复方苦参注射液调节磷脂酰肌醇3激酶——丝氨酸/苏氨酸激酶信号通路对肺腺癌细胞生物学行为的影响

Mechanism of inhibition of lung adenocarcinoma metastasis by compound kushen injection through regulation of PI3K-AKT signaling pathway

目的探索复方苦参注射液抑制肺腺癌转移的可能的作用机制。方法①利用中药系统药理学数据库与分析平台(TCMSP)数据库、在线人类孟德尔网(OMIM)和Genecard数据库、STRING软件检索复方苦参注射液所含的2种成分苦参与土茯苓的活性成分及对应靶标,及其与肺腺癌重合的潜在靶基因、治疗肺腺癌的作用靶点,并对关键靶点进行基因本体(GO)和京都基因和基因组百科全书(KEGG)富集分析获知其潜在作用机制。②通过MTT比色法、Transwell、细胞划痕、流式细胞术等体外实验检测苦参注射液对人肺腺癌A549细胞增殖、迁移侵袭、凋亡能力的影响;蛋白印迹法检测磷脂酰肌醇3激酶(PI3K)、丝氨酸/苏氨酸激酶(AKT)、哺乳动物雷帕霉素靶蛋白(mTOR)、磷酸化(p)-PI3K、p-AKT、p-mTOR的表达。结果①复方苦参注射液作用于肺腺癌共包含2味中药、58种有效活性成分、127个靶基因,KEGG通路富集分析涉及脂质与动脉粥样硬化、PI3K—AKT信号通路等生物学通路;②体外实验证实复发苦参注射液对肺腺癌A549增殖的抑制作用呈剂量依赖性,并可减弱A549细胞的迁移侵袭能力,增强细胞凋亡率,这一过程可能是通过抑制PI3K—AKT磷酸化水平,进而抑制该通路的促癌作用。结论复方苦参注射液的活性成分大豆抗毒素、芸豆球蛋白、甘草素、刺芒柄花素等,可能通过作用于丝裂原活化蛋白激酶1(MAPK1)、TP53、MAPK3、AKT1、JUN、TNF等靶点,通过PI3K—AKT信号通路调控肿瘤微环境抑制肺腺癌的转移发生。

Objective To explore the possible mechanism of action of compound kushen injection in inhibiting lung adenocarcinoma metastasis.Methods①The active ingredients and corresponding targets of the two ingredients of compound kushen injection,kushen and hyoscyamine,and their potential target genes overlapping with lung adenocarcinoma,and the action targets for the treatment of lung adenocarcinoma were searched using TCMSP database,OMIM and Genecard databases,and STRING software,and GO and KEGG enrichment analyses of key targets were performed to understand their potential mechanisms of action.②The effects of compound kushen injection on the proliferation,migration invasion and apoptosis ability of human lung adenocarcinoma A549 cells were detected by MTT,Transwell,cell scratching and flow cytometry in vitro assays.The expression of PI3K,AKT,mTOR,p-PI3K,p-AKT and p-mTOR were detected by Western blot.Results The action of compound kushen injection on lung adenocarcinoma contained a total of 2 Chinese herbs,58 active ingredients and 127 target genes,and the KEGG pathway enrichment analysis involved biological pathways such as lipid and atherosclerosis,PI3K—AKT signaling pathway.In vitro experiments confirmed that compound kushen injection inhibited the proliferation of lung adenocarcinoma A549 in a dose-dependent manner and attenuated the migratory invasive ability of A549 cells and enhanced the rate of apoptosis,a process that may be through the inhibition of PI3K-AKT phosphorylation levels,which in turn inhibited the pro-cancer effect of this pathway.Conclusion The active ingredients of compound kushen injection,such as soy antitoxin,brassinosteroid,glycyrrhizin,and prickly mangosteen,may inhibit the metastasis occurrence of lung adenocarcinoma by acting on MAPK1,TP53,MAPK3,AKT1,JUN,TNF and other targets,and regulating the tumor microenvironment through PI3K/AKT pathway.