摘要
目的:观察健脾复方胃肠安对人胃癌MKN45细胞自噬的影响,探讨其抗肿瘤的作用机制。方法:体外培养人胃癌MKN45细胞,采用细胞活力检测法(CCK-8)检测不同质量浓度胃肠安(250,500,1000,2000 mg·L^-1)作用体外培养的人胃癌细胞MKN45细胞,共孵育24,48,72 h,检测细胞增殖活力的改变;采用吖啶橙(AO)染色和单丹磺胺戊二胺(MDC)染色观察胃肠安对胃癌MKN45细胞自噬小体及自噬囊泡的变化;实时荧光定量聚合酶链式反应(Real-time PCR)和蛋白免疫印迹法(Western blot)分别检测微管相关蛋白1轻链3(LC3),酵母ATG6同源物(Beclin1),泛素结合蛋白-1(p62),自噬相关基因5(ATG5),自噬相关基因7(ATG7)mRNA和蛋白的表达。结果:CCK-8实验显示,与空白组比较,胃肠安(500,1000,2000 mg·L^-1)可明显抑制MKN45细胞的增殖能力(P<0.05,P<0.01);AO和MDC染色显示,与空白组比较,随着胃肠安浓度的增加,自噬小体和自噬囊泡含量逐渐增加;Real-time PCR和Western blot结果显示,与空白组比较,胃肠安(1000 mg·L^-1)组细胞中自噬相关蛋白LC3-Ⅱ,Beclin1,ATG5,ATG7 mRNA和蛋白表达升高(P<0.05),p62表达下降(P<0.05,P<0.01)。结论:胃肠安可诱导人胃癌MKN45细胞自噬,其机制涉及上调自噬相关基因Beclin1,ATG5,ATG7 mRNA和蛋白表达,下调p62 mRNA和蛋白表达,促进自噬标志蛋白LC3-I向LC3-Ⅱ转化。
Objective:To explore the effect of Weichang’an(WCA)on the autophagy of human gastric cancer MKN45 cells and its possible anti-cancer mechanism.Method:MKN45 cells were cultured in vitro and incubated with different concentrations(250,500,1000,2000 mg·L^-1)of WCA for 24,48,72 h.Cell counting kit-8(CCK-8)assay was used to detect the cell proliferation.AO/EB Dyeing(AO)staining and monodansylcadaverin(MDC)staining were used to observe the changes of the effect of WCA on autophagosome and autophagic vesicles in gastric cancer MKN45 cells at 48 h.Real-time polymerase chain reaction(Real-time PCR)and Western blot were used to detect microtubule-associated protein 1 light chain 3(LC3),Beclin1,sequestosome1(p62),human autophagy-related gene 5(ATG5),human autophagy-related gene 7(ATG7)mRNA and protein expression levels.Result:WCA showed a dose-and-time-dependent growth inhibition at the concentration above 1000 mg·L^-1.Compared with the blank group,WCA(500,1000,2000 mg·L^-1)significantly inhibited the proliferation of MKN45 cells(P<0.05,P<0.01).AO staining and MDC staining showed that autophagosomes and autophagic sacs increased with the rise of WCA concentration compared with the blank group.Real-time PCR and Western blot showed that the expressions of autophagy-related proteins LC3-Ⅱ,Beclin1,ATG5,ATG7,mRNA and protein increased gradually after WCA(1000 mg·L^-1)intervention,while p62 expression decreased(P<0.05,P<0.01).Conclusion:WCA induces the autophagy of human gastric cancer MKN45 cells in a time-and dose-dependent manner in vitro,which may be related to the up-regulation of LC3-Ⅱ,Beclin1,ATG5 and ATG7 as well as the down-regulation of p62 and LC3-Ⅰ.
作者
李朝燕
陈伟霞
秦梦梦
李佳
徐燕
赵爱光
LI Zhao-yan;CHEN Wei-xia;QIN Meng-meng;LI Jia;XU Yan;ZHAO Ai-guang(Longhua Hospital,Shanghai University of Traditional Chinese Medicine(TCM),Shanghai 200032,China;Henan Province Hospital of TCM,Zhengzhou 450002,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2020年第1期71-77,共7页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家重点研发计划项目(2017YFC1700605)
国家“重大新药创制”科技重大专项(2017ZX09304-001)
国家中医临床研究基地业务建设科研专项(JDZX2015068)
上海中医药大学附属龙华医院龙医学者(育苗计划)项目(LYTD-44)
关键词
健脾
胃肠安
胃癌
自噬
MKN45细胞
invigorating spleen
Weichang’an
gastric cancer
autophagy
MKN45 cells
作者简介
第一作者:李朝燕,博士,主治医师,从事中西医结合防治消化道恶性肿瘤研究,E-mail:lizhaoyan2008@hotmail.com;通信作者:赵爱光,博士,主任医师,从事中西医结合防治消化道恶性肿瘤研究,E-mail:aiguangzhao@qq.com。
