目的 利用网络药理学技术和实验研究方法探究黄芩活性成分调控铁死亡逆转肿瘤耐药的药效物质基础、潜在靶标及作用机制。方法 通过中药系统药理学数据库与分析平台（TCMSP），以生物利用度（OB）≥30%且类药性（DL）≥0.18的条件筛选黄芩活性成分，使用UniProt数据库获得黄芩活性成分的对应靶点基因。在GeneCards、OMIM数据库中搜集肿瘤耐药的相关靶点。利用Cytoscape 3.7.0软件将获得的黄芩活性成分与肿瘤耐药的交集靶点，绘制“活性成分–作用靶点”网络，并借助CytoHubba插件获得活性成分度（degree）值排名，并分析关键核心靶点。通过DAVID数据库在线分析功能进行基因本体论（GO）功能和京都基因与基因组百科全书（KEGG）通路富集分析。利用FerrDb数据库获取铁死亡过程中的调控基因，将其与黄芩活性成分参与逆转肿瘤耐药性的靶点基因取交集，获得黄芩通过调控铁死亡过程逆转肿瘤多药耐药性的靶点基因。利用Discovery Studio软件进行化合物和核心靶点的分子对接。取处于指数生长期的ZR-75-30和HeLa细胞，加入不同终浓度（12.5、25.0、50.0、100.0、200.0 μmol/L）的汉黄芩素培养48 h，分别计算汉黄芩素联合铁死亡抑制剂（Fer-1 1 μmol/L）、诱导剂（Erastin 20 μmol/L和RSL3 8 μmol/L）对肿瘤细胞生长的抑制率，检测汉黄芩素对肿瘤细胞ROS活力的影响。采用Western blotting法验证肿瘤蛋白53（TP53）的表达变化情况。结果 共筛选出黄芩中包括汉黄芩素、β-谷甾醇、黄芩素、豆甾醇、金合欢素等在内的32个活性成分，以及前列腺素内过氧化物合酶2（PTGS2）、TP53、花生四烯酸-12-脂加氧酶（ALOX12）等在内15个作用靶标。通过介导白细胞介素-17（IL-17）、低氧诱导因子-1（HIF-1）、磷脂酰肌醇3-激酶（PI3K）-蛋白激酶B（Akt）、肿瘤坏死因子（TNF）、C型凝集素受体、神经营养因子等信号通路调控铁死亡，从而发挥逆转肿瘤多药耐药的作用。细胞增殖实验的结果表明，汉黄芩素表现出抑制肿瘤细胞ZR-75-30和HeLa细胞增殖的作用。与汉黄芩素组比较，汉黄芩素与Fer-1联用后细胞抑制率有所下降；与Erastin和RSL-3联用后对细胞抑制作用明显增强（P＜0.05、0.01）。与对照组相比，100 μmol/L汉黄芩素可以显著促进ZR-75-30和HeLa细胞ROS含量以及HeLa细胞TP53蛋白表达的升高（P＜0.05）。汉黄芩素与Fer-1联用时ROS含量、TP53蛋白表达明显下降；与Erastin和RSL-3联用后ROS含量、TP53蛋白表达明显升高（P＜0.05、0.01）。结论 黄芩调控铁死亡逆转肿瘤耐药具有多成分、多靶点、多通路的作用特点，揭示了其药效物质和作用机制。

Objective To explore the pharmacodynamic material basis, potential targets and mechanism of the active ingredients of Scutellaria baicalensis regulating iron death to reverse tumor resistance based on network pharmacology and experimental methods. Methods To screen the active components of Scutellaria baicalensis through TCMSP datebase according to bioavailability (OB) ≥30% and medicine-like (DL) ≥ 0.18, and the corresponding target genes of active components of Scutellaria baicalensis were obtained by UniProt database. Related targets of drug resistance were collected from GeneCards and OMIM databases. Cytoscape 3.7.0 software was used to determine the intersection targets of the active ingredients of Scutellaria baicalensis and tumor resistance, plot the “active ingredients -target” network, and obtain the degree ranking of active ingredients by CytoHubba plugin, and analyze the key core targets. The GO function and the KEGG pathway enrichment were analyzed through the online analysis function of the DAVID database. FerrDb database was used to obtain the regulatory genes in the process of iron death, and their intersection with the target genes of Scutellaria baicalensis active ingredients involved in reversing tumor resistance was selected. Finally, the target gene of Scutellaria baicalensis was obtained to reverse multidrug resistance by regulating the process of iron death. The Discovery Studio software was used for molecular docking of compounds and core targets. ZR-75-30 and HeLa cells at exponential growth stage were cultured for 48 h with different final concentrations (12.5, 25.0, 50.0, 100.0, 200.0 μmol/L) of wogonin. The inhibitory rates of wogonin combined with iron death inhibitor (Fer-1 1 μmol/L), inducer (Erastin 20 μmol/L and RSL3 8 μmol/L) on tumor cell growth were calculated, and the effects of wogonin on ROS activity of tumor cells were detected. The expression of tumor protein 53 (TP53) was verified by Western blotting. Results A total of 32 active ingredients including wogonin, β-sitosterol, baicalein, stigmasterol, and acacetin were selected from Scutellaria baicalensis, and 15 targets including PTGS2, TP53 and ALOX12 were selected. It regulates iron death by mediating IL-17, HIF-1, PI3K-Akt, TNF, C-type lectin receptor, tumor necrosis factor, neurotrophin, and other signaling pathways, thus playing a role in reversing tumor multidrug resistance. The results of cell proliferation experiment showed that wogonin could inhibit the proliferation of ZR-75-30 and HeLa cells. Compared with wogonin group, the cell inhibition rate of wogonin combined with Fer-1 was decreased,and the inhibitory effect of Erastin and RSL-3 on cells was significantly enhanced (P < 0.05, 0.01). Compared with the control group, 100 μmol/L wogonin significantly increased the ROS contents of ZR-75-30 and HeLa cells and the expression of TP53 protein in HeLa cells (P < 0.05). When wogonin was combined with Fer-1, ROS content and TP53 protein expression were decreased significantly. When wogonin was combined with Erastin and RSL-3, ROS content and TP53 protein expression were significantly increased after treatment (P < 0.05, 0.01). Conclusion The regulation of Scutellaria baicalensis on iron death and reversal of tumor resistance has the characteristics of multi-component, multi-target and multi-pathway action, revealing its pharmacodynamic substances and mechanism of action.

R966

教育部“春晖计划”合作科研项目（14）；陕西省卫生健康科研基金项目（2021E022）；陕西省自然科学基础研究计划面上项目（2021JM-489）；国际合作专项—大连与日本神户合作项目（2022YF19WZ046）；西安医学院校级科研项目（2021DXS57）