目的 基于UPLC-Q-Exactive Orbitrap-MS技術(shù)、網(wǎng)絡(luò)藥理學(xué)、分子對接、分子動力學(xué)模擬和實驗驗證，探究人參固本丸的物質(zhì)基礎(chǔ)及延緩皮膚衰老的作用機制。方法 根據(jù)正、負(fù)離子模式下質(zhì)譜信息、質(zhì)荷比、二級碎片離子等信息結(jié)合相關(guān)文獻，推測鑒定化學(xué)成分。采用網(wǎng)絡(luò)藥理學(xué)方法，通過Swiss Target Prediction數(shù)據(jù)庫獲取人參固本丸的作用靶點，Genecards數(shù)據(jù)庫篩選皮膚衰老靶點，取交集靶點，利用蛋白質(zhì)-蛋白質(zhì)相互作用（PPI）和京都基因與基因組百科全書（KEGG）分析核心靶點的關(guān)鍵通路。根據(jù)網(wǎng)絡(luò)藥理學(xué)結(jié)果，選擇P值排名第1的HIF-1信號通路及其對應(yīng)的人參固本丸中化合物進行分子對接及分子動力學(xué)模擬。應(yīng)用H₂O₂誘導(dǎo)的HaCaT細胞皮膚衰老模型，CCK-8法評價人參固本丸（25、50、100、200、400、600 μg·mL^-1）及其含藥血清（1%、5%、10%、20%、40%）對HaCaT細胞存活率的影響；給予人參固本丸（50、100、200 μg·mL^-1）后，DCFH-DA避光染色檢測活性氧（ROS）含量，檢測細胞劃痕愈合率，實時熒光定量PCR（RT-qPCR）法檢測衰老相關(guān)基因、炎性衰老相關(guān)分泌表型基因、HIF-1信號通路中核心靶點的mRNA水平。結(jié)果 從人參固本丸中鑒定出95個化合物；網(wǎng)絡(luò)藥理學(xué)分析出12個核心靶點，篩選出HIF-1信號通路及對應(yīng)的核心靶點蛋白激酶B（AKT1）、絲裂原激活的蛋白激酶（MAPK3）、信號傳導(dǎo)及轉(zhuǎn)錄激活蛋白3（STAT3）、表皮生長因子受體（EGFR）、B淋巴細胞瘤-2（BCL2）、白細胞介素（IL）-6、低氧誘導(dǎo)因子1A（HIF1A）。分子對接和分子動力學(xué)模擬驗證活性成分5, 7-二羥基-6, 8, 4'-三甲氧基黃酮、（＋）-松脂醇、亞油酸、偽原薯蕷皂苷、N-對香豆酰酪胺和HIF-1信號通路核心靶點親和力良好，構(gòu)象穩(wěn)定。體外實驗表明，與模型組比較，人參固本丸可促進HaCaT細胞增殖、提高愈合率、降低ROS水平（P＜0.05、0.01），人參固本丸組MAPK3、STAT3、IL-6、HIF1A、AKT1、腫瘤壞死因子（TNF）-α、p53、p21、p16 mRNA水平表達顯著降低（P＜0.05、0.01），表皮生長因子受體（EGFR）、B淋巴細胞瘤-2（BCL2）mRNA水平表達升高（P＜0.05、0.01）。結(jié)論 人參固本丸中的化學(xué)成分可能通過調(diào)控AKT1、MAPK3、STAT3、EGFR、BCL2、IL-6、HIF1A靶點，降低ROS、炎癥水平，延緩皮膚衰老。

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

國家自然科學(xué)基金資助項目（82074127）；吉林省發(fā)展和改革委員會項目（2023C027-2）