基于网络药理学及实验验证探讨去甲汉黄芩素促进口腔鳞状细胞癌细胞凋亡的作用机制

Mechanism of norwogonin induces apoptosis in oral squamous cell carcinoma cells through network pharmacology and experimental validation

目的 采用网络药理学结合实验验证方法探索去甲汉黄芩素对口腔鳞状细胞癌的抗肿瘤活性及靶点。方法 采用网络药理学方法分析去甲汉黄芩素及口腔鳞状细胞癌的靶点。使用STRING和Cytoscape软件构建蛋白相互作用(PPI)网络。基因本体(GO)和京都基因和基因组百科全书(KEGG)富集分析生物学功能。采用分子对接和CETSA验证去甲汉黄芩素与其关键靶标之间的结合能力。MTT和吖啶橙/溴化乙锭(AO/EB)法检测去甲汉黄芩素对口腔鳞状细胞癌细胞增殖以及凋亡的影响。免疫蛋白印迹法检测相关蛋白的表达。结果 去甲汉黄芩素与口腔鳞状细胞癌共有71个重叠靶点;分别涉及磷脂酰肌醇3-激酶(PI3K)/蛋白激酶(Akt)、氧化应激以及凋亡通路中相关基因;其中前5位的Hub基因为表皮生长因子受体(EGFR)、雌激素受体1(ESR1)、非受体酪氨酸激酶(SRC)、前列腺素内过氧化物酶2(PTGS2)、基质金属蛋白酶9(MMP9)。分子对接结果显示,去甲汉黄芩素与EGFR的结合力最强(-6.6 kcal/mol);CETSA实验和Kaplan-Meier生存曲线结果显示,EGFR对口腔鳞状细胞癌患者生存影响最大。实验验证结果显示,去甲汉黄芩素以剂量相关性性地降低口腔鳞状细胞癌细胞活性,IC50为15μmol/L。AO/EB染色分析显示,去甲汉黄芩素诱导口腔鳞状细胞癌细胞发生程序性凋亡来抑制癌细胞的活力,且与Bax水平的增加和Bcl-2水平的降低相关。结论 去甲汉黄芩素可能通过调控EGFR的表达进而影响PI3K/Akt、氧化应激以及凋亡途径诱导口腔鳞状细胞癌细胞凋亡。

Objective To investigate the antitumor activity and target of norwogonin in oral squamous cell carcinoma by network pharmacology combined with experimental verification.Methods The target of norwogonin and oral squamous cell carcinoma was analyzed by network pharmacology.Build a protein interaction(PPI)network using STRING and Cytoscape software.GO and KEGG enrichment analyze biological functions.Molecular docking and CETSA were used to verify the binding ability between norwogonin and its key targets.MTT and acridine orange/ethidium bromide(AO/EB)methods were used to detect the effects of norwogonin on the proliferation and apoptosis of oral squamous cell carcinoma cells.The expression of related proteins was detected by Immunowestern blotting.Results A total of 71 overlapping targets between norwogonin and oral squamous cell carcinoma,implicated in the PI3K/Akt,oxidative stress,and apoptosis signaling pathways,respectively.The top five Hub genes identified in this study are EGFR,ESR1,SRC,PTGS2,and MMP9.The molecular docking results showed that the binding force between norwogonin and EGFR was the strongest(-6.6 kcal/mol).The results of CETSA test and Kaplan-Meier survival curve showed that EGFR had the greatest impact on the survival of patients with oral squamous cell carcinoma.The experimental results showed that the activity of oral squamous cell carcinoma was decreased dose-dependent with IC50 of 15μmol/L.AO/EB staining analysis showed that norwogonin induced programmed apoptosis of oral squamous cell cancer cells to inhibit cancer cell viability,which was associated with increased Bax levels and decreased Bcl-2 levels.Conclusions Norwogonin may affect PI3K/Akt,oxidative stress and apoptosis pathway to induce apoptosis of oral squamous cell carcinoma cells by regulating EGFR expression.