目的 运用网络药理学方法和分子对接技术探讨滋肾健脾化瘀片（山萸肉、三七、黄芪、葛根、鸡血藤、 生地黄）治疗糖尿病视网膜病变（DR）的作用机制，并通过体外实验进行验证。方法 （1）利用中药系统药理学 数据库与分析平台（TCMSP）及 BATMAN-TCM 数据库筛选滋肾健脾化瘀片的有效成分及其对应的靶点蛋白。利 用 GeneCards、OMIM 及 TTD 数据库检索 DR 疾病相关靶点。利用 VENNY 2.1.0 平台对药物活性成分靶点与 DR 疾病相关靶点取交集（共同靶点），即为滋肾健脾化瘀片治疗 DR 的潜在作用靶点。构建“中药-活性成分- 共同靶点”网络，筛选出滋肾健脾化瘀片治疗 DR 的关键活性成分。将共同靶点导入 STRING 数据库，获取 PPI 网络关系，并筛选出核心靶点。运用 Metascape 平台对共同靶点进行 GO 功能及 KEGG 通路富集分析。将 关键活性成分及核心靶点通过 Autodock 4 软件进行分子对接验证。（2）制备滋肾健脾化瘀片含药血清及空白血 清。将人视网膜微血管内皮细胞（HRmECs）随机分成 5 组：对照组（低糖 DMEM 培养基+10% 空白血清）、高糖 组（高糖 DMEM 培养基+10% 空白血清）及滋肾健脾化瘀片含药血清低、中、高剂量组（高糖 DMEM 培养基 +10% 低、中、高剂量含药血清），培养 48 h 后进行检测。采用 CCK-8 法检测 HRmECs 细胞增殖活性； RT-qPCR 法检测 HRmECs 细胞中 IL-1β、AKT1、VEGFA、TP53 mRNA 表达水平。结果 （1）共筛选出滋肾健 脾化瘀片治疗 DR 的潜在作用靶点（共同靶点）74 个；9 个关键活性成分包括槲皮素、芒柄花黄素、毛地黄黄 酮、β 谷甾醇、山柰酚、毛蕊异黄酮、γ-氨基丁酸、豆甾醇、异鼠李亭；12 个核心靶点：IL-1β、PPARG、 NOS3、CXCL8、IL-6、AKT1、TNF、INS、EGF、VEGFA、TP53、PTGS2。GO 功能及 KEGG 富集分析显示核 心靶点主要参与了炎症反应、蛋白质磷酸化的正调控、细胞迁移等生物过程，以及 NF-κB 信号通路、 AGE-RAGE 信号通路、HIF-1 通路、TNF 通路、PI3K-AKT 通路等。关键活性成分与核心靶点均具有较好的 结合亲和力。（2）与对照组比较，高糖组 HRmECs 细胞活性显著降低（P＜0.01）；细胞中 VEGFA、TP53、IL-1β mRNA 表达水平显著升高（P＜0.01），AKT1 mRNA 表达水平显著降低（P＜0.01）。与高糖组比较，滋肾健脾化 瘀片含药血清低、中、高剂量组的 HRmECs 细胞活性均显著升高（P＜0.05，P＜0.01），并呈浓度依赖性；含药 血清高剂量组细胞的 VEGFA、TP53、IL-1β mRNA 表达水平显著降低（P＜0.05，P＜0.01），而 AKT1 mRNA 表达水平显著升高（P＜0.01）。结论 滋肾健脾化瘀片可能通过槲皮素、山柰酚、毛地黄黄酮等多种活性成 分，作用于 IL-1β、IL-6、VEGFA 等核心靶点，以及 NF-κB 信号通路、AGE-RAGE 信号通路、PI3K-AKT 通 路等关键通路，从而发挥对 DR 的治疗作用。

Objective To explore the mechanism of Zishen Jianpi Huayu Tablets （Corni Fructus， Notoginseng Radix et Rhizoma， Astragali Radix， Puerariae Lobatae Radix， Spatholobi Caulis， Rehmanniae Radix） in the treatment of diabetic retinopathy （DR） by means of network pharmacology and molecular docking technique，and verified by in vitro experiments. Methods The active components of Zishen Jianpi Huayu Tablets and their corresponding target proteins were screened using the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform （TCMSP） and the BATMAN-TCM database. Drug target proteins were converted to their corresponding gene names through the UniProt database. DR-related targets were searched using "diabetic retinopathy" as a keyword in GeneCards， DrugBank， OMIM， and TTD databases. Common targets between the disease and the drug were identified using the Venny tool. These common targets were analyzed using the String database， a protein-protein interaction （PPI） network was constructed. Topological heterogeneity analysis was performed using Cytoscape 3.9.1 to select core targets and create a PPI network diagram. These common targets were entered into the Metascape database for Gene Ontology （GO） function analysis and Kyoto Encyclopedia of Genes and Genomes （KEGG） pathway analysis to identify potential action pathways. Molecular docking of the main active components and core targets was performed using Auto Dock tools software， followed by further experimental validation. The CCK-8 assay was used to assess the effect of Zishen Jianpi Huayu Tablet medicated serum on the cell viability of Human Retinal Microvascular Endothelial Cells （HRmECs） under high glucose conditions，and RTqPCR was used to measure the expression of IL-1β， AKT1， VEGFA， and TP53 mRNA in HRmECs. Results （1） The effective components and corresponding target proteins of Zishen Jianpi Huayu Tablets were screened by Traditional Chinese Medicine System Pharmacology Database and Analysis Platform （TCMSP） and BATMAN-TCM database. The disease-related targets of DR were searched by GeneCards，OMIM and TTD databases. The use of VENNY platform for drug active components target and DR disease-related target to take intersection （common target），that is，Zishen Jianpi Huayu Tablets in the treatment of DR potential target. The network of "drugs-active components-common targets" was constructed to screen out the key active components of Zishen Jianpi Huayu Tablets in the treatment of DR. Import the common target into STRING database， obtain the PPI network relationship，and screen out the core target. Metascape platform was used to analyze the GO function and KEGG pathway enrichment of the common targets. The key active components and core targets were verified by Autodock 4 software for molecular docking. （2） The drug-containing serum and blank serum of Zishen Jianpi Huayu Tablets was prepared. Human retinal microvascular endothelial cells （HRmECs） were randomly divided into 5 groups：the control group （low-sugar DMEM medium + 10% blank serum），high-glucose group （high-sugar DMEM medium + 10% blank serum） and Zishen Jianpi Huayu Tablets containing low-，medium- and high- dose serum （high-sugar DMEM medium + 10% low-，medium- and high- dose drug containing serum） were detected after 48 hours of culture. The proliferative activity of HRmECs cells was detected by CCK-8 method，and the mRNA expressions of IL-1β， AKT1， VEGFA and TP53 in HRmECs cells were detected by RT-qPCR method. Conclusion Zishen Jianpi Huayu Tablets may act on core targets such as IL-1β，IL-6 and VEGFA，as well as key pathways such as NF-κB signaling pathway， AGE-RAGE signaling pathway and PI3K-AKT pathway through various active components such as quercetin，kaempferol and rehmannia flavonoids，so as to play a therapeutic role in DR.

R285.5；R857.3

国家自然科学基金项目（82174441）；广东省中医药管理局科研项目（20241092）