目的 利用網(wǎng)絡(luò)藥理學(xué)技術(shù)和實驗研究方法探究黃芩活性成分調(diào)控鐵死亡逆轉(zhuǎn)腫瘤耐藥的藥效物質(zhì)基礎(chǔ)、潛在靶標(biāo)及作用機制。方法 通過中藥系統(tǒng)藥理學(xué)數(shù)據(jù)庫與分析平臺（TCMSP），以生物利用度（OB）≥30%且類藥性（DL）≥0.18的條件篩選黃芩活性成分，使用UniProt數(shù)據(jù)庫獲得黃芩活性成分的對應(yīng)靶點基因。在GeneCards、OMIM數(shù)據(jù)庫中搜集腫瘤耐藥的相關(guān)靶點。利用Cytoscape 3.7.0軟件將獲得的黃芩活性成分與腫瘤耐藥的交集靶點，繪制“活性成分–作用靶點”網(wǎng)絡(luò)，并借助CytoHubba插件獲得活性成分度（degree）值排名，并分析關(guān)鍵核心靶點。通過DAVID數(shù)據(jù)庫在線分析功能進行基因本體論（GO）功能和京都基因與基因組百科全書（KEGG）通路富集分析。利用FerrDb數(shù)據(jù)庫獲取鐵死亡過程中的調(diào)控基因，將其與黃芩活性成分參與逆轉(zhuǎn)腫瘤耐藥性的靶點基因取交集，獲得黃芩通過調(diào)控鐵死亡過程逆轉(zhuǎn)腫瘤多藥耐藥性的靶點基因。利用Discovery Studio軟件進行化合物和核心靶點的分子對接。取處于指數(shù)生長期的ZR-75-30和HeLa細(xì)胞，加入不同終濃度（12.5、25.0、50.0、100.0、200.0 μmol/L）的漢黃芩素培養(yǎng)48 h，分別計算漢黃芩素聯(lián)合鐵死亡抑制劑（Fer-1 1 μmol/L）、誘導(dǎo)劑（Erastin 20 μmol/L和RSL3 8 μmol/L）對腫瘤細(xì)胞生長的抑制率，檢測漢黃芩素對腫瘤細(xì)胞ROS活力的影響。采用Western blotting法驗證腫瘤蛋白53（TP53）的表達變化情況。結(jié)果 共篩選出黃芩中包括漢黃芩素、β-谷甾醇、黃芩素、豆甾醇、金合歡素等在內(nèi)的32個活性成分，以及前列腺素內(nèi)過氧化物合酶2（PTGS2）、TP53、花生四烯酸-12-脂加氧酶（ALOX12）等在內(nèi)15個作用靶標(biāo)。通過介導(dǎo)白細(xì)胞介素-17（IL-17）、低氧誘導(dǎo)因子-1（HIF-1）、磷脂酰肌醇3-激酶（PI3K）-蛋白激酶B（Akt）、腫瘤壞死因子（TNF）、C型凝集素受體、神經(jīng)營養(yǎng)因子等信號通路調(diào)控鐵死亡，從而發(fā)揮逆轉(zhuǎn)腫瘤多藥耐藥的作用。細(xì)胞增殖實驗的結(jié)果表明，漢黃芩素表現(xiàn)出抑制腫瘤細(xì)胞ZR-75-30和HeLa細(xì)胞增殖的作用。與漢黃芩素組比較，漢黃芩素與Fer-1聯(lián)用后細(xì)胞抑制率有所下降；與Erastin和RSL-3聯(lián)用后對細(xì)胞抑制作用明顯增強（P＜0.05、0.01）。與對照組相比，100 μmol/L漢黃芩素可以顯著促進ZR-75-30和HeLa細(xì)胞ROS含量以及HeLa細(xì)胞TP53蛋白表達的升高（P＜0.05）。漢黃芩素與Fer-1聯(lián)用時ROS含量、TP53蛋白表達明顯下降；與Erastin和RSL-3聯(lián)用后ROS含量、TP53蛋白表達明顯升高（P＜0.05、0.01）。結(jié)論 黃芩調(diào)控鐵死亡逆轉(zhuǎn)腫瘤耐藥具有多成分、多靶點、多通路的作用特點，揭示了其藥效物質(zhì)和作用機制。

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“春暉計劃”合作科研項目（14）；陜西省衛(wèi)生健康科研基金項目（2021E022）；陜西省自然科學(xué)基礎(chǔ)研究計劃面上項目（2021JM-489）；國際合作專項—大連與日本神戶合作項目（2022YF19WZ046）；西安醫(yī)學(xué)院校級科研項目（2021DXS57）