瑞香素对胃癌细胞的抑制作用及其机制

Inhibitory effect of daphnetin on gastric cancer cells and its mechanism

目的探讨瑞香素(Dap)对胃癌细胞的抑制作用及其机制。方法通过MTT实验检测浓度为0、30、60、90、120、150μmol/L的Dap处理胃癌AGS细胞48 h后对细胞存活率的影响。将AGS细胞分为A~E组,分别用0、30、60、90、120、150μmol/L的Dap培养48 h,通过光镜和结晶紫染色观察各组细胞数量和细胞形态的变化情况。DCF染色评估Dap对A~D组AGS细胞内活性氧(ROS)水平的影响。JC-1染色评估Dap对A~D组AGS细胞内线粒体膜电位(MMP)水平的影响。采用Western blot实验检测A~D组AGS细胞内LC3和P62蛋白的表达水平。通过Ad-mCherry-GFP-LC3B染色检测AGS细胞内自噬流的变化。结果Dap以浓度依赖性的方式显著抑制AGS细胞的存活率(F=321.50,t=12.44~34.77,P<0.05),处理48 h时AGS细胞的IC 50约为90μmol/L。Dap处理48 h后,随Dap浓度的增加,A~E组AGS细胞数量逐渐减少,扭曲和皱缩形状的AGS细胞逐渐增多。随着Dap浓度的升高,A~D组AGS细胞内ROS水平逐渐增高。C和D组AGS细胞中JC-1绿色/红色荧光强度比值显著高于A组(F=10.92,t=3.09、4.96,P<0.05)。Western blot实验结果表明,B~D组与A组相比,细胞内LC3B/LC3A比值显著增高(F=36.46,t=3.17~9.78,P<0.05),P62的表达也显著升高(F=109.90,t=12.38~16.78,P<0.05)。Ad-mCherry-GFP-LC3B染色发现,Dap可抑制自噬小体与溶酶体的融合,导致自噬流阻断。结论Dap能够抑制胃癌细胞的增殖,其作用机制可能与诱导胃癌细胞内ROS大量产生以及阻断自噬流有关。

Objective To explore the inhibitory effect of daphnetin(Dap)on gastric cancer cells and its mechanism.Methods Gastric cancer AGS cells were treated with Dap at concentrations of 0,30,60,90,120,and 150μmol/L,and the effect on cell viability was evaluated after 48 h using the MTT assay.AGS cells were divided into groups A to E,and each group was treated with Dap at concentrations of 0,30,60,90,120,and 150μmol/L,respectively,for 48 h.Each group was observed for changes in cell count and morphology using optical microscopy and crystal violet staining.DCF staining was used to evaluate the effect of Dap on intracellular reactive oxygen species(ROS)levels in AGS cells in groups A to D,while JC-1 staining was used to evaluate the effect of Dap on the level of mitochondrial membrane potential(MMP)in AGS cells in groups A to D.Western blot was conducted to measure the expression levels of LC3 and P62 proteins in AGS cells in groups A to D,and changes in autophagic flux in AGS cells were measured by Ad-mCherry-GFP-LC3B staining.Results Dap significantly inhibited AGS cell viability in a concentration-dependent manner(F=321.50,t=12.44-34.77,P<0.05),with an IC 50 of approximately 90μmol/L at 48 h of treatment.After 48 h of Dap treatment,the number of AGS cells in groups A to E gradually decreased,and the number of twisted and wrinkled AGS cells gradually increased with the increase in Dap concentration.Intracellular ROS in AGS cells in groups A to D also gradually increased with the increase in Dap concentration.The ratio of JC-1 green/red fluorescence intensity in AGS cells in groups C and D was significantly higher than that in group A(F=10.92,t=3.09,4.96,P<0.05).Western blot revealed that groups B to D had a significant increase in the ratio of LC3B/LC3A(F=36.46,t=3.17-9.78,P<0.05)and in the expression of P62(F=109.90,t=12.38-16.78,P<0.05)compared to group A.Ad-mCherry-GFP-LC3B staining revealed that Dap inhibited the fusion of autophagosomes with lysosomes,resulting in the blockage of autophagic flux.Conclusion Dap can inhibit the proliferation of gastric cancer cells,and its mechanism of action may be related to inducing significant production of ROS within gastric cancer cells and blocking the autophagic flux.