基于网络药理学与体外实验探讨香荆芥酚诱导鼻咽癌细胞凋亡和周期阻滞的作用机制

Exploration of the Mechanisms of Apoptosis and Cell Cycle Arrest Induced by Carvacrol in Nasopharyngeal Carcinoma Cells Based on Network Pharmacology and In Vitro Experiments

摘要:

【】 目的:探讨香荆芥酚(Carvacrol,CV)对人鼻咽癌CNE2细胞活力、增殖、凋亡和周期的影响,并探索CV诱导CNE2细胞凋亡和周期阻滞的可能作用机制。 方法:运用网络药理学预测CV作用于鼻咽癌的潜在靶点,用R软件对潜在靶点进行KEGG通路富集分析；使用CB-Dock2平台将CV与靶点蛋白进行分子对接,并预测结合能力；使用Gromacs2022.3软件进行分子动力学模拟,以评估CV与靶点蛋白相互作用的稳定性。用0(对照组)、10、20、30、40、50、60 μg/mL的CV作用于CNE2细胞48 h后,通过CCK-8法检测CV对CNE2细胞存活率的影响。用0(对照组)、30、40、50 μg/mL的CV作用于CNE2细胞48 h后,通过集落形成实验和流式细胞术检测CV对CNE2细胞增殖、凋亡和周期的影响,通过Western Blot法检测CV对CNE2细胞内PIK3CA、p-PI3K、p-AKT、Bcl-2、Bax、Caspase-3和CDK2蛋白表达的影响。 结果:经网络药理学预测,CV作用鼻咽癌存在137个共有靶点,KEGG富集分析结果显示CV作用鼻咽癌可能涉及PI3K/AKT等信号通路；分子对接结果显示PI3K、AKT、PIK3CA、Bcl-2、Caspase-3、CDK2与CV具有较好的结合亲和力。分子动力学模拟进一步表明CV与CDK2结合较稳定。CCK-8结果显示,与对照组比较,随着CV浓度的增加,CNE2细胞存活率显著降低。与对照组比较,经CV作用后的CNE2细胞增殖能力降低,细胞凋亡能力增加,细胞周期G0/G1期延长,PIK3CA、p-PI3K、p-AKT、Bcl-2和CDK2蛋白表达均明显降低,Bax和Caspase-3蛋白表达均明显升高。差异均具有统计学意义(P＜0.05)。 结论:CV抑制CNE2细胞活力和增殖,并诱导细胞凋亡和阻滞细胞周期于G0/G1期。CV诱导CNE2细胞凋亡和周期阻滞可能与PI3K/AKT信号通路相关的PIK3CA、p-PI3K、p-AKT、Bcl-2、CDK2蛋白表达降低及Bax、Caspase-3蛋白表达升高有关。

关键词:香荆芥酚;鼻咽癌;网络药理学;分子对接;凋亡;周期;PI3K/AKT

Abstract:

【】? Objective: To investigate the effects of carvacrol (CV) on the viability, proliferation, apoptosis, and cell cycle of human nasopharyngeal carcinoma CNE2 cells, and to explore the potential mechanisms underlying CV-induced apoptosis and cell cycle arrest in CNE2 cells.??? Methods: Network pharmacology was used to predict the potential targets of CV in nasopharyngeal carcinoma. KEGG pathway enrichment analysis was performed on these potential targets using R software. Molecular docking of CV with target proteins was conducted on the CB-Dock2 platform to predict binding affinity. Molecular dynamics simulations were carried out using Gromacs2022.3 software to assess the stability of the interaction between CV and the target proteins.After treating CNE2 cells with 0 (control group), 10, 20, 30, 40, 50, and 60 μg/mL CV for 48 hours, the effect of CV on the cell viability was assessed using the CCK-8 assay.After treating CNE2 cells with 0 (control group), 30, 40, and 50 μg/mL CV for 48 hours, colony formation assays and flow cytometry were used to detect the effects of CV on CNE2 cell proliferation, apoptosis, and cell cycle.The expression levels of PIK3CA, p-PI3K, p-AKT, Bcl-2, Bax, Caspase-3, and CDK2 proteins in CNE2 cells were detected by Western blotting to evaluate the effects of CV.?? Results: Network pharmacology predictions indicated that CV affects nasopharyngeal carcinoma through 137 common targets. KEGG pathway enrichment analysis suggested that CV may involve several signaling pathways, including PI3K/AKT. Molecular docking results revealed that PI3K, AKT, PIK3CA, Bcl-2, Caspase-3, and CDK2 exhibit strong binding affinity with CV. Molecular dynamics simulations further demonstrated that CV forms a stable binding with CDK2. CCK-8 assay results showed that, compared to the control group, the cell viability of CNE2 cells significantly decreased with increasing CV concentrations. Compared to the control group, CV treatment reduced the proliferative capacity of CNE2 cells, increased their apoptosis rate, extended the G0/G1 phase of the cell cycle, and notably decreased the expression levels of PIK3CA, p-PI3K, p-AKT, Bcl-2, and CDK2 proteins. In contrast, the expression levels of Bax and Caspase-3 proteins were significantly elevated. All differences were statistically significant (P < 0.05).?? Conclusion: CV inhibits the viability and proliferation of CNE2 cells, induces apoptosis, and causes cell cycle arrest at the G0/G1 phase. The apoptosis and cell cycle arrest induced by CV in CNE2 cells may be associated with a reduction in the expression of PIK3CA, p-PI3K, p-AKT, Bcl-2, and CDK2 proteins, as well as an increase in the expression of Bax and Caspase-3 proteins, which are related to the PI3K/AKT signaling pathway.

Key words:Carvacrol; Nasopharyngeal Carcinoma; Network Pharmacology; Molecular Docking; Apoptosis; Cycle; PI3K/AKT