胃肠安诱导胃癌MKN45细胞自噬的机制

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。

Mechanism of Weichang' an on Autophagy of MKN45 Cells

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.