幽门螺杆菌对GES-1细胞自噬及凋亡的影响

Effect of Helicobacter pylori on autophagy and apoptosis in GES-1 cells

目的研究幽门螺杆菌(Hp)对胃上皮细胞GES-1自噬与凋亡的影响。方法Hp与自噬抑制剂3-MA、广谱凋亡抑制剂Z-VAD-FMK处理后的GES-1细胞共培养,流式细胞术检测细胞凋亡情况,Western blot检测LC3B-Ⅱ、Caspase-3及PARP蛋白的表达。结果3-MA处理后,与空白对照组相比,Hp组LC3B-Ⅱ蛋白相对表达量显著增高(t=7.54,P<0.01);3-MA+Hp组与Hp组相比,LC3B-Ⅱ蛋白相对表达量显著降低(t=-3.24,P<0.05);与空白对照组相比,Hp组、3-MA组GES-1细胞凋亡率均显著增加(t值分别为-9.71和-3.25,P<0.01或P<0.05)。Z-VAD-FMK处理后,与空白对照组相比,Hp组GES-1细胞凋亡率显著增加,(t=-9.29,P<0.01);Hp+Z-VAD-FMK组较Hp组GES-1细胞凋亡率显著下降(t=-8.47,P<0.01);与空白对照组相比,Hp组、Z-VAD-FMK组的LC3B-Ⅱ蛋白相对表达量均显著增加(t值分别为-12.98和-11.28,均P<0.01)。3-MA与Z-VAD-FMK共处理后,3-MA+Hp+Z-VAD-FMK组较3-MA+Z-VAD-FMK组和Hp+Z-VAD-FMK组的GES-1细胞凋亡率显著增加(t值分别为-4.91和-9.64,P<0.05或P<0.01);3-MA+Hp+Z-VAD-FMK组与3-MA+Z-VAD-FMK组相比,Caspase-3蛋白相对表达量显著增加,PARP蛋白相对表达量显著下降(t值分别为-5.16和3.02,均P<0.05);3-MA+Hp+Z-VAD-FMK组较Hp+Z-VAD-FMK组LC3B-Ⅱ蛋白相对表达量显著下降(t=4.63,P<0.05)。结论幽门螺杆菌既能上调GES-1细胞自噬也能诱导其凋亡,并通过增加Caspase-3表达的同时降低PARP的表达以增强自噬抑制引起的细胞凋亡。而在抑制凋亡后,幽门螺杆菌可能诱导GES-1细胞发生自噬性死亡。

Objective To investigate the relationship between Helicobacter pylori on GES-1 autophagy and apoptosis in gastric epithelial cells.Methods H.pylori was co-cultured with GES-1 cells treated with autophagy inhibitor 3-MA and the PanCaspase apoptosis inhibitor Z-VAD-FMK.Flow cytometry was used to detect the rate of apoptosis and Western blotting was used to detect the expression of LC3 B-II,Caspase-3,and PARP.Results After treatment with 3-MA,the relative expression of LC3 B-II in the Hp group increaseds significantly increased compared to that in the blank control group(t=7.54,P<0.01).The relative expression of LC3 B-II decreased in the 3-MA+Hp group compared to the Hp group(t=-3.24,P<0.05).The rate of apoptosis in GES-1 cells in the Hp group and 3-MA group both increased compared to that in the blank control group(t=-9.71,-3.25,P<0.01,P<0.05).After treatment with Z-VAD-FMK,the rate of apoptosis in GES-1 cells in the Hp group increased significantly compared to that in the blank control group(t=-9.29,P<0.01).The rate of apoptosis significantly decreased in GES-1 cells in the Hp+Z-VAD-FMK group compared to that in the Hp group(t=-8.47,P<0.01).The relative expressions of LC3 B-II in the Hp group and Z-VAD-FMK group both significantly increased compared to that the blank control group(t=-12.98,-11.28,P<0.01).After treatment with Z-VAD-FMK treated,the rate of apoptosis in GES-1 cells in the3-MA+Z-VAD-FMK group and Hp+Z-VAD-FMK group both increased compared to that in the 3-MA+Hp+Z-VAD-FMK group(t=-4.91,-9.64,P<0.05,P<0.01).The relative expression of Caspase-3 increased,and the relative expression of PARP decreased in the 3-MA+Hp+Z-VAD-FMK group compared to expression in the 3-MA+Z-VAD-FMK group(t=-5.16,3.02,P<0.05).T,the relative expressions of LC3 B-II decreased in the 3-MA+Hp+Z-VAD-FMK group compared to that in the Hp+Z-VAD-FMK group(t=4.63,P<0.05).Conclusion H.pylori can both up-regulate autophagy and induce apoptosis in GES-1 cells.H.pylori can enhance apoptosis caused by inducing the inhibition of autophagy by increasing Caspase-3 expression and decreasing PARP expression.After suppressing apoptosis,H.pylori may induce autophagic death in GES-1 cells.