摘要
目的探讨吴茱萸碱通过丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路对人胃癌细胞的抑制作用。方法应用MTT法对SGC-7901细胞活性进行评定。以8、16、32μmol/L吴茱萸碱作用胃癌SGC-7901细胞(记为A1组,A2组,A3组),另设置相同浓度吴茱萸碱处理后的细胞,加入p38抑制剂(记为B1组,B2组,B3组),对照组用细胞培养液处理作为空白对照。Annexin V-FITC/PI法观察细胞凋亡,PI染色法观察细胞周期,Western blot检测凋亡相关蛋白Bcl-2、cleaved-caspase-3,划痕愈合实验评价细胞迁移抑制情况,蛋白质印迹法检测c-dc2、cdc25C、cyclin B1及p-ERK、p-p38蛋白水平表达。结果吴茱萸碱能够阻碍SGC-7901细胞增殖,细胞活性随着浓度和时间的增加而降低,24、48 h的IC50分别是16.78、7.28μmol/L。与对照组(0.06%)比较,A组 SGC-7901细胞凋亡比例(18.72%、31.08%、52.17%)增加,G2/M细胞周期阻滞,与A组比较,B组SGC-7901细胞凋亡比例减少,G2/M细胞周期阻滞减弱,G2/M期调控蛋白cdc2、cdc25C、cyclin B1水平下调(P均<0.05)。划痕愈合实验表明SGC-7901细胞的细胞划痕愈合率伴随吴茱萸碱浓度升高而降低;与A组(59.13%、23.15%、18.31%)比较,B组细胞划痕愈合率(63.17%、55.27%、34.08%)升高(P<0.05)。Western blot结果表明吴茱萸碱处理Bcl-2、cleaved-caspase-3水平上调,SGC-7901细胞MAPK通路蛋白p-ERK水平下调,p-p38表达水平上调(P<0.05)。与A组比较,B组cdc2、cdc25C、cyclin B1表达上调,Bcl-2、cleaved-caspase-3水平显著下降,MAPK通路蛋白p-ERK水平上调,p-p38水平下调(P<0.05)。结论吴茱萸碱对胃癌细胞有抑制作用,其机制是通过MAPK信号通路下调p-p38蛋白、上调凋亡蛋白实现。
Objective To investigate the anti-tumor effect of evodiamine on gastric cancer cells via the MAPK signaling pathway.Methods MTT assay was used to assess the cell viability of SGC-7901 cells after the treatment of 8,16 and 32μmol/L evocarpine(denoted as A1 group,A2 group,A3 group).The effect of p38 inhibitor on the above cells(denoted as B1 group,B2 group,B3 group)were studied.The blank control cell group was the cells treated with culture medium.Annexin V-FITC/PI was used to observe cell apoptosis,PI staining to observe cell cycle,Western blotting to measure apoptosis-related proteins Bcl-2 and cleaved-caspase-3,and scratch healing test to evaluate the inhibitory effect on cell migration.Western blotting was employed to detect the expression levels of c-dc2,cdc25C,cyclin B1,and p-ERK and p-p38.Results Evodiamine negatively inhibited the proliferation of SGC-7901 cells in a dose-dependent and time-dependent manner.The IC50 values at 24 and 48 h were 16.78 and 7.28μmol/L,respectively.Compared with the control group(0.06%),the apoptotic rate of SGC-7901 cells was increased in groups A1,A2 and A3(18.72%,31.08%and 52.17%),and the cells arrested at G2/M phase.The proportion of apoptotic cells in group B was decreased,there were less cells arrested at G2/M phase,and the expression of G2/M phase regulatory proteins CDC2,CDC25C and Cyclin B1 were down-regulated when compared with group A(all P<0.05).The scratch healing test showed that the healing rate of SGC-7901 cells was decreased with the increase of evodiamine concentration.Compared with groups A1,A2 and A3(59.13%,23.15%and 18.31%),the rate was increased in groups B1,B2 and B3(63.17%,55.27%,34.08%,P<0.05).Western blot results indicated that evodiamine treatment induced the up-regulation of Bcl-2 and cleaved caspase-3,down-regulation of p-ERK,and obvious elevation of p-p38(P<0.05).Compared with group A,the expression levels of CDC2,CDC25C and Cyclin B1 were increased,those of Bcl-2 and cleaved caspase-3 were significantly decreased,while that of p-ERK was up-regulated and that of p-p38 was down-regulated in group B(P<0.05).Conclusion Evodiamine shows anti-tumor effect on gastric cancer cells,and it is realized by the down-regulation of p-p38 protein and up-regulation of apoptosis-related proteins through MAPK signaling pathway.
作者
千维娜
李治
方瑜
姜伊娜
肖海娟
QIAN Weina;LI Zhi;FANG Yu;JIANG Yina;XIAO Haijuan(Department of Oncology,Affiliated Hospital,Shaanxi University of Traditional Chinese Medicine,Xianyang,Shaanxi Province,712000,China;School of Pharmacy,Shaanxi University of Traditional Chinese Medicine,Xianyang,Shaanxi Province,712000,China;School of Basic Medicine,Shaanxi University of Traditional Chinese Medicine,Xianyang,Shaanxi Province,712000,China)
出处
《第三军医大学学报》
CAS
CSCD
北大核心
2021年第16期1535-1542,共8页
Journal of Third Military Medical University
基金
国家自然科学基金面上项目(82074255)
陕西省自然科学基础研究计划项目(2020JQ-859)。
