目的 基于網(wǎng)絡(luò)藥理學(xué)和實(shí)驗(yàn)驗(yàn)證探討知母Anemarrhenae Rhizoma鹽制后治療糖尿病認(rèn)知障礙（diabetes cognitive impairment，DCI）的增效機(jī)制，為闡釋鹽知母炮制增效的科學(xué)內(nèi)涵、臨床合理用藥提供科學(xué)依據(jù)。方法 中藥系統(tǒng)藥理學(xué)數(shù)據(jù)庫(kù)分析平臺(tái)（traditional Chinese medicine systems pharmacology database and analysis platform，TCMSP）聯(lián)合課題組前期化學(xué)成分分析研究結(jié)果獲得知母中主要活性成分，采用Swiss Target Prediction數(shù)據(jù)庫(kù)預(yù)測(cè)成分的潛在靶點(diǎn)，從人類(lèi)基因數(shù)據(jù)庫(kù)（Gene cards）和在線人類(lèi)孟德?tīng)栠z傳數(shù)據(jù)庫(kù)（online Mendelian Inheritance in man，OMIM）的Genemap獲得2型糖尿?。╰ype 2 diabetes mellitus，T2DM）和阿爾茨海默?。ˋlzheimer’s disease，AD）疾病的相關(guān)靶點(diǎn)，利用Venny數(shù)據(jù)庫(kù)獲取成分與疾病的交集靶點(diǎn)，并通過(guò)STRING數(shù)據(jù)庫(kù)和Cytoscape 3.8.2軟件分析并構(gòu)建蛋白質(zhì)-蛋白質(zhì)相互作用（protein-protein interaction，PPI）網(wǎng)絡(luò)。借助Metascape平臺(tái)進(jìn)行基因本體（gene ontology，GO）功能及京都基因與基因組百科全書(shū)（Kyoto encyclopedia of genes and genomes，KEGG）通路富集分析，利用Cytoscape 3.8.2構(gòu)建“成分-靶點(diǎn)-通路”網(wǎng)絡(luò)。采用高糖高脂飼料聯(lián)合鏈脲佐菌素（streptozotocin，STZ）誘導(dǎo)糖尿病大鼠模型，造模成功后，隨機(jī)選取48只分為模型組、生知母皂苷組（176.4 mg/kg）、鹽知母皂苷組（176.4 mg/kg）、生知母組（2.52 g/kg）、鹽知母組（2.52 g/kg）和吡拉西坦（500 mg/kg）組，每組8只，另取8只生理狀態(tài)大鼠作為對(duì)照組，除對(duì)照組外，其余各給藥組每日上午ig三氯化鋁（aluminum chloride，AlCl₃，500 mg/kg），構(gòu)建糖尿病認(rèn)知障礙（diabetes cognitive impairment，DCI）模型，各給藥組每日下午ig給藥（10 mL/kg），對(duì)照組、模型組ig等體積蒸餾水，1次/d，連續(xù)4周。觀察各組大鼠毛發(fā)的變化情況、測(cè)定大鼠體質(zhì)量和血糖；采用Morris水迷宮法檢測(cè)大鼠的認(rèn)知能力；采用蘇木素-伊紅（hematoxylin eosin，HE）染色觀察腦組織海馬區(qū)病理變化；采用ELISA法檢測(cè)大鼠腦組織中腫瘤壞死因子-α（tumor necrosis factor-α，TNF-α）、白細(xì)胞介素-6（interleukin-6，IL-6）、IL-1β炎癥因子的水平，采用實(shí)時(shí)熒光定量聚合酶鏈?zhǔn)綌U(kuò)增反應(yīng)（reverse transcription-polymerase chain reaction，RT-PCR）、Western blotting法檢測(cè)大鼠腦組織中c-Jun氨基末端激酶（c-Jun N-terminal kinase，JNK）、TNF-α mRNA和蛋白的表達(dá)。結(jié)果 共篩選出知母活性成分潛在作用靶點(diǎn)120個(gè)，知母與疾病T2DM和AD三者之間的交集靶點(diǎn)61個(gè)，初步推測(cè)知母治療T2DM與AD的核心成分為知母皂苷BⅡ、知母皂苷AⅢ以及山奈酚等；核心靶點(diǎn)主要是TNF、蛋白激酶B（protein kinase B，Akt）、腫瘤抑制基因（tumor protein P53，TP53）、前列腺素內(nèi)過(guò)氧化物合成酶2（prostaglandin-endoperoxide synthase 2，PTGS2）和雌激素受體alpha基因（estrogen receptor alpha gene，ESR1）等；糖尿病年齡信號(hào)通路可能為主要的調(diào)控通路。動(dòng)物實(shí)驗(yàn)證明，與模型組比較，各給藥組均可改善大鼠的皮毛、體質(zhì)量、血糖的變化（P＜0.01），顯著縮短大鼠的逃避潛伏期（P＜0.01），減輕大鼠海馬組織病變，降低大鼠腦組織中TNF-α、IL-6、IL-1β的表達(dá)水平（P＜0.05、0.01）。生、鹽知母及其總皂苷組均顯著降低大鼠腦組織中JNK、TNF-α mRNA和蛋白的表達(dá)水平（P＜0.01），且鹽知母皂苷組和鹽知母組的改善效果分別優(yōu)于生知母皂苷組及生知母組（P＜0.05、0.01）。結(jié)論 生知母、鹽知母均可不同程度的改善DCI，且鹽制后作用更為顯著，其機(jī)制可能與調(diào)控JNK/TNF-α信號(hào)通路有關(guān)。

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

國(guó)家自然科學(xué)基金項(xiàng)目（81102810）;國(guó)家中醫(yī)藥管理局特色炮制技術(shù)規(guī)律挖掘項(xiàng)目（GZY-KJS-2022-049）