和胃降逆胶囊对胃腺癌细胞凋亡的影响及其机制

Effect of Hewei Jiangni capsules on apoptosis of gastric adenocarcinoma cells and its mechanism

目的以核转录因子-κB/上皮间充质转化(NF-κB/EMT)信号通路为基础,探讨和胃降逆含药血清对胃腺癌AGS细胞增殖和凋亡的影响及作用机制。方法制备不同药物剂量(3,6,12 g/kg)的和胃降逆含药血清,作为含药血清低、中、高剂量组,不含药物的血清作为空白对照组。采用不同药物剂量的和胃降逆含药血清分别干预AGS细胞24,48,72 h后,用CCK-8法观察细胞增殖;DAPI染色法检测细胞凋亡;划痕试验观察细胞迁移;实时定量聚合酶链反应法(RT-qPCR)检测NF-κB、EMT标志物相关蛋白E-钙黏蛋白(E-cadherin)、N-钙黏蛋白(N-cadherin)、波形蛋白(Vimentin)mRNA水平;蛋白免疫印迹法(Western blot)检测细胞内NF-κB、E-cadherin、Vimentin相关信号通路蛋白的表达。结果同一时间点,与空白对照组比较,和胃降逆含药血清组细胞活力降低(P<0.01);同一含药血清剂量组48,72 h与24 h比较细胞活力降低(P<0.05)。干预48 h,与空白对照组比较,不同剂量含药血清组AGS细胞凋亡率剂量依赖性增加(P<0.05)。与空白对照组比较,和胃降逆含药血清组划痕愈合速度均减慢,迁移率降低(P<0.05);干预24 h,与空白对照组比较,高剂量组细胞迁移降低(P<0.01),干预48 h,中、高剂量组细胞迁移率降低(P<0.01)。与空白对照组比较,和胃降逆含药血清组AGS细胞中NF-κB、Vimentin的mRNA水平剂量依赖性下调(P<0.05),E-cadherin的mRNA水平剂量依赖性上调(P<0.01)。与空白对照组比较,和胃降逆含药血清组AGS细胞中NF-κB、Vimentin的蛋白表达剂量依赖性下调,E-cadherin的蛋白表达剂量依赖性上调(P<0.05)。结论和胃降逆胶囊可能通过抑制NF-κB活化、调控下游EMT相关蛋白的表达介导胃腺癌细胞AGS的迁移和侵袭,进而抑制AGS细胞增殖并促进其凋亡。

Objective To observe the effects of serum containing Hewei Jiangni on proliferation and apoptosis of gastric cancer cells(AGS)and its mechanism based on NF-κB/EMT signaling pathway.Methods Serums containing low,medium,and high concentrations of Hewei Jiangni(3,6,and 12 g/kg)were prepared,and the blank serum was used as control group.AGS cells were intervened with serums containing different concentrations of Hewei Jiangni for 24,48,72 h respectively.The proliferation of AGS cells was detected by CCK-8 kit.The apoptosis of AGS cells was detected by DAPI staining.The migration of AGS cells was observed by Scratch test.The mRNA levels of NF-κB and EMT marker proteins E-cadherin and Vimentin were detected by quantitative real-time polymerase chain reaction(RT-qPCR).The protein expressions of NF-κB,E-cadherin,and Vimentin were detected by Western blot.Results At the same time points,compared with control group,the viability of cells was dose-dependently decreased in Hewei Jiangni groups(P<0.01).Compared with 24 h,the viability of AGS cells was decreased in Hewei Jiangni groups at 48,72 h(P<0.05).At 48 h,compared with control group,the apoptosis of AGS cells was dose-dependently increased in Hewei Jiangni groups(P<0.05).Compared with control group,the scratch healing speed was slowed down,and the rate of cell migration was reduced in Hewei Jiangni groups(P<0.05).Compared with control group,the migration was decreased at 24 h in high dose group(P<0.01),and was also reduced at 48 h in medium and high dose groups(P<0.01).Compared with control group,the mRNA levels of NF-κB and Vimentin in AGS cells were dose-dependently downregulated in Hewei Jiangni groups(P<0.05),while E-cadherin mRNA was dose-dependently upregulated(P<0.01).Compared with control group,the protein levels of NF-κB and Vimentin in AGS cells were dose-dependently downregulated while the E-cadherin proein expression was dose-dependently upregulated in Hewei Jiangni groups(P<0.05).Conclusion Hewei Jiangni can mediate the migration and invasion of AGS cells by inhibiting the activation of NF-κB,and regulating the expressions of downstream EMT-related proteins,thus inhibiting the proliferation and promoting the apoptosis of AGS cells.