多伞阿魏体外抗胃癌活性筛选、细胞凋亡及周期阻滞机制

Mechanism of Uygur Medicine Root of Ferula ferulaeoides in Resisting Gastric Cancer Activity and Inducing Cell Apoptosis and Cell Cycle Arrest in Vitro

目的：研究确定新疆特色维族药多伞阿魏体外抗胃癌活性部位及其敏感胃癌细胞系,并探讨多伞阿魏诱导胃癌细胞凋亡和细胞周期阻滞情况,为其进一步在抗胃癌方面的研究、开发与应用提供实验依据。方法：采用四甲基偶氮唑盐（MTT）比色法检测不同质量浓度多伞阿魏不同提取物（挥发油,95%乙醇提取物,及其石油醚部位,三氯甲烷部位,乙酸乙酯部位,正丁醇部位,水部位）分别对5种胃癌细胞系（AGS,MKN-45,BGC-823,MGC-803,SGC-7901）的增殖抑制作用;采用Hoechst33258荧光染色法观察多伞阿魏挥发油和三氯甲烷部位对胃癌细胞AGS和SGC-7901的影响情况;并应用细胞流式仪检测多伞阿魏不同提取部位作用胃癌细胞AGS和SGC-7901的凋亡和周期阻滞影响情况。结果：与空白组比较,多伞阿魏挥发油,95%乙醇提取物,石油醚部位,三氯甲烷部位,乙酸乙酯部位对5种胃癌细胞均有不同程度的增殖抑制作用（P〈0.05）,并呈现浓度依赖关系。挥发油对胃癌细胞AGS呈现出较强的增殖抑制作用,其半数抑制浓度（IC50）为（7.98±2.62）mg·L^-1,三氯甲烷部位对5种胃癌细胞系均具有较好的敏感性,对胃癌细胞SGC-7901最为敏感,其IC50为（8.73±0.55）mg·L^-1,而正丁醇部位和水部位未呈现出明显的细胞增殖抑制作用;多伞阿魏挥发油和三氯甲烷部位作用胃癌细胞AGS和SGC-7901后,细胞核被Hoechst33258染色呈亮蓝色,且随着药物浓度的增加蓝色荧光越强;流式细胞凋亡检测结果显示,多伞阿魏不同提取部位诱导胃癌细胞AGS和SGC-7901发生不同程度的凋亡（P〈0.05）,且随着药物浓度的增加,细胞总凋亡率明显增高;流式细胞周期检测结果显示,与空白组比较,多伞阿魏挥发油使胃癌细胞AGS的周期发生明显改变,细胞休眠期/DNA复制前期（G0/G1期）细胞比例增高,DNA复制期（S期）细胞比例降低,DNA复制后期/有丝分裂期（G2/M期）比例降低（P〈0.05）;与空白组比较,多伞阿魏三氯甲烷部位使胃癌细胞SGC-7901的周期也发生显著改变,G0/G1期细胞比例降低,S期细胞比例增高,G2/M期比例降低（P〈0.05）。结论：多伞阿魏挥发油对胃癌细胞AGS呈现出较强的细胞毒活性,三氯甲烷部位对5种胃癌细胞均具有较好的增殖抑制作用,尤其对胃癌细胞SGC-7901最为敏感;多伞阿魏各提取部位诱导胃癌细胞AGS和SGC-7901主要发生晚期凋亡,而多伞阿魏乙酸乙酯部位诱导胃癌细胞AGS主要发生细胞早期凋亡;多伞阿魏挥发油能够将胃癌细胞AGS阻滞于G0/G1期,阻止细胞进入S期及G2/M期;伞阿魏三氯甲烷部位将胃癌SGC-7901细胞周期阻滞于S期。研究表明多伞阿魏挥发油和三氯甲烷部位具有较好的抗胃癌活性作用,具有潜在的研究价值和开发利用空间,并为多伞阿魏体内抗胃癌及其抗胃癌机制研究提供了一定的科学依据。

Objective：To study and determined the active fraction of Uygur medicine Ferula ferulaeoides and its sensitive gastric cancer cell line in vitro,and explore the effect of F.ferulaeoides in inducing the apoptosis and cell cycle arrest of gastric cancer cells,in order to provide experimental basis for further studies.Method：The3-（4,5）-dimethylthiahiazo（-z-yl）-3,5-di-phenytetrazoliumromide（MTT） method was used to detect the inhibition effects of different extracts（with volatile oil,95% ethanol extract,petroleum ether,chloroform,ethyl acetate,n-butanol,and water fraction） from F.ferulaeoides on 5 kinds of gastric cancer cell lines（AGS,MKN-45,BGC-823,MGC-803 and SGC-7901）,Hoechst33258 fluorescent staining was used to observe the effect of the volatile oil and the chloroform fraction of F.ferulaeoides on the gastric cancer cell lines AGS and SGC-7901,the effects of different extract parts of F.ferulaeoides on the apoptosis of gastric cancer cell line SGC-7901 were detected by flow cytometry,the effect of volatile oil and chloroform fraction of F.ferulaeoides on cell cycle arrest of gastric cancer cell lines AGS and SGC-7901 were detected by flow cytometry.Result：Volatile oil,95% ethanol extract and its petroleum ether,chloroform and ethyl acetate fraction on 5 kinds of gastric cancer cells had different proliferation inhibition effects（P〈0.05） in a dose-dependent manner.The chloroform fraction had a good sensitivity to the five kinds of gastric cancer cell lines,with the highest sensitivity in gastric cancer cell lines SGS-7901,and the median inhibitory concentration（IC50） was（8.73±0.55） mg·L-1,and the volatile oil had a strong inhibitory effect on gastric cancer cell AGS,and IC50 was（7.98±2.62） mg·L-1.The n-butanol and water fractions did not show a significant inhibition effect on cell proliferation,the cell nucleus of gastric cancer cell lines AGS and SGC-7901 was stained by Hoechst 33258,and the blue fluorescence was stronger with the increase of drug concentration.The results of flow cytometry showed that different extract parts of F.ferulaeoides induced the apoptosis of gastric cancer cells AGS and SGC-7901 to varying degrees,and the total apoptotic rate was significantly increased with the increase of drug concentration.The results of flow cytometry showed that the AGS cycle of gastric cancer cells significantly changed,the proportion of G0/G1-phase cells increased,while the proportion of S phase cells decreased,the proportion of G2/M phase decreased compared with control group（P〈0.05）.The cell cycle of SGC-7901 significantly changed too,while the proportion of G0/G1 phase cells was decreased,the proportion of S phase cells increased,and the ratio of G2/M phase（P〈0.05）,with significant differences compared with the control group（P〈0.05）.Conclusion：The volatile oil of F.ferulaeoides had a high inhibitory effect on the growth of gastric cancer cell SGC-7901,the chloroform fraction of F.ferulaeoides had a high inhibitory effect on the growth of five kinds of gastric cancer cells,especially for gastric cancer cell SGC-7901,late apoptosis of gastric cancer cells AGS and SGC-7901 was induced by different extract parts of F.ferulaeoides,while early apoptosis of gastric cancer cells AGS was induced by the ethyl acetate fraction of F.ferulaeoides.The cell cycle of gastric cancer cell line SGC-7901 was arrested in S phase and G2/M phase.The cell cycle of gastric cancer SGC-7901 was arrested in S phase.The results show that the volatile oil and the chloroform fraction of F.ferulaeoides had a good anti-gastric cancer activity,and provide scientific basis for further studies on the anti-gastric cancer mechanism of F.ferulaeoides in vivo.