荷叶碱抑制Akt/mTOR/4EBP1-糖酵解通路抗胆管癌细胞增殖作用研究

Effect of nuciferine against the proliferation of cholangiocarcinoma cells through Akt/mTOR/4EBP1-glycolytic pathway

本研究基于网络药理学探究荷叶碱抑制人肝内胆管癌细胞HuCCT1增殖的分子机制。采用MTT比色法、克隆形成实验检测荷叶碱对HuCCT1细胞增殖的影响;采用比色法、分光光度法检测荷叶碱对糖酵解生化指标的影响;通过网络构建和富集分析预测荷叶碱作用于肝内胆管癌的潜在靶点及通路,以此为基础探究荷叶碱影响HuCCT1细胞增殖的可能机制。结果表明荷叶碱能抑制HuCCT1增殖,显著降低细胞葡萄糖消耗及乳酸生成,并呈时间和剂量依赖性;网络药理学分析显示荷叶碱主要通过PI3K/Akt、MAPK、HIF-1信号通路影响肝内胆管癌生长、运动;聚合酶链式反应(PCR)和蛋白免疫印迹(Western blot)结果显示(30、60、120μmol/L)荷叶碱能降低低氧诱导因子-1α(HIF-1α)、己糖激酶2(HK2)和丙酮酸激酶2(PKM2)的mRNA水平和蛋白表达,同时下调p-Akt^(Thr308)、p-mTOR、p-4EBP1蛋白表达。综上,荷叶碱可能通过影响Akt/mTOR/4EBP1信号级联调控的糖酵解活性抑制HuCCT1细胞增殖。

The study explores the molecular mechanism of nuciferine in inhibiting the proliferation of human intrahepatic cholangiocarcinoma cell HuCCT1 by network pharmacology.The MTT colorimetric and clone formation assay was used to measure the proliferation activity of HuCCT1 cells.After nuciferine treatment,the glycolytic biochemical indexes were detected by colorimetric and spectrophotometric assay.The potential targets and pathways of nuciferine in intrahepatic cholangiocarcinoma were predicted by constructing a network and the enrichment analysis results.And further investigation of nuciferine inhibiting HuCCT1 cell proliferation is being explored based on it.It is shown that nuciferine improved HuCCT1 proliferation in a time-and dose-dependent manner and significantly reduced cellular glucose consumption and lactate production.Network pharmacological analysis indicated that nuciferine affected intrahepatic cholangiocarcinoma growth and motility mainly through PI3K/Akt,MAPK,and HIF-1 signaling pathways.Data of polymerase chain reaction(PCR)and Western blot demonstrated that nuciferine was able to downregulate the mRNA and protein expression of hypoxia-inducible factor-1α(HIF-1α),hexokinase 2(HK2),pyruvate kinase 2(PKM2),as well as protein expression of p-Akt ^(Thr308),p-mTOR and p-4EBP1.In conclusion,nuciferine may inhibit the proliferation of HuCCT1 cells through the adjustment of glycolytic activity regulated by the Akt/mTOR/4EBP1 signaling cascade.