目的 基于网络药理学与体外实验探讨高良姜素对口腔鳞状细胞癌的作用机制。方法 通过SwissTargetPrediction数据库筛选高良姜素潜在作用靶点，结合GeneCards与OMIM数据库获取口腔鳞状细胞癌相关靶点；通过构建药物–疾病靶点蛋白相互作用（PPI）网络以识别核心靶点；使用R软件包clusterProfiler进行富集分析；通过生存和诊断价值分析确定核心靶点；利用基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析对关键靶点、通路进行可视化。通过体外实验验证高良姜素对CAL-27细胞的细胞增殖、凋亡、迁移和侵袭，Western blotting法检测的相关蛋白表达水平。结果 网络药理学分析共得到59个高良姜素抗口腔鳞状细胞癌相互作用靶点；经PPI网络拓扑分析筛选得到前列腺素内过氧化物合酶2（PTGS2）、糖原合成酶激酶3β（GSK3B）、雌激素受体α（ESR1）、多聚ADP核糖聚合酶1（PARP1）、表皮生长因子受体（EGFR）、髓细胞白血病1（MCL1）、Src酪氨酸蛋白激酶（SRC）、基质金属蛋白酶9（MMP9）8个关键靶点。GO和KEGG富集分析显示，磷脂酰肌醇3-激酶/蛋白激酶B（PI3K/Akt）信号通路被确定为高良姜素抗口腔鳞状细胞癌作用的主要靶向通路。使用R软件根据预后和诊断价值进一步得到核心基因GSK3B、ESR1、PARP1；KEGG检索发现，核心靶点GSK3B位于PI3K/Akt信号通路下游。CCK-8与Transwell试验实验显示，高良姜素能够有效抑制CAL-27细胞增殖、侵袭与迁移；流式细胞术结果显示，高良姜素以浓度相关性方式促进CAL-27细胞凋亡（P＜0.01）。蛋白免疫印迹分析结果显示，高良姜素组CAL-27细胞中p-PI3K/PI3K、p-Akt/Akt和p-GSK3B/GSK3B蛋白表达均较对照组显著下降（P＜0.05、0.01）；加入PI3K激活剂740Y-P后，与高良姜素组比较，高良姜素＋740Y-P组p-PI3K、p-Akt、p-GSK3B和凋亡相关蛋白水平被有效逆转（P＜0.05）。结论 PI3K/Akt/GSK3B信号通路可能是高良姜素发挥抗口腔鳞状细胞癌作用的关键途径。

Objective To explore the mechanism of action of galangin on oral squamous cell carcinoma based on network pharmacology and in vitro experiments. Methods The potential targets of galangin were screened through the SwissTargetPrediction database, and the related targets of oral squamous cell carcinoma were obtained by combining the GeneCards and OMIM databases. To identify core targets by construct a drug-disease target protein interaction (PPI) network. Enrichment analysis was conducted using the R software package clusterProfiler. The core targets were determined through survival and diagnostic value analysis. Key targets and pathways were visualized using GO and KEGG enrichment analysis. The cell proliferation, apoptosis, migration and invasion of CAL-27 cells by galangin were verified through in vitro experiments, and the expression levels of related proteins were detected by Western blotting. Results A total of 59 interaction targets of galangin against oral squamous cell carcinoma were obtained through network pharmacological analysis. Through PPI network topology analysis and screening, a total of 8 key targets such as PTGS2, GSK3B, ESR1, PARP1, EGFR, MCL1, SRC, and MMP9 were obtained. GO and KEGG enrichment analyses revealed that the PI3K/Akt signaling pathway was identified as the primary targeted pathway for the anti-oral squamous cell carcinoma effect of galangin. The core genes GSK3B, ESR1, and PARP1 were further obtained based on prognosis and diagnostic value using R software. KEGG search revealed that the core target GSK3B is located downstream of the PI3K/Akt signaling pathway. The CCK-8 and Transwell assay experiments showed that galangin could effectively inhibit the proliferation, invasion and migration of CAL-27 cells. The results of flow cytometry showed that galangin promoted apoptosis of CAL-27 cells in a concentration-dependent manner (P < 0.01). The results of Western blotting analysis showed that the protein expressions of p-PI3K/PI3K, p-Akt/Akt and p-GSK3B/GSK3B in CAL-27 cells of the galangin group were significantly lower than those of the control group (P < 0.05, 0.01). After the addition of PI3K activator 740Y-P, compared with the galangin group, the levels of p-PI3K, p-Akt p-GSK3B and apoptosis-related proteins in the galangin + 740Y-P group were effectively reversed (P < 0.05). Conclusion The PI3K/Akt/GSK3B signaling pathway may be the key way for galangin to exert its anti-oral squamous cell carcinoma effect.

R287.7

国家自然科学基金资助项目（81373537）