目的 基于网络药理学和实验验证探讨知母Anemarrhenae Rhizoma盐制后治疗糖尿病认知障碍（diabetes cognitive impairment，DCI）的增效机制，为阐释盐知母炮制增效的科学内涵、临床合理用药提供科学依据。方法 中药系统药理学数据库分析平台（traditional Chinese medicine systems pharmacology database and analysis platform，TCMSP）联合课题组前期化学成分分析研究结果获得知母中主要活性成分，采用Swiss Target Prediction数据库预测成分的潜在靶点，从人类基因数据库（Gene cards）和在线人类孟德尔遗传数据库（online Mendelian Inheritance in man，OMIM）的Genemap获得2型糖尿病（type 2 diabetes mellitus，T2DM）和阿尔茨海默病（Alzheimer’s disease，AD）疾病的相关靶点，利用Venny数据库获取成分与疾病的交集靶点，并通过STRING数据库和Cytoscape 3.8.2软件分析并构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络。借助Metascape平台进行基因本体（gene ontology，GO）功能及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，利用Cytoscape 3.8.2构建“成分-靶点-通路”网络。采用高糖高脂饲料联合链脲佐菌素（streptozotocin，STZ）诱导糖尿病大鼠模型，造模成功后，随机选取48只分为模型组、生知母皂苷组（176.4 mg/kg）、盐知母皂苷组（176.4 mg/kg）、生知母组（2.52 g/kg）、盐知母组（2.52 g/kg）和吡拉西坦（500 mg/kg）组，每组8只，另取8只生理状态大鼠作为对照组，除对照组外，其余各给药组每日上午ig三氯化铝（aluminum chloride，AlCl₃，500 mg/kg），构建糖尿病认知障碍（diabetes cognitive impairment，DCI）模型，各给药组每日下午ig给药（10 mL/kg），对照组、模型组ig等体积蒸馏水，1次/d，连续4周。观察各组大鼠毛发的变化情况、测定大鼠体质量和血糖；采用Morris水迷宫法检测大鼠的认知能力；采用苏木素-伊红（hematoxylin eosin，HE）染色观察脑组织海马区病理变化；采用ELISA法检测大鼠脑组织中肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）、白细胞介素-6（interleukin-6，IL-6）、IL-1β炎症因子的水平，采用实时荧光定量聚合酶链式扩增反应（reverse transcription-polymerase chain reaction，RT-PCR）、Western blotting法检测大鼠脑组织中c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）、TNF-α mRNA和蛋白的表达。结果 共筛选出知母活性成分潜在作用靶点120个，知母与疾病T2DM和AD三者之间的交集靶点61个，初步推测知母治疗T2DM与AD的核心成分为知母皂苷BⅡ、知母皂苷AⅢ以及山奈酚等；核心靶点主要是TNF、蛋白激酶B（protein kinase B，Akt）、肿瘤抑制基因（tumor protein P53，TP53）、前列腺素内过氧化物合成酶2（prostaglandin-endoperoxide synthase 2，PTGS2）和雌激素受体alpha基因（estrogen receptor alpha gene，ESR1）等；糖尿病年龄信号通路可能为主要的调控通路。动物实验证明，与模型组比较，各给药组均可改善大鼠的皮毛、体质量、血糖的变化（P＜0.01），显著缩短大鼠的逃避潜伏期（P＜0.01），减轻大鼠海马组织病变，降低大鼠脑组织中TNF-α、IL-6、IL-1β的表达水平（P＜0.05、0.01）。生、盐知母及其总皂苷组均显著降低大鼠脑组织中JNK、TNF-α mRNA和蛋白的表达水平（P＜0.01），且盐知母皂苷组和盐知母组的改善效果分别优于生知母皂苷组及生知母组（P＜0.05、0.01）。结论 生知母、盐知母均可不同程度的改善DCI，且盐制后作用更为显著，其机制可能与调控JNK/TNF-α信号通路有关。

Objective To explore the efficiency enhancing mechanism of salt-processing Anemarrhenae Rhizoma in the treatment of diabetes cognitive impairment (DCI) based on network pharmacology and experimental validation, providing a scientific basis for elucidating the scientific connotation of the synergy of salt-processing Anemarrhenae Rhizoma and the rational clinical use of drugs. Methods The main active components of Anemarrhenae Rhizoma were obtained by the preliminary chemical composition analysis research results of our research group combined with the traditional Chinese medicine systematic pharmacology database platform (TCMSP), and the potential targets of components were predicted by the Swiss Target Prediction database. The related targets of type 2 diabetes mellitus (T2DM) and Alzheimer’s diseases (AD) were obtained from Ggenemap of the human gene database (Gene cards) and the online Mendelian inheritance in man database (OMIM), and the intersection targets of components and diseases were obtained by Venny database. The protein-protein interaction (PPI) network was analyzed and constructed through the STRNG database and Cytoscape 3.8.2 software. Gene ontology (GO) function and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis were performed by Metascape platform. Cytoscape 3.8.2 was used to construct an “ingredients-targets-pathway” network. In this study, a diabetic rat model was established through a high-fat, high-sugar diet combined with streptozotocin (STZ) induction. After successful modeling, a total of 48 diabetic rats were randomly assigned to the following groups: model group, timosaponins of Anemarrhenae Rhizoma group (176.4 mg/kg), timosaponins of salt-processed Anemarrhenae rhizoma group (176.4 mg/kg), Anemarrhenae Rhizoma group (2.52 g/kg), salt-processed Anemarrhenae Rhizoma group (2.52 g/kg) and piracetam (500 mg/kg) group, with eight rats in each group. Another eight normal rats were taken as the control group. Except for the control group, all treatment groups received 500 mg/kg of AlCl₃ via intragastric administered every morning to establish DCI model, all treatment groups were given intragastric (10 mL/kg) daily afternoon, the control group and the model group were given same volume of distilled water intragastric once a day for a duration of four weeks. Changes in fur condition, body weight, and blood glucose levels of the rats were observed and measured. Morris water maze experiment was used to test the cognitive ability of the rats. Hematoxylin eosin (HE) staining was employed to observe pathological changes in the hippocampal region of brain tissue. Additionally, the levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β were detected by ELISA. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting were employed to detect the expression of c-Jun N-terminal kinase (JNK), TNF-α mRNA and protein in rat brain tissue. Results A total of 120 potential targets of active ingredients of Anemarrhenae Rhizoma were screened, and 61 common intersection targets between Anemarrhenae Rhizoma and T2DM and AD were identified. Preliminary evidence suggests that the core components of Anemarrhenae Rhizoma for treating T2DM and AD are Anemarrhenae Rhizoma timosaponin BⅡ, Anemarrhenae Rhizoma timosaponin AⅢ and kaempferol. Core targets include TNF, protein kinase B (Akt), tumor protein P53 (TP53), prostaglandin-endoperoxide synthase 2 (PTGS2) and estrogen receptor alpha gene (ESR1), etc. Diabetic age signaling pathway may be the main regulatory pathway. In animal experiments, compared to the model group, each drug treatment group could improve the changes in the fur, body weight, and blood glucose of rats (P < 0.01), significantly shorten the escape latency of rats (P < 0.01), alleviate hippocampal tissue lesions, and reduce the levels of TNF-α, IL-6 and IL-1β in rat brain tissue (P < 0.05, 0.01). Anemarrhenae Rhizoma, salt-processed Anemarrhenae Rhizoma and their total timosaponins groups significantly reduced the JNK and TNF-α mRNA and protein expression levels in the brain tissue (P < 0.01), with the salt-processed Anemarrhenae Rhizoma group and the timosaponins of salt-processed Anemarrhenae Rhizoma group showing better improvement compared to the Anemarrhenae Rhizoma group and the timosaponins of Anemarrhenae Rhizoma group (P < 0.05, 0.01). Conclusion Both Anemarrhenae Rhizoma and salt-processed Anemarrhenae Rhizoma can improve DCI, and salt-processed Anemarrhenae Rhizoma shows more significant effects. Its mechanism may be related to the regulation of JNK/TNF-α signaling pathway.

R285.5

国家自然科学基金项目（81102810）;国家中医药管理局特色炮制技术规律挖掘项目（GZY-KJS-2022-049）