目的 探讨补阳还五汤加入水蛭对脑梗死大鼠学习记忆及自噬相关蛋白Beclin 1和LC3-Ⅱ表达的影响。方法 采用颈动脉注射微栓子的方法制备大鼠脑梗死模型，筛选模型后随机分为模型组、补阳还五汤加水蛭低、中、高剂量组、补阳还五汤原方组，另设假手术组。分组后连续14天各组灌胃给予相应药物治疗。巴恩斯迷宫观察进入目标洞的潜伏期和探究非目标洞的错误次数，苏木素-伊红（HE）染色观察脑梗死边缘区组织形态及细胞结构变化；免疫组化定性检测Beclin 1、LC3-Ⅱ蛋白表达，免疫蛋白印迹法（Western blot）半定量检测Beclin 1、LC3-Ⅱ的表达。结果 巴恩斯结果显示，与假手术组比较，模型组到达目标洞的潜伏期明显延长，进入错误洞的次数增加；与经过模型手术的实验鼠相比，补阳还五汤加水蛭低、中、高、补阳还五汤原方组治疗的大鼠成功进入目标洞的潜伏期显著缩短；进入错误洞的次数分别减少。HE染色显示，梗死边缘区脑组织损伤明显，海马区神经元密度显著减少，残存神经元出现核固缩、噬伊红现象。免疫组化显示，经过模型手术的脑梗死致模大鼠海马神经细胞中Beclin 1、LC3-Ⅱ与假手术组相比，棕黄色颗粒呈现高表达状态，补阳还五汤加水蛭低、中、补阳还五汤原方组药物治疗组Beclin 1棕黄色颗粒呈现阳性表达显著降低，补阳还五汤加水蛭低、中、高、补阳还五汤组LC3-Ⅱ棕黄色颗粒呈现阳性表达比模型组明显减弱。免疫蛋白印迹结果呈现，与假手术组比经造模手术大鼠脑组织中Beclin 1和LC3-Ⅱ蛋白含量显著升高。与模型组相比，补阳还五汤加水蛭各治疗组及原方药物治疗组Beclin 1含量显著下降，LC3-Ⅱ的含有量明显下降。结论 补阳还五汤加水蛭可通过下调脑梗死大鼠脑组织内LC3-Ⅱ和Beclin 1蛋白表达，抑制脑组织受损后激活的自噬过度，降低脑梗死体积，改善大鼠因脑梗而带来的学习记忆等障碍，逐瘀的治疗作用也优于补阳还五汤原方，对脑神经功能有维持及保护作用

Objective To explore the effect of Buyang Huanwu Decoction added with leech on learning and memory and the expression of autophagy-related proteins Beclin-1 and LC3-Ⅱ in rats with cerebral infarction.Methods The rat cerebral infarction model was prepared by injecting microembolism into the carotid artery. After screening the models, they were randomly divided into a model group, Buyang Huanwu Decoction plus low, medium and high dose groups of Leech, and Buyang Huanwu Decoction original prescription group. mock surgical group. After all rats were divided into groups, each group was given corresponding medication by gavage for 14 consecutive days. Barnes maze to observe the latency of entering the target hole and the number of errors in exploring non-target holes, hematoxylin-eosin (HE) staining to observe the changes in the tissue morphology and cell structure of the marginal zone of cerebral infarction; The expression of Beclin 1, LC3-Ⅱ protein was qualitatively detected by immunohistochemistry, and the expression of Beclin 1, LC3-Ⅱ was semi-quantitatively detected by Western blot.Results Barnes results showed that compared with the sham operation group, the latency period for the model group to reach the target hole was significantly longer, and the number of times to enter the wrong hole increased; Compared with the experimental rats undergoing model surgery, the rats treated with Buyanghuanwutang plus leech low, medium, high, and Buyanghuanwutang original prescriptions successfully entered the target hole significantly shorter latency; the number of times to enter the wrong hole was reduced. HE staining showed that the brain tissue in the marginal area of infarction was obviously damaged, the density of neurons in the hippocampus was significantly reduced, and the remaining neurons showed pyknosis and eosin phagocytosis. Immunohistochemistry showed that the expression of Beclin 1, LC3-Ⅱ in hippocampal neurons of model rats with cerebral infarction induced by model operation was higher than that of sham operation group. The positive expression of Beclin 1 brown particles in the drug treatment group of Buyang Huanwu Decoction group was significantly reduced, and the positive expression ratio model of LC3-Ⅱ brown particles in Buyang Huanwu Decoction plus leech low, medium, high, and Buyang Huanwu Decoction group. The group was significantly weakened. The results of western blotting showed that the protein content of Beclin 1 and LC3-Ⅱ in the brain tissue of the model-operated rats was significantly higher than that in the sham-operated group. Compared with the model group, the Beclin 1 content of Buyang Huanwu Decoction plus leech treatment group and the original drug treatment group decreased significantly, and the content of LC3-Ⅱ decreased significantly.Conclusion Buyang Huanwu Decoction plus leech can down-regulate the expression of LC3-Ⅱ and Beclin 1 protein in brain tissue of rats with cerebral infarction, inhibit excessive autophagy activated after brain tissue damage, reduce cerebral infarction volume, and improve rats with cerebral infarction The treatment effect of removing blood stasis caused by obstacles such as learning and memory is also better than the original prescription of Buyang Huanwu Decoction, which can maintain and protect brain nerve function.

R285.5

承德医学院科技创新培育项目（ky2021032）：中医药干预在中枢神经损伤与修复中应用，负责人：吴晓光；河北省级重点学科病理学与病理生理学项目资助（2013），负责人：李红玉；河北省中医药重点学科中药药理学项目自助（2021），负责人：商亚珍。