目的 基于UPLC-Q-Exactive Orbitrap-MS技术、网络药理学、分子对接、分子动力学模拟和实验验证，探究人参固本丸的物质基础及延缓皮肤衰老的作用机制。方法 根据正、负离子模式下质谱信息、质荷比、二级碎片离子等信息结合相关文献，推测鉴定化学成分。采用网络药理学方法，通过Swiss Target Prediction数据库获取人参固本丸的作用靶点，Genecards数据库筛选皮肤衰老靶点，取交集靶点，利用蛋白质-蛋白质相互作用（PPI）和京都基因与基因组百科全书（KEGG）分析核心靶点的关键通路。根据网络药理学结果，选择P值排名第1的HIF-1信号通路及其对应的人参固本丸中化合物进行分子对接及分子动力学模拟。应用H₂O₂诱导的HaCaT细胞皮肤衰老模型，CCK-8法评价人参固本丸（25、50、100、200、400、600 μg·mL^-1）及其含药血清（1%、5%、10%、20%、40%）对HaCaT细胞存活率的影响；给予人参固本丸（50、100、200 μg·mL^-1）后，DCFH-DA避光染色检测活性氧（ROS）含量，检测细胞划痕愈合率，实时荧光定量PCR（RT-qPCR）法检测衰老相关基因、炎性衰老相关分泌表型基因、HIF-1信号通路中核心靶点的mRNA水平。结果 从人参固本丸中鉴定出95个化合物；网络药理学分析出12个核心靶点，筛选出HIF-1信号通路及对应的核心靶点蛋白激酶B（AKT1）、丝裂原激活的蛋白激酶（MAPK3）、信号传导及转录激活蛋白3（STAT3）、表皮生长因子受体（EGFR）、B淋巴细胞瘤-2（BCL2）、白细胞介素（IL）-6、低氧诱导因子1A（HIF1A）。分子对接和分子动力学模拟验证活性成分5, 7-二羟基-6, 8, 4'-三甲氧基黄酮、（＋）-松脂醇、亚油酸、伪原薯蓣皂苷、N-对香豆酰酪胺和HIF-1信号通路核心靶点亲和力良好，构象稳定。体外实验表明，与模型组比较，人参固本丸可促进HaCaT细胞增殖、提高愈合率、降低ROS水平（P＜0.05、0.01），人参固本丸组MAPK3、STAT3、IL-6、HIF1A、AKT1、肿瘤坏死因子（TNF）-α、p53、p21、p16 mRNA水平表达显著降低（P＜0.05、0.01），表皮生长因子受体（EGFR）、B淋巴细胞瘤-2（BCL2）mRNA水平表达升高（P＜0.05、0.01）。结论 人参固本丸中的化学成分可能通过调控AKT1、MAPK3、STAT3、EGFR、BCL2、IL-6、HIF1A靶点，降低ROS、炎症水平，延缓皮肤衰老。

Objective Based on UPLC-Q-Exactive Orbitrap-MS technology, network pharmacology, molecular docking, molecular dynamics simulation, and experimental validation, to explore the components and delay skin aging mechanism of Renshen Guben Pills. Methods According to the mass spectrum information, mass-to-charge ratio, secondary fragment ions and other information in positive and negative ion modes, combined with related literature, the compounds in Renshen Guben Pills were speculated and identified. Using the method of network pharmacology, the target of chemical components of Renshen Guben Pills was obtained through SwissTargetPrediction database, and the target of skin aging was obtained through Genecards database, and the two targets were mapped to take the intersection target. Using PPI and KEGG to analyze the key pathways of core targets. Molecular docking and molecular dynamics simulation were used to confirm the relationship between chemical components and core targets. Using the H₂O₂-induced HaCaT cell skin aging model, the effects of Renshen Guben Pills (25, 50, 100, 200, 400, 600 μg·mL^-1) and their drug-containing serum (1%, 5%, 10%, 20%, 40%) on the survival rate of HaCaT cells were evaluated by the CCK-8 method. After administration of Renshen Guben Pills (50, 100, 200 μg·mL^-1), the content of ROS was detected by DCFH-DA staining in the dark, and the cell scratch healing rate was also measured. The mRNA levels of aging-related genes, inflammatory aging-related secretory phenotypes and core targets in HIF-1 signaling pathway were detected by qRT-PCR. Results The results showed that 95 compounds were identified from Renshen Guben Pills, 12 core targets were identified through network pharmacology analysis, and the HIF-1 signaling pathway and its corresponding core targets AKT1, MAPK3, STAT3, EGFR, BCL2, IL-6, and HIF1A were selected. Molecular docking and molecular dynamics simulations confirmed that the active ingredients 5, 7-dihydroxy-6, 8, 4'-trimethoxyflavone, (+)-pinoresinol, linoleic acid, pseudostellarin glycoside, N-p-coumaroyltyrosine, and the HIF-1 signaling pathway core targets had good affinity and stable conformation. In vitro experiments showed that compared with the model group, Renshen Guben Pills could promote the proliferation of HaCaT cells, increase the healing rate, and reduce the level of ROS (P < 0.05, 0.01). The levels of mitogen-activated protein kinase 3 (MAPK3), signal transducer and activator of transcription 3 (STAT3), interleukin (IL)-6, hypoxia-inducible factor 1A (HIF1A), protein kinase B (AKT1), tumor necrosis factor (TNF)-α, p53, p21 and p16 mRNA expression were significantly lower in the Renshen Guben Pills group (P< 0.05, 0.01), while the expression of epidermal growth factor receptor (EGFR) and B-cell lymphoma 2 (BCL2) mRNA was higher (P < 0.05, 0.01). Conclusion The chemical components in Renshen Guben Pills may regulate AKT1, MAPK3, STAT3, EGFR, BCL2, IL-6, and HIF1A targets, reduce ROS and inflammation levels, and delay skin aging.

R285.5

国家自然科学基金资助项目（82074127）；吉林省发展和改革委员会项目（2023C027-2）