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  

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

作　　者：周慧 张雨晴 甘超 范喜瑞 戚之琳[1,2] 齐世美[1,2] ZHOU Hui;ZHANG Yuqing;GAN Chao;FAN Xirui;QI Zhilin;QI Shimei(Key Laboratory of Biologically Active Biomacromolecules,Wannan Medical College,Wuhu 241002,China;Department of Biochemistry and Molecular Biology,Wannan Medical College,Wuhu 241002,China)

机构地区：[1]皖南医学院活性生物大分子重点实验室,安徽芜湖241002 [2]皖南医学院生物化学与分子生物学教研室,安徽芜湖241002

出　　处：《南方医科大学学报》2023年第3期412-419,共8页Journal of Southern Medical University

基　　金：安徽省大学生创新创业训练计划(S202110368008,201910368011,201910368022);安徽高校自然科学研究项目重大项目(KJ2019ZD31,KJ2020ZD54);皖南医学院重点科研项目培育基金(WK2018Z09);活性生物大分子研究安徽省重点实验室项目(1306C083008)。

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、300μg/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、300μg/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、10μmol/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200μg/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)(30μmol/L)或圣草次苷(100、200、300μg/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50μg/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300μg/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300μg/mL刺激30 min时ROS表达水平较高,NAC(30μmol/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。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 Nacetyl-cysteine(NAC,30μmol/L)and eriocitrin(100,200,and 300μg/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 50μg/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 300μg/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 hepatocell

关 键 词：圣草次苷 SMMC-7721 ROS MAPKS 

分 类 号：R285[医药卫生—中药学]