南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (3): 412-419.doi: 10.12122/j.issn.1673-4254.2023.03.11

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圣草次苷抑制肝细胞癌SMMC-7721细胞的增殖和迁移：基于激活ROS/MAPKs信号轴

周 慧，张雨晴，甘 超，范喜瑞，戚之琳，齐世美   

  1. 皖南医学院活性生物大分子重点实验室，生物化学与分子生物学教研室，安徽 芜湖 241002
  • 出版日期:2023-03-20 发布日期:2023-03-20

Eriocitrin suppresses proliferation and migration of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPK pathway

ZHOU Hui, ZHANG Yuqing, GAN Chao, FAN Xirui, QI Zhilin, QI Shimei   

  1. Key Laboratory of Biologically Active Biomacromolecules, Department of Biochemistry and Molecular Biology, Wannan Medical College, Wuhu 241002, China
  • Online:2023-03-20 Published:2023-03-20

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摘要： 目的 探讨圣草次苷通过ROS/MAPKs信号轴调控肝癌SMMC-7721细胞增殖和迁移的作用机制。方法 分别用0、25、50、100、150、200、250、300 μg/mL圣草次苷处理肝癌SMMC-7721细胞系24 h后，CCK-8法检测细胞活性；不同浓度的圣草次苷（100、200、300 μg/mL）处理细胞后，运用划痕实验分别在24、48、72 h检测划痕愈合率，Transwell实验检测细胞迁移率，克隆形成实验检测细胞增殖能力，DAPI染色观察细胞核形态变化，Western blot检测侵袭蛋白E-cadherin、N-cadherin、MMP-2、MMP-9、凋亡因子 PARP 和 Pro-caspase 3 以及 MAPKs 通路 p-JNK、p-P38、p-ERK 信号分子；用（分别为 2、5、10 μmol/L）ERK 抑制剂U0126、JNK抑制剂SB203580和P38抑制剂SP600125预先处理细胞2 h，再用200 μg/mL圣草次苷处理细胞24 h，Western blot检测凋亡蛋白PARP的切割情况；分别用N-乙酰-半胱氨酸（NAC）（30 μmol/L）或圣草次苷（100、200、300 μg/mL）单独或共处理细胞后，通过DCFH-DA荧光探针法分别在15、30、60 min检测内源性活性氧（ROS）水平。结果 圣草次苷在50 μg/mL范围内，对细胞活力基本无影响（P>0.05）；圣草次苷能够显著抑制SMMC-7721细胞的划痕愈合（P<0.01），细胞迁移（P<0.01）和克隆形成（P<0.01），在300 μg/mL浓度时作用最显著；圣草次苷明显减少侵袭蛋白N-cadherin、MMP-2、MMP-9的蛋白表达量（P<0.01），上调E-cadherin的蛋白表达量（P<0.05）；圣草次苷诱导SMMC-7721细胞发生显著的凋亡形态学变化，Pro-caspase 3表达降低，PARP切割增多（P<0.01）；圣草次苷在300 μg/mL刺激30 min时ROS表达水平较高，NAC（30 μmol/L）处理后，细胞内ROS表达水平下降明显；圣草次苷能够显著诱导MAPKs信号途径JNK、P38和ERK的磷酸化（P<0.01），U0126 和SB203580能够增强圣草次苷诱导的PAPR切割，而SP600125逆转圣草次苷诱导的PARP切割。结论 圣草次苷通过促进细胞内ROS的合成，诱导MAPKs信号通路的活化，调控肝癌细胞系SMMC-7721的增殖、迁移和凋亡。

关键词: 圣草次苷；SMMC-7721；ROS；MAPKs

Abstract: Objective To investigate the role of the ROS/MAPK signaling axis in mediating the inhibitory effect of eriocitrin on proliferation and migration of hepatocellular carcinoma SMMC-7721 cells. Methods SMMC-7721 cells were treated with different concentrations of eriocitrin for 24 h, and the changes in cell viability were detected with CCK-8 assay. The migration and invasion abilities of the treated cells were evaluated using Transwell and scratch healing assays, the cell proliferation was assessed with colony- forming assay, and changes in nuclear morphology were observed with DAPI staining. Western blotting was performed to examine the changes in the expressions of E-cadherin, N-cadherin, MMP-2, MMP-9, PARP, Pro-caspase 3, p-JNK, p-P38, and p-ERK. The effect of eriocitrin on PARP cleavage in SMMC-7721 cells pretreated with ERK, JNK and P38 inhibitors (U0126, SB203580 and SP600125, respectively) was detected using Western blotting. The effect of treatment with N-acetyl-cysteine (NAC, 30 μmol/L) and eriocitrin (100, 200, and 300 μg/mL), alone or in combination, on reactive oxygen species (ROS) levels in the cells was examined using a DCFH-DA fluorescent probe. Results Eriocitrin below 50 μg/mL did not produce significant effect on the viability of SMMC-7721 cells (P>0.05). Treatment with eriocitrin significantly inhibited scratch healing, migration, and colony formation of the cells (P<0.01), reduced the protein expressions of N-cadherin, MMP-2, and MMP-9 (P<0.01), and up-regulated E-cadherin protein expression (P<0.05). Eriocitrin-treated SMMC-7721 cells showed obvious apoptotic morphologies with decreased Pro- caspase 3 expression and increased PARP cleavage (P<0.01) and phosphorylation levels of JNK, P38, and ERK (P<0.01); Eriocitrin-induced PAPR cleavage was obviously enhanced by U0126 and SB203580 but attenuated by SP600125. Treatment with 300 μg/mL eriocitrin for 30 min significantly increased ROS level in the cells, and this effect was obviously suppressed by NAC. Conclusion Eriocitrin can suppress the proliferation and migration and promote apoptosis of hepatocellular carcinoma SMMC-7721 cells by promoting ROS production and activating the MAPKs signaling pathway.

Key words: eriocitrin; SMMC-7721; reactive oxygen species; MAPKs