目的 运用转录组学测序（transcriptome sequencing，RNA-Seq）和生物信息学技术对酸枣仁皂苷A（jujuboside A，JuA）改善淀粉样前体蛋白（amyloid precursor protein，APP）/早老蛋白1（pesenilin 1，PS1）双转基因小鼠认知功能的潜在机制进行分析，并采用qRT-PCR与Western blotting实验进行验证。方法 7月龄APP/PS1双转基因小鼠随机分为模型组及JuA低、中、高剂量（5、10、20 mg/kg，ip）组和多奈哌齐（0.65 mg/kg，ig）组，每日给药1次，连续21 d。采用Morris水迷宫实验检测小鼠的学习和记忆能力；苏木素-伊红（HE）、尼氏染色法检测脑组织病理损伤；RNA-Seq检测小鼠海马组织差异表达基因，对差异基因进行基因本体（gene ontology，GO）功能和京都基因与基因组百科全书（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析，筛选JuA治疗阿尔茨海默病（Alzheimer disease，AD）可能干预的信号通路，并对其中的核心富集基因进行qRT-PCR与Western blotting实验验证。结果 水迷宫实验结果显示，JuA组小鼠的学习记忆能力显著高于模型组（P<0.05、0.01），高剂量组作用最佳。HE和尼氏染色结果显示，与模型组比较，JuA组小鼠皮层和海马区神经元损伤明显减轻，尼氏体数量明显增多（P<0.01），说明JuA对AD小鼠脑组织有明显保护作用。RNA-Seq分析结果显示，模型组相较于对照组脑中明显上调的基因有218个，下调123个；JuA组相较于模型组明显上调基因有66个，下调28个；模型组中包括葡萄糖激酶（glucokinase，Gck）在内的5个差异基因经JuA干预后表达趋势被逆转。GO分析发现JuA治疗后差异基因主要富集在离子转运调控、细胞骨架、核受体活性等方面。KEGG通路分析发现JuA治疗后主要富集于2型糖尿病、细胞黏附因子、白细胞介素-17（interleukin-17，IL-17）信号通路和转化生长因子-β（transforming growth factor-β，TGF-β）信号通路；筛选模型组vs对照组、JuA高剂量组vs模型组共同调节的与JuA治疗AD相关性最高的前10条通路，发现主要集中于代谢疾病和炎症通路；Gck基因在这些通路上高频出现，说明其可能是JuA干预AD的重要靶点之一，qRT-PCR与Western blotting实验也验证了对Gck基因的筛选分析结果。结论 JuA能明显减轻AD小鼠脑组织病理损伤，提高学习记忆能力，其作用机制可能与调控炎症反应与代谢紊乱有关，其中上调Gck mRNA与蛋白表达维持葡萄糖稳态可能是JuA抗AD的重要机制之一。

Objective To analyze potential mechanism of jujuboside A (JuA) on improving cognitive function of amyloid precursor protein (APP)/pesenilin 1 (PS1) double transgenic mice using transcriptome sequencing (RNA-Seq) and bioinformatics techniques, and verify by qRT-PCR and Western blotting. Method Seven month-old APP/PS1 double transgenic mice were randomly divided into model group, JuA low-, medium-, high-dose (5, 10, 20 mg/kg, ip) groups and donepezil (0.65 mg/kg, ig) group. The drug was administered once a day for 21 consecutive days. Morris water maze experiment was used to detect the learning and memory abilities of mice; HE and Nissl staining methods were used to detect pathological damage in brain tissue; RNA-Seq was used to detect the differentially expressed genes in mouse hippocampus, gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed on the differentially expressed genes, and the signal pathways that JuA might interfere with Alzheimer disease (AD) were screened, and the core enrichment genes were verified by qRT-PCR and Western blotting. Results The results of the water maze experiment showed that the learning and memory abilities of mice in JuA group were significantly higher than those of model group (P < 0.05, 0.01), and the high-dose group had the best effect. HE and Nissl staining results showed that compared with model group, the neuronal damage in cortex and hippocampus of mice in JuA group mice was significantly reduced, and the number of Nissl bodies was significantly increased (P < 0.01), indicating that JuA had a significant protective effect on the brain tissue of AD mice. The results of RNA-Seq analysis showed that 218 genes were significantly up-regulated and 123 genes were down-regulated in model group compared with control group; Compared to model group, 66 genes were significantly upregulated in JuA high-dose group and 28 genes were downregulated; The expression trend of five differential genes including glucokinase (Gck) in model group was reversed after JuA intervention. GO analysis found that the differential genes after JuA treatment were mainly enriched in ion transport regulation, cytoskeleton, nuclear receptor activity, etc. KEGG pathway analysis revealed that JuA treatment was mainly enriched in type 2 diabetes, cell adhesion factor, interleukin-17 (IL-17) and transforming growth factor-β (TGF-β) signaling pathways; Screening the top 10 pathways that were jointly regulated by model group vs control group and JuA high-dose group vs model group and had the highest correlation with JuA treatment of AD, found that they mainly focused on metabolic disorders and inflammatory pathway; Gck gene appeared frequently in these pathways, indicating that it may be one of the important targets for JuA intervention in AD. qRT-PCR and Western blotting experiments had also verified the screening analysis results. Conclusion JuA can significantly alleviate the pathological damage of brain tissue in AD mice and improve the ability of learning and memory. Its mechanism may be related to the regulation of metabolic disorders and inflammation related pathways, and the upregulation of Gck mRNA and protein expression to maintain glucose homeostasis may be one of the important mechanisms of its anti-AD effect.

R285.5

大连市中心医院登峰计划项目（2022ZZ201）；辽宁省教育厅一般项目（L201945）