基于网络药理学及体外实验探讨过氧麦角甾醇对非小细胞肺癌A549细胞的作用机制  

Mechanism of ergosterol peroxide on non-small cell lung cancer A549 cells based on network pharmacology and in vitro experiment

作　　者：任文康 罗然 王璐 郭晓珊 林宇[1] 卜明 REN Wen-kang;LUO Ran;WANG Lu;GUO Xiao-shan;LIN Yu;BU Ming(College of Pharmacy,Qiqihar Medical Institute,Qiqihar Heilongjiang 161006)

机构地区：[1]齐齐哈尔医学院药学院,黑龙江齐齐哈尔161006

出　　处：《中南药学》2024年第11期2871-2879,共9页Central South Pharmacy

基　　金：齐齐哈尔医学院研究生创新基金项目(No.QYYCX2022-31);黑龙江省自然科学基金联合引导项目(No.LH2022H113)。

摘　　要：目的基于网络药理学结合体外实验,探究过氧麦角甾醇(EP)对人非小细胞肺癌A549细胞凋亡的作用,并分析其可能机制。方法采用网络药理学对EP治疗人非小细胞肺癌的关键靶点进行筛选,并通过Metascape数据库对其关键靶点进行GO和KEGG通路富集分析;用0(空白对照)、2.5、5、10、20、40、80μmol·L^(-1)的EP作用于A549细胞24、48和72 h,采用MTT法检测各组细胞的增殖活性。以0(空白对照)、10、20、40μmol·L^(-1)的EP作用于A549细胞48 h后,AO/EB染色法和Annexin V-FITC/PI双染法检测各组细胞的凋亡率;JC-1染色法观察A549细胞的线粒体膜电位变化;DCFH-DA染色法观察细胞的活性氧水平。Western blot法检测各组细胞PI3K/AKT/mTOR信号通路相关蛋白表达变化。结果经网络药理学预测,得出EP治疗人非小细胞肺癌存在170个共有靶点,GO富集分析结果显示EP治疗人非小细胞肺癌主要涉及的生物过程有蛋白质的磷酸化、磷酸化的正向调节、磷酸转移酶活性、蛋白丝氨酸激酶活性等,KEGG富集分析结果显示EP治疗人非小细胞肺癌的前列腺癌信号通路、人乳头状瘤病毒感染信号通路、PI3K-AKT信号通路等;MTT结果显示EP显著抑制A549细胞增殖,呈现时间和剂量相关性;与空白对照组相比,经EP作用后A549细胞的总凋亡率、活性氧水平显著升高,线粒体的膜电位显著下降;此外,p-PI3K、p-AKT、p-mTOR蛋白表达水平显著降低(P<0.05)。结论EP可抑制人非小细胞肺癌A549细胞增殖,并诱导其凋亡,其作用机制可能与PI3K/AKT/mTOR通路介导线粒体凋亡途径有关。Objective To determine the effect of ergosterol peroxide(EP)on the apoptosis of human nonsmall cell lung cancer A549 cells based on network pharmacology and in vitro experiment,and related mechanism.Methods The key targets of EP treatment in human non-small cells were screened by network pharmacology.The GO and KEGG pathways were enriched by Metascape database.A549 cells were treated with 0(the control),2.5,5,10,20,40,and 80μmol·L^(-1) EP for 24,48 and 72 h,and the proliferation of the cells was detected by MTT.A549 cells were treated with 0(control),10,20 and 40μmol·L^(-1) EP for 48 h,and the apoptosis rate was detected by AO/EB staining and Annexin V-FITC/PI double staining.The changes in mitochondrial membrane potential in A549 cells were observed by JC-1 staining.The reactive oxygen species levels were observed by DCFH-DA staining.The expression of PI3K/AKT/mTOR signaling pathway was detected by Western blot.Results There were 170 common targets in EP treatment of human non-small cell lung cancer predicted by network pharmacology.GO enrichment analysis showed that EP treatment of human non-small cell lung cancer mainly involved biological processes such as protein phosphorylation,positive regulation of phosphorylation,phosphotransferase activity,and protein serine kinase activity,etc.KEGG enrichment analysis showed that EP treated human non-small cell lung cancer related pathways included prostate cancer signaling pathway,human papillomavirus infection signaling pathway,PI3K-AKT signaling pathway,etc.MTT showed that EP obviously inhibited A549 cell proliferation in a time-and dose-dependent manner.Compared with the blank control group,the total apoptosis rate and reactive oxygen species levels of A549 cells were significantly increased after the EP treatment,while the membrane potential of the mitochondria was much decreased.In addition,the protein expression levels of p-PI3K,p-AKT and p-mTOR were significantly decreased(P<0.05).Conclusion EP can inhibit the proliferation and induce the apoptosis of human

关 键 词：过氧麦角甾醇 非小细胞肺癌 A549细胞 网络药理学 细胞凋亡 活性氧 线粒体膜电位 

分 类 号：R286[医药卫生—中药学]