甘草甜素体外诱导肝癌细胞MHCC97-H自噬性死亡的实验

Glycyrrhizin induces autophagic cell death in MHCC97-H cell line in vitro

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）检测白噬相关蛋白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。2mmol／L浓度的甘草甜素处理细胞48h，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（176．74-14．5比410．0±32．1），细胞侵袭迁移率较低（g1．0％±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。

Objective 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 concen- tration and time were selected with 50% cell viability. MHCC97-H cell plate clone formation assay and inva- sion-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 sig- nificantly 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 en- hanced 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 he observed in MHCC97-H ceils 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.