度增加，线粒体丰富，突触前致密物增多，突触后膜增厚。ELISA结果显示，与Control组比较，Model组cAMP蛋白表达明显减少（P<0.01）；与Model组比较，海马区DPH组和XST-H组cAMP含量明显增加（P<0.05），皮质区DPH组、XST-M组和XST-H组cAMP含量明显升高（P<0.05）。免疫组化结果显示，与Control组比较，Model组SYN、GAP-43、PSD-95蛋白表达均呈明显下降趋势（P<0.01）。与Model组相比，其余各组以上蛋白表达均有不同程度的增加（P<0.05）。Western Blot结果显示，与Control组比较，Model组PSD-95、GAP-43、CREB、PKA和SYN蛋白表达明显减少（P<0.05）；与Model组比较，药物干预后PSD-95、GAP-43、CREB、PKA和SYN表达水平不同程度增加（P<0.05）。结论：XST能够改善阿尔兹海默病模型小鼠学习记忆能力，其作用机制可能与激活cAMP/PKA/CREB信号通路、修复突触可塑性相关。

Objective: To investigate the effects of Xuesaitong soft capsule(XST) on the cognitive functions of scopolamine-induced Alzheimer′s disease in mice and the modulation of synaptic plasticity. Methods: A total of 90 male Kunming mice were randomly divided into the blank(Control) group, the model(Model) group, the donepezil hydrochloride(DPH) group, and the Xuesaitong low, medium, and high dosage(XST-L, XST-M, XST-H) groups. The Alzheimer′s disease model was prepared by intraperitoneal injection of scopolamine, and the spatial cognitive memory function of mice was detected by Morris water maze. Hemoxylin-eosin staining(H&E) was used to observe the morphological changes in the CA1 and cortical areas of the mouse hippocampus. Transmission electron microscopy was used to observe ultrastructural changes in the hippocampus. ELISA was used to determine changes in the content of cyclic adenosine monophosphate(cAMP) in brain tissue. Immunohistochemical staining was used to detect the expression levels of synaptophysin(SYN), growth-associated protein-43(GAP-43), and postsynaptic densifier-95(PSD-95) proteins. Western blot was performed to detect the expression of PSD-95, GAP-43, SYN, cyclic-AMP response binding protein(CREB), and protein kinase A(PKA) in the hippocampus and cortical regions. Results: The results of Morris water maze showed that compared to the Control group, the mice in the Model group had a significantly longer mean escape latency, showed a decrease in the number of traversing platforms(P<0.05), and had lengthy and disorganised swimming trajectories. Compared to the Model group, the average escape latency of mice treated with XST was significantly shorter, the number of traversing platforms increased(P<0.05), and the exploratory trajectories were brief and clear. H&E staining results showed that the cell layer of the Model group was thinned, the neurons were loosely arranged and disordered, the cell membranes of some cells were ruptured, the nuclei of the cells were deeply stained, the chromatin was cleaved, and the protrusions disappeared to varying degrees. Compared to the Model group, the above pathological changes were improved to different degrees in each medication group. The results of transmission electron microscope observation showed that the synaptic structure was incomplete, the synaptic gap was blurred, the synaptic vesicles were reduced, and the presynaptic dense material was reduced in the Model group. The synaptic structure was relatively complete, the synaptic gap was clear, the synaptic vesicles were increased, the mitochondria were enriched, the presynaptic dense material was increased, and the postsynaptic membrane was thickened after the drug intervention. ELISA results showed that cAMP protein expression was significantly reduced in the Model group compared to the Control group(P<0.01); cAMP contents were significantly increased in the hippocampal region DPH and XST-H groups compared to the Model group(P<0.05), and cAMP contents in the cortical region of the DPH, XST-M, and XST-H groups were significantly elevated(P<0.05). Immunohistochemical results showed that the protein expression of SYN, GAP-43, and PSD-95 in the Model group showed a significant decreasing trend compared to the Control group(P<0.01). Compared to the Model group, the expression of the above proteins in the remaining groups increased to different degrees(P<0.05). Western blot results showed that PSD-95, GAP-4C3, CREB, PKA, and SYN protein expression was significantly reduced in the Model group compared to the Control group(P<0.05); and the expression levels of PSD-95, GAP-4C3, CREB, PKA, and SYN were increased to varying degrees after pharmacological interventions compared to the Model group(P<0.05). Conclusion: XST improves cognitive functions in Alzheimer′s disease modeled mice, and its mechanism of action may be related to the activation of cAMP/PKA/CREB signaling pathway and repair of synaptic plasticity.