摘要目的 观察甘草甜素在体外对肝癌细胞MHCC97-H的抑制作用,探索其相关机制.方法 体外培养肝癌细胞MHCC97-H,加入不同浓度的甘草甜素,观察不同时间点细胞存活率的变化,摸索50％细胞存活的处理浓度和时间.建立细胞平板克隆和侵袭迁移实验模型,观察甘草甜素处理对MHCC97-H细胞增殖和侵袭迁移能力的影响.对培养细胞进行吖啶橙染色,观察自噬小体的形成情况,并利用自噬抑制剂3-MA和自噬关键基因Atg7-siRNA抑制细胞自噬活性,检测细胞存活率.利用蛋白印迹(Western-blot)检测自噬相关蛋白LC3B的变化以及信号通路mTOR和ERK1/2的活化情况.结果 甘草甜素对MHCC97-H细胞活性具有显著抑制作用.2 mmol/L浓度的甘草甜素处理细胞48 h,细胞存活率约为50％.甘草甜素处理组细胞克隆球数量明显少于未处理组(176.7±14.5比410.0±32.1),细胞侵袭迁移率较低(41.0％±3.8％比100％).甘草甜素处理细胞胞质中自噬小体形成明显增加,LC3B-Ⅱ表达增强,LC3B-Ⅱ/Ⅰ比值增大,P62降解加快.同时,可检测到p-mTOR表达减少和p-ERK1/2表达增加.自噬抑制后,细胞存活率高于对照组(3-MA:64.3％比45.9％;Atg7-siRNA:67.7％比47.1％).结论 甘草甜素具有增强肝癌细胞自噬活性,诱导细胞自噬性死亡的能力,具备极大的临床应用潜力.

abstractsObjective To investigate the inhibitory effect of Glycyrrhizin in MHCC97-H cell line in vitro and explore the relevant mechanism.Methods MHCC97-H cells were cultured in vitro and treated with Glycyrrhizin in different concentrations and then cell viability was assayed at different time points.The concentration and time were selected with 50％ cell viability.MHCC97-H cell plate clone formation assay and invasion-migration experiment were also performed to study the tumor-suppressor efficacy of Glycyrrhizin.Acridine orange staining was used to evaluate the formation of autophagic vacuoles.Meanwhile,3-MA and Atg7-siRNA were both employed to avoid the autophagy activation in MHCC97-H cells and cell viability was reassessed.Western-blot was carried out to study the expression of autophagic proteins of LC3B,p-mTOR and p-ERK1/2.Results It showed Glycyrrhizin significantly inhibited MHCC97-H cell viability and the concentration and time at 50％ cell viability were 2 mmol/L and 48 h respectively.Clone number in Glycyrrhizin group was significantly smaller than that in the control group (176.7 ± 14.5 vs.410.0 ± 32.1).Invasion-migration rate was also lower in Glycyrrhizin group compared with the control group (41.0％ ±3.8％ vs.100％).Autophagic vacuoles was increased in MHCC97-H cells when treated with Glycyrrhizin and expression of LC3B-Ⅱ was enhanced and LC3B-Ⅱ/I Ratio was increased,at the same time degradation of P62 was accelerated.Reduced p-mTOR in concurrence with upregulated p-ERK1/2 could be observed in MHCC97-H cells administered with Glycyrrhi-zin.Cell groups additionally treated with 3-MA or Atg7-siRNA exerted higher cell viability (64.3％ vs.45.9％ and 67.7％ vs.47.1％,respectively).Conclusion Glycyrrhizin can induce excessive autophagy in hepatocellular carcinoma cells to cause autophagic cell death and exhibit great potential in clinical application.