目的 探究治疗闭角型青光眼中药的特点、用药规律和作用机制。方法 检索中国知网、万方、维普、中国生物医学文献数据库及古今医案云平台收录的有关中药治疗闭角型青光眼的文献和处方，通过数据挖掘分析确定核心处方；采用网络药理学，利用TCMSP与BATMAN-TCM 2.0数据库筛选口服生物利用度≥30%且类药性≥0.18的活性成分，经UniProt数据库统一注释靶点，并与GeneCards、TTD、OMIM、CTD 4大数据库整合去重获得的疾病靶点取交集，得到药效靶点，在Cytoscape 3.10.2中构建“核心处方-活性成分-药效靶点”互作网络。随后将药效靶点导入Metascape平台（限定人类物种，以P＜0.05为阈值）进行基因本体（gene ontology，GO）（生物过程、分子功能、细胞组分）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，并可视化前20条显著通路及功能；同时利用String数据库构建蛋白质相互作用（protein-protein interaction，PPI）网络，通过Cytoscape MCODE插件（cut-off＝0.05）筛选核心靶点，以解析核心处方治疗闭角型青光眼的潜在分子机制。通过分子对接考察活性成分与核心靶点的结合能。采用连二亚硫酸钠诱导建立小鼠视网膜神经节细胞RGC缺氧损伤模型，运用实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction，qRT-PCR）技术检测上述活性成分对核心靶点mRNA表达的影响；使用ELISA法检测上述活性成分对肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）水平的影响；使用免疫荧光检测上述活性成分对磷酸化蛋白激酶B（phosphorylated protein kinase B，p-AKT）、核因子-κB（nuclear factor-κB，NF-κB）蛋白水平的影响。结果 闭角型青光眼使用频数排名前3位的中药为茯苓、当归、柴胡，核心证候以肝郁气滞与肝肾两虚为主，核心处方为牡丹皮-当归-栀子-柴胡-菊花-白术-甘草-白芍-茯苓；聚类分析得到3个聚类簇，分别为柴胡疏肝散加减、益精补阳还五汤加减及杞菊地黄丸加减。网络药理学分析表明，核心处方主要通过调控TNF、磷脂酰肌醇-3-羟激酶（phosphatidylinositol-3-hydroxykinase，PI3K）-AKT、丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）等信号通路，作用于AKT1、TNF-α、NF-κB等关键靶点，参与炎症反应、氧化应激等生物学过程，从而干预青光眼病理进程。通过分子对接确定槲皮素和木犀草素为核心处方干预闭角型青光眼的核心成分，并进行体外实验验证。结果显示，木犀草素各剂量（5、10、20 μmol/L）组均可上调AKT1 mRNA表达（P＜0.01），中、高剂量组可下调NF-κB、TNF-α mRNA表达（P＜0.05），并显著升高p-AKT蛋白表达，降低NF-κB蛋白及TNF-α水平（P＜0.05）；槲皮素中、高剂量（40、80 μmol/L）组能显著上调AKT1 mRNA表达，下调NF-κB、TNF-α mRNA表达（P＜0.05），低（20μmol/L）、中、高剂量组均可升高p-AKT蛋白表达，降低NF-κB蛋白表达（P＜0.05），中、高剂量组可显著降低TNF-α水平（P＜0.05）。结论 运用数据挖掘技术系统梳理古今治疗闭角型青光眼的文献与医案，从用药频次、性味归经、功效类别、关联规则及核心组合等维度，层层解析“高频-核心-差异”三维特征，提炼核心方剂并揭示其配伍规律。网络药理学分析进一步证实核心方剂通过“多成分-多靶点-多通路”协同调控氧化应激、炎症反应及细胞凋亡等关键生物过程，体现中药复方整体调节的治疗优势，为临床辨证用药提供了科学依据，具有一定的指导意义。体外实验证实，在缺氧损伤模型中，木犀草素和槲皮素能上调AKT1 mRNA及p-AKT蛋白表达，抑制NF-κB、TNF-α mRNA及蛋白表达，有效降低炎症及细胞凋亡相关因子水平，通过“抑炎-促存活”的双向机制对RGC细胞发挥保护作用。

Objective To explore the characteristics, medication patterns, and the mechanisms of action of traditional Chinese medicines for treating angle-closure glaucoma. Methods Literature and prescriptions related to the treatment of angle-closure glaucoma with traditional Chinese medicine were retrieved from databases including CNKI, Wanfang, VIP, China Biology Medicine (CBM), and the Ancient and Modern Medical Case Cloud Platform. Through data mining and analysis, core prescriptions were identified. Subsequently, a network pharmacology approach was employed. Active ingredients with oral bioavailability (OB) ≥ 30% and drug-likeness (DL) ≥ 0.18 were screened using the TCMSP and BATMAN-TCM 2.0 databases. Targets were uniformly annotated via the UniProt database and intersected with disease targets obtained by integrating and deduplicating data from four major databases (GeneCards, TTD, OMIM, and CTD) to identify pharmacodynamic targets. A “core prescription-active ingredient-pharmacodynamic target” interaction network was constructed using Cytoscape 3.10.2. The pharmacodynamic targets were then imported into the Metascape platform (restricted to human species, with a threshold of P < 0.05) for gene ontology (GO) (biological process, molecular function, cellular component) and Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis, with the top 20 significant pathways and functions visualized. Additionally, a protein-protein interaction (PPI) network was constructed using the String database, and core targets were screened using the Cytoscape MCODE plugin (cut-off = 0.05) to elucidate the potential molecular mechanisms underlying the core prescriptions in treating angle-closure glaucoma. The binding energy between the active ingredient and the core target was investigated through molecular docking. Sodium dithionite was used to induce hypoxic injury in mouse retinal ganglion cells (RGCs) to establish an in vitro model. Real-time fluorescence quantitative polymerase chain reaction (qRT-PCR) was employed to detect the effects of these active components on mRNA expression of core target genes. ELISA was used to measure the impact of these active components on tumor necrosis factor-α (TNF-α) levels. Immunofluorescence staining was applied to evaluate the effects of these active components on phosphorylated protein kinase B (p-AKT) and nuclear factor-κB (NF-κB) protein expression. Results The top three most frequently used traditional Chinese medicines for angle-closure glaucoma were Fuling (Poria), Danggui (Angelicae Sinensis Radix), and Chaihu (Bupleuri Radix). The core syndromes were mainly liver qi stagnation and liver-kidney deficiency. A core prescription consisted of Mudanpi (Moutan Cortex)-Angelicae Sinensis Radix-Zhizi (Gardeniae Fructus)-Bupleuri Radix-Juhua (Chrysanthemi Flos)-Baizhu (Atractylodis Macrocephalae Rhizoma)-Gancao (Glycyrrhizae Radix et Rhizoma)-Baishao (Paeoniae Radix Alba)-Poria. Cluster analysis yielded three clusters: modified Chaihu Shugan San (柴胡疏肝散), modified Yijing Buyang Huanwu Decoction (益精补阳还五汤), modified Qiju Dihuang Pills (杞菊地黄丸). Network pharmacology analysis showed that the core prescription regulated key targets such as AKT1, TNF-α, NF-κB by modulating signalling pathways such as TNF, phosphatidylinositol-3-hydroxykinase (PI3K)-Akt, and MAPK (mitogen-activated protein kinase), thereby participating in biological processes such as inflammatory response, and oxidative stress, and thus intervening in the pathological process of glaucoma. Quercetin and luteolin were identified as the core components of the core prescription for the intervention of angle-closure glaucoma through molecular docking, and in vitro experiments were conducted for verification. The results showed that all luteolin dose groups (5, 10, 20 μmol/L) upregulated AKT1 mRNA expression (P < 0.01), while the medium and high dose groups downregulated NF-κB and TNF-α mRNA expression (P < 0.05), significantly increased p-AKT protein expression, and decreased NF-κB protein and TNF-α levels (P < 0.05). The medium and high dose groups of quercetin (40, 80 μmol/L) also significantly upregulated AKT1 mRNA expression and downregulated NF-κB and TNF-α mRNA expression (P < 0.05). All low (20 μmol/L), medium, and high dose groups of quercetin increased p-AKT protein expression and decreased NF-κB protein expression (P < 0.05), while the medium and high dose groups significantly reduced TNF-α expression (P < 0.05). Conclusion Data mining techniques were systematically employed to analyze ancient and modern literature and medical records concerning the treatment of angle-closure glaucoma. From multiple dimensions including medication frequency, nature-flavor-meridian tropism, efficacy categories, association rules, and core combinations, the “high-frequency-core-differential” three-dimensional characteristics were progressively analyzed to extract core prescriptions and reveal their compatibility patterns. Network pharmacology analysis further confirmed that the above high-frequency prescriptions synergistically regulate key biological processes such as oxidative stress, inflammatory response, and apoptosis through a “multi-component-multi-target-multi-pathway” mechanism, reflecting the holistic therapeutic advantages of traditional Chinese medicine compound formulas, thereby providing scientific evidence for clinical syndrome differentiation and medication, with certain guiding significance. In vitro experiments simultaneously validated that, in the hypoxic injury model, both luteolin and quercetin significantly upregulated AKT1 mRNA and p-AKT protein expression, inhibited NF-κB and TNF-α mRNA and protein expression, effectively reduced levels of inflammation and apoptosis-related factors, and exerted protective effects on RGCs through a bidirectional mechanism of “anti-inflammatory and pro-survival” effects.

R285

中山市中医药传承创新发展科研专项（2024B3014）