基于网络药理学及体外实验探讨过氧麦角甾醇对乳腺癌MCF-7细胞的作用机制

Mechanism of ergosterol peroxide on MCF-7 breast cancer cells based on network pharmacology and in vitro experiments

基于网络药理学方法探究过氧麦角甾醇(EP)抗乳腺癌的靶点及作用机制,并通过体外实验验证。采用网络药理学对EP作用靶点进行筛选,构建靶点网络及蛋白-蛋白互作(PPI)网络,对EP抗乳腺癌潜在的作用靶点及相关通路进行预测。采用MTT法检测EP对乳腺癌MCF-7细胞的增殖抑制活性,克隆形成实验检测EP对MCF-7细胞增殖影响;采用流式细胞术和激光共聚焦显微镜检测细胞的凋亡情况、线粒体膜电位及活性氧水平的变化情况;通过Western blot法检测EP对B细胞淋巴瘤(Bcl-2)、Bcl-2相关X蛋白(Bax)、细胞色素C(Cyt C)、半胱氨酸蛋白酶蛋白(caspase)-7、活化的caspase-7(cleaved caspase-7)、磷脂酰肌醇3-激酶(PI3K)、丝氨酸-苏氨酸激酶B(AKT)蛋白表达水平的影响情况。结果显示,经网络药理学预测,得出EP与乳腺癌有173个共同靶点;KEGG富集分析结果显示EP治疗乳腺癌结果主要在癌症通路、PI3K-AKT信号通路、细胞衰老信号通路、病毒致癌作用等信号通路;MTT结果显示,与对照组相比,EP组MCF-7细胞的活性明显降低,呈时间-浓度依赖趋势;EP能够抑制乳腺癌细胞MCF-7集落形成;采用10、20、40μmol·L^(-1)的EP作用MCF-7细胞24 h后,MCF-7细胞总凋亡率均显著升高,线粒体膜电位均显著降低,活性氧水平均显著升高;此外,MCF-7细胞经过EP处理后,细胞内Cyt C、Bax、cleaved caspase-7蛋白的表达水平上升,p-PI3K、p-AKT、Bcl-2蛋白的表达水平下降。研究表明,EP可抑制乳腺癌MCF-7细胞增殖,减少集落形成,其机制可能与PI3K-AKT通路介导线粒体凋亡途径有关。

This study investigated the effects of ergosterol peroxide(EP)on the proliferation and apoptosis of MCF-7 breast cancer cells,explored its possible mechanisms of action,and verified the effects and mechanisms by in vitro experiments.Network pharmaco-logy was used to screen the target proteins of EP and construct target networks and protein-protein interaction(PPI)networks to predict the potential target proteins and related pathways involved in EP anti-breast cancer effects.The MTT assay was performed to measure the inhibitory effect of EP on MCF-7 cell proliferation,and the colony formation assay was used to assess the cell cloning ability.Flow cytometry and laser confocal microscopy were employed to evaluate cell apoptosis,mitochondrial membrane potential and reactive oxygen species(ROS)levels.Western blot analysis was conducted to examine the expression levels of B-cell lymphoma 2(Bcl-2),Bcl-2-associated X protein(Bax),cytochrome C(Cyt C),caspase-7,cleaved caspase-7,phosphatidylinositol 3-kinase(PI3K),and se-rine/threonine kinase B(AKT)in MCF-7 cells treated with EP.The results of network pharmacology prediction yielded 173 common targets between EP and breast cancer;the results of Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis showed that EP treatment for breast cancer mainly affected the signaling pathways such as cancer pathway,PI3K-AKT signaling pathway,cellular senescence signaling pathway,and viral carcinogenesis pathway;and the MTT assay results showed that the viability of MCF-7 cells in the EP group was significantly lower than that in the control group,exhibiting a time-and concentration-dependent trend,and EP can inhibit colony formation of MCF-7 breast cancer cells.Treatment with 10,20,and 40μmol·L^(-1)EP for 24 h resulted in a significant increase in the total apoptosis rate of MCF-7 cells,a significant decrease in mitochondrial membrane potential,and a significant increase in ROS levels.In addition,treatment with EP led to an upregulation of Cyt C,Bax,and cleaved caspase-7 protein expression,and a downregulation of p-PI3K,p-AKT,and Bcl-2 protein expression in MCF-7 cells.Studies have shown that EP inhibits MCF-7 breast cancer cell proliferation and reduces colony formation by a mechanism that may be related to the PI3K-AKT pathway mediating the mitochondrial apoptotic pathway.