目的 通过整合转录组学数据与机器学习算法预测帕金森病抑郁（dPD）铁自噬的分子机制，并采用网络药理学与动物实验进一步揭示刺五加-百合（AS-LB）通过调控铁自噬干预dPD的分子机制。方法 基于基因表达综合数据库（GEO）中帕金森病数据集，整合Genecards中抑郁及铁自噬基因集取交集，获得dPD铁自噬核心靶基因，并对核心基因进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析。基于中药系统药理学平台数据库（TCMSP）收集AS-LB的化学成分，并整合本课题组前期采用UPLC-Q-TOF-MS分析鉴定的AS-LB成分，确定AS-LB的活性成分；基于核心靶基因，筛选AS-LB调控dPD铁自噬的活性成分。将60只雄性C57BL/6小鼠随机分为6组（n＝10）：对照组、模型组、普拉克索（阳性药，0.5 mg·kg_-1）组、AS-LB（刺五加与百合质量比4∶ 1）组、AS-LB（2∶ 1）组、AS-LB（1∶ 1）组，AS-LB剂量为5.07 g·kg_-1，除对照组外，采用ip 30 mg·kg_-1 1-甲基-4-苯基-1,2,3,6-四氢吡啶（MPTP）结合慢性不可预知应激（CUMS）构建dPD模型小鼠，造模完成后连续给药4周；进行爬杆、旷场、蔗糖水偏好行为学实验；采用苏木精-伊红（HE）染色观察小鼠脑组织黑质、海马区域病理学变化； ELISA法检测脑组织中多巴胺（DA）、5-羟色胺（5-HT）、去甲肾上腺素（NE）、肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-6、IL-1β含量； Western blotting法检测脑组织铁自噬相关蛋白核受体共激活因子4（NCOA4）、苄氯素1（BECN1）、醛脱氢酶1家族成员A1（ALDH1A1）、自噬相关基因7（ATG7）、α-突触核蛋白（SNCA）、铁蛋白轻链（FTL）、多聚嘧啶区结合蛋白1（PTBP1）的表达。结果 筛选出1 908个帕金森病差异表达基因（DEGs）、17 107个抑郁相关基因、42个铁自噬基因，三者取交集获得dPD铁自噬相关的7个核心靶基因（NCOA4、ATG7、BECN1、FTL、PTBP1、SNCA、ALDH1A1），富集分析结果显示，这些核心靶基因主要定位于自噬体、自噬溶酶体等细胞组分，参与铁离子的反应等生物过程，影响亚铁离子结合、Atg12的激活酶活性等分子功能，进而调控铁死亡、自噬、凋亡、神经退行性疾病等通路。网络药理学结果进一步显示，AS-LB可调控dPD铁自噬途径的活性成分为刺五加苷B、王百合苷B。动物实验结果表明，与模型组比较，AS-LB可通过减缓dPD小鼠黑质、海马区域神经元损伤，显著缩短小鼠爬杆时间（P＜0.01），显著延长旷场总距离（P＜0.01），显著升高蔗糖水偏好率（P＜0.01），显著升高DA、5-HT、NE水平（P＜0.05、0.01），显著降低炎症因子水平（P＜0.05、0.01），显著回调铁自噬蛋白NCOA4、ATG7、BECN1、FTL、PTBP1、SNCA、ALDH1A1表达水平（P＜0.05、0.01），其中以AS-LB 2∶ 1组效果最优。结论 AS-LB通过介导NCOA4、ATG7、BECN1、FTL、PTBP1、SNCA、ALDH1A1调控铁自噬途径干预dPD疾病进程，刺五加苷B、王百合苷B可能为其发挥效用的主要活性成分。

Objective To explore the molecular mechanism of ferritinophagy in depression in Parkinson's disease (dPD) by integrating transcriptomic data with machine learning algorithms. Furthermore, it investigates the molecular mechanism by which the combination of Acanthopanax senticosus-Lilium brownie (AS-LB) regulates ferritinophagy to intervene in dPD through network pharmacology and animal experiments. Methods Based on the Parkinson’s disease dataset in the Gene Expression Omnibus (GEO) database, the intersection of the depression and iron autophagy gene sets from Genecards was obtained to acquire the core target genes of dPD iron autophagy. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted on the core genes. The active components of AS-LB were collected from the Traditional Chinese Medicine Systems Pharmacology Database (TCMSP), and combined with the components identified by UPLC-Q-TOF-MS in our previous study, to determine the active components of AS-LB. Based on the core target genes, the active components of AS-LB regulating dPD iron autophagy were screened. Sixty male C57BL/6 mice were randomly divided into six groups (n = 10): The control group, the model group, the pramipexole (positive drug, 0.5 mg·kg^-1) group, the AS-LB (Acanthopanax senticosus to Lilium brownii mass ratio 4∶ 1) group, the AS-LB (2∶ 1) group, and the AS-LB (1∶ 1) group. The dose of AS-LB was 5.07 g·kg^-1. Except for the control group, the dPD model mice were constructed by intraperitoneal injection of 30 mg·kg^-1 MPTP combined with chronic unpredictable stress (CUMS), and the mice were continuously treated for 4 weeks. Behavioral experiments such as pole climbing, open field, and sucrose preference were conducted. HE staining was used to observe the pathological changes in the substantia nigra and hippocampus regions of the mouse brain. The contents of dopamine (DA), 5-hydroxytryptamine (5-HT), norepinephrine (NE), tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-1β in the brain tissue were detected by ELISA. The expressions of iron autophagy-related proteins nuclear receptor coactivator 4 (NCOA4), beclin 1 (BECN1), aldehyde dehydrogenase 1 family member A1 (ALDH1A1), autophagy-related gene 7 (ATG7), α-synuclein (SNCA), ferritin light chain (FTL), and polypyrimidine tract-binding protein 1 (PTBP1) in the brain tissue were detected by Western blotting. Results A total of 1 908 differentially expressed genes (DEGs) in Parkinson's disease, 17 107 depression-related genes, and 42 iron autophagy genes were screened. The intersection of the three sets yielded 7 core target genes (NCOA4, ATG7, BECN1, FTL, PTBP1, SNCA, ALDH1A1) related to dPD iron autophagy. Enrichment analysis showed that these core target genes were mainly located in cellular components such as autophagosomes and autolysosomes, participated in biological processes such as iron ion reactions, and affected molecular functions such as ferrous ion binding and Atg12 activating enzyme activity, thereby regulating pathways such as ferroptosis, autophagy, apoptosis, and neurodegenerative diseases. Network pharmacology results further indicated that the active components of AS-LB regulating the dPD iron autophagy pathway were acanthoside B and lirioside B. The animal experiment results showed that compared with the model group, AS-LB could alleviate the neuronal damage in the substantia nigra and hippocampus regions of dPD mice, significantly reduce the pole climbing time (P < 0.01), significantly increase the total distance in the open field (P < 0.01), significantly increase the sucrose preference rate (P < 0.01), significantly increase the levels of DA, 5-HT, and NE (P < 0.05, 0.01), significantly reduce the levels of inflammatory factors (P < 0.05, 0.01), and significantly restore the expression levels of iron autophagy proteins NCOA4, ATG7, BECN1, FTL, PTBP1, SNCA, and ALDH1A1 (P < 0.05, 0.01), with the AS-LB 2∶ 1 group showing the best effect. Conclusion AS-LB regulates the ferritinophagy pathway through the mediation of NCOA4, ATG7, BECN1, FTL, PTBP1, SNCA, and ALDH1A1 to intervene in the progression of dPD. Acanthopanax senticosus saponin B and Lily glycoside B may be the main active components responsible for their therapeutic effects.

R285.5

国家自然科学基金资助项目（82274125）；黑龙江省博士后基金面上项目（LBH-Z25301）