铁筷子挥发油对慢性脑缺血致血管性认知功能障碍大鼠的作用及机制研究

目的 探究苗药铁筷子腊梅Chimonanthus praecox及山腊梅C. nitens的干燥根]挥发油对慢性脑缺血（chronic cerebral hypoperfusion，CCH）致血管性认知功能障碍（vascular cognitive impairment，VCI）大鼠的作用及机制。方法 采用改良的双侧颈总动脉结扎（bilateral common carotid artery occlusion，BCCAO）法建立CCH模型，将造模成功大鼠随机分为模型组及铁筷子挥发油高、中、低剂量（80、40、20 mg/kg）组和丁苯酞（63 mg/kg）组，另设假手术组，给予相应药物干预28 d，采用水迷宫实验检测各组大鼠学习记忆能力；采用旷场实验、Y迷宫实验检测各组大鼠自主活动性和新奇事物探索能力；采用苏木素-伊红（HE）染色、Nissl染色、TUNEL染色观察各组大鼠大脑皮层及海马CA1区神经细胞结构变化、尼氏小体以及凋亡细胞数量变化；采用ELISA法检测各组大鼠血清中脑源性神经营养因子（brain-derived neurotrophic factor，BDNF）水平；采用比色法检测各组大鼠海马组织中乙酰胆碱酯酶（acetylcholinesterase，AChE）和乙酰胆碱转移酶（cholineacetyltransferase，ChAT）的活性；采用Western blotting法检测各组大鼠海马组织中BDNF、酪氨酸激酶受体B（tyrosine kinase receptor B，TrkB）、磷酸化TrkB（phosphorylated TrkB，p-TrkB）、磷脂酰肌醇-3-激酶（phosphatidylin-ositol-3-kinase，PI3K）、蛋白激酶B（Akt）、p-Akt和半胱氨酸天冬氨酸蛋白酶-3（cystein-asparate protease-3，Caspase-3）蛋白表达。结果 与模型组比较，铁筷子挥发油组大鼠逃避潜伏期缩短（P＜0.05、0.01），穿越平台次数和跨格次数提高（P＜0.05、0.01），在新异臂所待时间延长（P＜0.05）；神经细胞结构的变性得到改善，脑组织中尼氏小体的数量提高（P＜0.05），细胞凋亡减少（P＜0.05）；血清中BDNF水平及海马组织中ChAT活性升高（P＜0.05、0.01），海马组织中AChE活性降低（P＜0.05）；海马组织中BDNF、TrkB、p-TrkB、PI3K和p-Akt蛋白表达水平均显著升高（P＜0.05、0.01），Caspase-3蛋白表达水平降低（P＜0.05）。结论 苗药铁筷子挥发油可以改善CCH致VCI大鼠认知功能障碍，其作用机制可能与激活BDNF/TrkB/PI3K/Akt信号通路有关。

Effect and mechanism of volatile oil of Chimonanthi Radix on vascular cognitive impairment rats induced by chronic cerebral hypoperfusion

Objective To explore the effect and mechanism of volatile oil of Miao medicine Tiekuaizi (Chimonanthi Radix) on chronic cerebral hypoperfusion (CCH)-induced vascular cognitive impairment (VCI) in rats. Methods CCH model was established by modified bilateral common carotid artery occlusion (BCCAO) method, rats with successful modeling were randomly divided into model group, volatile oil of Chimonanthi Radix high-, medium-and low-dose (80, 40, 20 mg/kg) groups and butylphthalide (63 mg/kg) group, and sham operation group was set up. Rats were given corresponding drug for 28 d, water maze test was used to test the learning and memory ability of rats in each group; Open field test and Y-maze test were used to test the autonomous activity and novelty exploration ability of rats in each group; Hematoxylin-eosin (HE) staining, Nissl staining, TUNEL staining were used to observe the changes in structure of nerve cells, Nissl bodies and number of apoptotic cells in cerebral cortex and hippocampus CA1 area of rats in each group; Brain-derived neurotrophic factor (BDNF) level in serum was detected by ELISA; Colorimetric method was used to detect the activities of acetylcholinesterase (AChE) and cholineacetyltransferase (ChAT) in hippocampus of rats in each group; Western blotting was used to detect BDNF, tyrosine kinase receptor B (TrkB), phosphorylated TrkB (p-TrkB), phosphatidylin-ositol-3-kinase (PI3K), protein Kinase B (Akt), p-Akt and cysteine ​​aspartate protease-3 (Caspase-3) protein expressions in hippocampus of rats in each group. Results Compared with model group, escape latency of rats in volatile oil of Chimonanthi Radix group was shortened (P < 0.05, 0.01), number of crossing platform and grid crossings were increased (P < 0.05, 0.01), time spent in new arm was prolonged (P < 0.05, 0.01), degeneration of nerve cell structure was improved, number of Nissl bodies in brain tissue was increased (P < 0.05), cell apoptosis was decreased (P < 0.05), BDNF level in serum and ChAT activity of in hippocampus were increased (P < 0.05, 0.01), AChE activity in hippocampus was decreased (P < 0.05), BDNF, TrkB, p-TrkB, PI3K and p-Akt protein expression levels in hippocampus were significantly increased (P < 0.05, 0.01), Caspase-3 protein expression level was decreased (P < 0.05). Conclusion Miao medicine volatile oil of Chimonanthi Radix can improve CCH-induced cognitive impairment in VCI rats, and its mechanism may be related to the activation of BDNF/TrkB/PI3K/Akt signaling pathway.