摘要
目的探讨刺梨果多糖对肺腺癌A549细胞增殖、凋亡的作用及机制。方法在0、2、4、6、8、10 g·L^(-1)刺梨果多糖干预A549细胞24、48、72 h后,采用CCK-8法检测A549细胞增殖情况。在0、2、6、10 g·L^(-1)刺梨果多糖干预A549细胞24、48 h后,采用细胞划痕实验测定A549细胞的迁移情况;Transwell实验测定A549细胞侵袭情况。在0、2、6、10 g·L^(-1)刺梨果多糖干预A549细胞48 h后,采用流式细胞术(PI单染法)测定A549细胞周期;流式细胞术(Annexin V-FITC/PI双染法)测定A549细胞的凋亡情况;Real-Time PCR法测定A549细胞的周期、凋亡相关基因表达水平;Western Blot法测定A549细胞的周期、凋亡相关蛋白表达水平。结果(1)干预24、48、72 h后,与对照组(0 g·L^(-1))比较,刺梨果多糖2、4、6、8、10 g·L^(-1)组的A549细胞生长受到显著抑制,细胞的存活率显著降低(P<0.05,P<0.01),刺梨果多糖干预48 h的IC50值为6.109 g·L^(-1),后续实验采用2、6、10 g·L^(-1)浓度作为刺梨果多糖低、中、高剂量。(2)干预48 h后,与对照组比较,刺梨果多糖2、6、10 g·L^(-1)组的A549细胞迁移距离显著增加(P<0.05,P<0.01),细胞侵袭数量显著减少(P<0.05,P<0.01);刺梨果多糖6、10 g·L^(-1)组的G0/G1期、G2/M期细胞比例均显著升高(P<0.05,P<0.01),S期的细胞比例显著降低(P<0.01);刺梨果多糖2、6、10 g·L^(-1)组A549细胞的G0/G1、G2/M期相关调控因子CDK-4、CDK-6、CyclinD1、CDK-1、CyclinB1 mRNA及蛋白表达水平均显著下调(P<0.05,P<0.01);A549细胞的凋亡率均显著升高(P<0.01);凋亡相关基因Caspase-3、Caspase-8、Caspase-9 mRNA表达显著上调(P<0.05,P<0.01);促凋亡Bax、Caspase-3、Caspase-8、Caspase-9蛋白表达显著上调(P<0.05,P<0.01),抗凋亡Bcl-2蛋白表达显著下调(P<0.05,P<0.01)。结论刺梨果多糖能够抑制A549细胞的增殖、迁移和侵袭,阻滞细胞周期于G0/G1和G2/M期,并通过线粒体通路和死亡受体通路诱导A549细胞凋亡。
To investigate the effect and mechanism of Rose roxburghii Tratt.fruits polysaccharide(RTFP)on proliferation and apoptosis of lung adenocarcinoma A549 cells.Methods After A549 cells were treated with 0,2,4,6,8,10 g·L^(-1)RTFP for 24,48,72 hours,the proliferation of A549 cells was detected by CCK-8 method.After A549 cells were treated with 0,2,6 and 10 g·L^(-1)RTFP for 24 and 48 hours,the migration of A549 cells was determined by cell scratch test.Transwell assay was used to determine the invasion of A549 cells.The cell cycle of A549 cells was determined by flow cytometry(PI single staining method)after A549 cells were treated with 0,2,6 and 10 g·L^(-1)RTFP for 48 hours.The apoptosis of A549 cells was detected by flow cytometry(Annexin V-FITC/PI double staining).The expression levels of cell cycle and apoptosis-related genes in A549 cells were determined by Real-Time PCR.The expression levels of cell cycle and apoptosis-related proteins in A549 cells were determined by Western Blot.Results(1)After 24,48 and 72 hours of intervention,compared with the control group(0 g·L^(-1)),the growth of A549 cells in the RTFP 2,4,6,8 and 10 g·L^(-1)groups was significantly inhibited,and the survival rate of cells was significantly decreased(P<0.05,P<0.01).The IC50 value of RTFP intervention for 48 hours was 6.109 g·L^(-1),and the concentration of 2,6 and 10 g·L^(-1)was used as the low-,medium-and high-dose groups of RTFP in subsequent experiments.(2)After 48 hours of intervention,compared with the control group,the migration distance of A549 cells in the RTFP 2,6 and 10 g·L^(-1)groups was significantly increased(P<0.05,P<0.01),and the number of cell invasion was significantly decreased(P<0.05,P<0.01).The proportion of cells in G0/G1 phase and G2/M phase was significantly increased(P<0.05,P<0.01),and the proportion of cells in S phase was significantly decreased(P<0.01)in the 6 and 10 g·L^(-1)groups.The mRNA and protein expressions of G0/G1 and G2/M phase-related regulatory factors CDK-4,CDK-6,CyclinD1,CDK-1 and CyclinB1 of A549 cells in the 2,6 and 10 g·L^(-1)groups of RTFP were significantly increased(P<0.05,P<0.01).The apoptosis rate of A549 cells was significantly increased(P<0.01).The mRNA expressions of apoptosis-related genes Caspase-3,Caspase-8 and Caspase-9 were significantly up-regulated(P<0.05,P<0.01).The protein expressions of pro-apoptotic Bax,Caspase-3,Caspase-8 and Caspase-9 were significantly upregulated(P<0.05,P<0.01),and the protein expressions of anti-apoptotic Bcl-2 was significantly downregulated(P<0.05,P<0.01).Conclusion RTFP could inhibit the proliferation,migration and invasion of A549 cells,block the cell cycle in G0/G1 and G2/M phases,and induce apoptosis of A549 cells through mitochondrial pathway and death receptor pathway.
作者
李倩倩
李自霖
刘涵
黄丽金
王涵
杨紫焰
陈贵元
张翠香
LI Qianqian;LI Zilin;LIU Han;HUANG Lijin;WANG Han;YANG Ziyan;CHEN Guiyuan;ZHANG Cuixiang(School of Basic Medicine,Dali University,Dali 671000 Yunnan,China;Yunnan Provincial Key Laboratory of Insect Biomedicine Research and Development,Dali 671000 Yunnan,China)
出处
《中药新药与临床药理》
CAS
CSCD
北大核心
2024年第4期484-492,共9页
Traditional Chinese Drug Research and Clinical Pharmacology
基金
国家自然科学基金项目(31860252)
云南省教育厅科研基金项目(2021J0337)
云南省昆虫生物医药研发重点实验室2022年度开放项目课题(AG2022006)
云南省自然科学基金高校联合面上项目(2017FH001-084)。
关键词
刺梨果多糖
肺腺癌A549细胞
细胞增殖
迁移
侵袭
细胞周期
细胞凋亡
Rose roxburghi Tratt.fruits polysaccharide e(RTFP)
lung cancer A549 cells
cell proliferation
migration
invasion
cell cycle
apoptosis
