Abstract: Background　Vascular dementia（VD）is one of the refractory neurological diseases，and its pathogenesis remains unclear. Therefore，it is crucial to explore key targets for the treatment of VD. Objective　To investigate the intervention effect of YFXFJZF（Yifei Xuanfei Jiangzhuo prescription）on vascular dementia（VD）rats based on the AMPK/mTOR/ULK1 pathway. Methods　A total of 70 8-week-old SPF-grade male SD rats were used，with 10 assigned to the normal group（K group）. The remaining rats were used to establish a VD rat model via bilateral common carotid artery ligation. After successful modeling，the rats were randomly divided into the VD model group（M group），VD model group+YFXFJZF group（Z group），VD model group+autophagy inhibitor group（Y group），VD model group+autophagy activator group（D group），VD model group+autophagy inhibitor group+YFXFJZF group（YZ group），and VD model group+autophagy activator group+YFXFJZF group（DZ group），with 10 rats in each group. The Z group was administered YFXFJZF at a dose of 1.2 g/kg via gavage. The K and M groups were given an equal volume of normal saline via gavage. Starting from day 15 of the experiment，the Y group was given intraperitoneal injections of 3-methyladenine（3-MA）at 1.5 mg/kg every other day for 14 days，and the D group was given intraperitoneal injections of rapamycin at 2 μg/kg every other day for 14 days. The intervention lasted for 28 days in total. After behavioral testing，blood samples and hippocampal tissues were collected from the rats. Hematoxylin-eosin（HE）staining was used to observe the pathological changes in the hippocampal tissue. Lead citrate staining（electron microscopy）was used to observe the condition of hippocampal neurons and autophagosomes. Western blotting was used to detect the protein expression of AMPK，p-AMPK，mTOR，p-mTOR，ULK1，p-S757-ULK1，LC3，and P62 in the hippocampus. Real-time quantitative PCR was used to detect the mRNA expression of AMPK，mTOR，ULK1，LC3，and P62 in the hippocampus. Data conforming to a normal distribution were expressed as（-x±s）. For comparisons among multiple groups with homogeneous variances，one-way analysis of variance was used，and the LSD method was used for pairwise comparisons between groups. Results　Behavioral testing showed that compared with the K group，the escape latency of VD rats in the M group was significantly prolonged，and the number of platform crossings was significantly reduced（P<0.05）. Compared with the M group，the escape latency was significantly shortened in the Z，Y，YZ，and DZ groups（P<0.05），and the number of platform crossings was significantly increased in the D group（P<0.05）. HE staining showed that the cells in the CA1 region of the hippocampus in the K group were neatly arranged with normal morphology and structure. The M group had severe damage，with deeper cell staining，unclear boundaries between nuclei and cytoplasm，cytoplasmic vacuolization，and irregular shapes. The D，DZ，and YZ groups showed some repair of the damaged hippocampal tissue compared with the M group，while the Z and Y groups showed more obvious recovery of the damaged hippocampal tissue. Lead citrate staining showed that the neuronal cell membranes in the CA1 region of the hippocampus in the K group were intact and smooth，with clear nucleoli，evenly distributed chromatin in the nuclei，and intact nuclear membranes. Compared with the K group，the neurons in the M group were severely damaged，with intact cell membranes，edematous cytoplasm，sparse and dissolved cytoplasmic contents，and markedly swollen organelles. Most mitochondria were significantly swollen，with intact membranes，large amounts of dissolved matrix，and broken or disappeared cristae. Compared with the M group，the neuronal damage was improved in the Z，Y，D，YZ，and DZ groups， with reduced autophagy levels. Western blotting results showed that the p-AMPK/AMPK ratio was higher in the M，Y，and D groups than in the K group，while it was lower in the Z，YZ，and DZ groups than in the M group. The p-mTOR/mTOR ratio was lower in the M，Y，D，and DZ groups than in the K group，while it was higher in the Z and YZ groups than in the M group. Thep-S757-ULK1/ULK1 ratio was higher in the M and D groups than in the K group，while it was lower in the Z，Y，YZ，and DZ groups than in the M group. The LC3II/LC3I ratio was higher in the M and D groups than in the K group，while it was lower in the Z， Y，YZ，and DZ groups than in the M group. P62 expression was lower in the M and D groups than in the K group，while it was higher in the Z，Y，YZ，and DZ groups than in the M group（P<0.05）. Real-time quantitative PCR results showed that the mRNA expression of AMPK，mTOR，LC3，P62，and ULK1 in the M group was significantly different from that in the K group （P<0.05）. There was no significant difference in the mRNA expression of AMPK，mTOR，LC3，P62，and ULK1 between the Y and D groups and the M group（P>0.05）. The mRNA expression of AMPK was lower in the Z，DZ，and YZ groups than in the M group（P<0.05）. The mRNA expression level of mTOR was lower in the Z，DZ，and YZ groups than in the M group （P<0.05）. The mRNA expression level of LC3 was lower in the Z，YZ，and DZ groups than in the M group（P<0.05）. The mRNA expression levels of P62 and ULK1 were lower in the Z，DZ，and YZ groups than in the M group（P<0.05）. Conclusion　YFXFJZF may regulate the level of autophagy in the hippocampus by modulating the AMPK/mTOR/ULK1 signaling pathway，protect the function of hippocampal neurons，and delay the progression of VD.

Key words: Dementia, vascular；Yifei Xuanfei Jiangzhuo prescription；AMP-activated protein kinase；Mechanistic target of rapamycin kinase；UNC-51-like kinase 1；Autophagy

摘要： 背景　血管性痴呆（VD）是难治性神经系统疾病之一，目前VD的发病机制不明确，因此探寻治疗VD的关键靶点至关重要。目的　探究益肺宣肺降浊方（YFXFJZF）基于腺苷酸活化蛋白激酶（AMPK）/哺乳动物雷帕霉素靶蛋白（mTOR）/Unc-51样激酶1（ULK1）通路对VD大鼠的干预作用。方法　70只8周龄SPF级SD雄性大鼠，其中10只作为正常组（K组），其余大鼠采用双侧颈总动脉分次结扎的造模方法建立VD大鼠模型。造模完成后将大鼠随机分成VD模型组（M组）、VD模型组+YFXFJZF组（Z组）、VD模型组+自噬抑制剂组（Y组）、VD模型组+自噬激动剂组（D组）、VD模型组+自噬抑制剂组+YFXFJZF组（YZ组）、VD模型组+自噬激动剂组+YFXFJZF组（DZ组），每组10只。Z组给予1.2 g/kg YFXFJZF灌胃，K组和M组给予等体积生理盐水灌胃，实验第15天开始，Y组给予腹腔注射3-甲基腺嘌呤1.5 mg/kg，隔日1次，共14 d。D 组给予雷帕霉素2 μg/kg腹腔注射，隔日1次，共14 d，一共干预28 d。进行行为学检测后采集大鼠血液样本和海马组织，苏木素-伊红（HE）染色观察海马组织病理形态学改变，枸橼酸铅染色（电镜）观察海马神经元、自噬小体情况。免疫印迹法（Western blotting）观察海马组织AMPK、磷酸化AMPK（p-AMPK）、mTOR、磷酸化mTOR（p-mTOR）、ULK1、磷酸化ULK1（Ser757）（p-S757-ULK1）、微管相关蛋白1轻链3（LC3）、P62蛋白表达，实时荧光定量PCR检测海马组织AMPK、mTOR、ULK1、LC3、P62 mRNA表达。符合正态分布的计量资料采用（x±s）表示，方差齐的数据多组间比较采用单因素方差分析，组间两两比较采用LSD法。结果　行为学检测结果示，与K组比较，M组VD大鼠逃避潜伏期明显延长，穿越平台次数显著减少（P <0.05）。与M组比较，Z组、Y组、YZ组、DZ组的逃避潜伏期显著缩短（P <0.05），与M组比较，D组逃避潜伏期缩短（P <0.05），穿越平台次数显著增加（P <0.05）。HE染色结果示，K组大鼠海马CA1区细胞排列整齐，细胞的形态、结构正常。M组受损严重，细胞染色加深，胞核与胞质界限不明，胞质空泡化，形态不规则。D组、DZ组、YZ组与M组比较，受损的海马组织有所修复；Z组、Y组中受损的海马组织恢复较明显。枸橼酸铅染色结果示，K组海马组织CA1区神经元胞膜完整光滑，核仁清晰可见，核内的染色质均匀分布且核膜保持完整形态；与K组比较，M组神经元细胞整体损伤，细胞膜完整，胞质出现水肿，稀疏、溶解，细胞器明显肿胀；线粒体大部分肿胀明显，膜完整，基质大量溶解，嵴断裂、消失；与M组比较，Z组、Y组、D组、YZ组、DZ组神经损伤有所改善，自噬水平降低。Western blotting结果示，M组、Y组、D组p-AMPK/AMPK高于K组，Z组、YZ组、DZ组p-AMPK/AMPK低于M组；M组、Y组、D组、DZ组p-mTOR/mTOR低于K组，Z组、YZ组p-mTOR/mTOR高于M组；M组、D组p-S757-ULK1/ULK1高于K组，Z组、Y组、YZ组、DZ组p-S757-ULK1/ULK1低于M组；M组、D组LC3Ⅱ/LC3Ⅰ高于K组，Z组、Y组、YZ组、DZ组LC3Ⅱ/LC3Ⅰ低于M组；M组、D组P62低于K组，Z组、Y组、YZ组、DZ组P62高于M组（P <0.05）。实时荧光定量PCR结果示，M组AMPK、mTOR、LC3、P62、ULK1mRNA与K组比较，差异有统计学意义（P <0.05）；Y组、D组的AMPK、mTOR、LC3、P62、ULK1 mRNA与M组比较，差异无统计学意义（P>0.05）；Z组、DZ组、YZ组AMPK mRNA表达量低于M组（P <0.05）；Z组、DZ组、YZ组mTORmRNA表达水平较M组降低（P <0.05）；Z组、YZ组LC3 mRNA表达水平较M组降低（P <0.05）；Z组、DZ组、YZ组P62、ULK1 mRNA表达水平较M组降低（P <0.05）。结论　YFXFJZF可能通过调控AMPK/mTOR/ULK1信号通路，调节海马神经区的自噬水平，保护海马神经元功能，延缓VD发展进程。

关键词: 痴呆, 血管性；益肺宣肺降浊方；腺苷酸活化蛋白激酶；哺乳动物雷帕霉素靶蛋白；Unc-51 样激酶 1；自噬