幽门螺杆菌CagA对胃上皮细胞线粒体功能的影响

Effect of Helicobacter pylori CagA on the mitochondrial function of gastric epithelial cells

目的评价幽门螺杆菌(Helicobacter pylori,Hp)细胞毒素相关蛋白CagA对胃上皮细胞线粒体功能的影响。方法用CagA基因失活突变Hp菌株(1004Hp△cagA)和野生型1004Hp菌株分别感染人胃上皮细胞GES-1和AGS 24 h,收集标本。Western blot检测各组细胞中CagA蛋白、线粒体融合分裂蛋白Mfn1、Mfn2、Fis1和Drp1的表达情况;加入JC-1荧光探针后,荧光显微镜观察各组细胞线粒体膜电位变化;ATP检测试剂盒检测各组细胞中ATP生成水平的变化。结果Western blot鉴定显示野生型1004Hp中有CagA的表达,而1004Hp△cagA无CagA的表达。1004Hp感染组与1004Hp△cagA感染组比较,线粒体融合蛋白Mfn1、Mfn2表达下降(P<0.05),线粒体分裂蛋白Fis1、Drp1表达上升(P<0.05),线粒体膜电位下降,且在GES-1和AGS两株细胞结果一致。1004Hp感染组和1004Hp△cagA中AGS和GES-1细胞ATP相对生成量分别为0.30±0.03、0.64±0.04和0.64±0.01、1.23±0.19,AGS中1004 Hp感染组较1004 Hp△cagA感染组ATP生成水平下降53.1%,GES-1中1004 Hp感染组较1004 Hp△cagA感染组ATP生成水平下降48.0%(P<0.05)。结论Hp/cagA影响线粒体动态平衡,诱发功能障碍,可能是Hp引起胃部病理变化的原因之一。

Objective To evaluate the effect of the Helicobacter pylori cytotoxin-related protein CagA on the mitochondrial function of gastric epithelial cells.Methods A CagA gene-inactivated mutant H.pylori strain(1004 H.pylori△cagA)and a wild-type 1004 H.pylori strain were used to infect GES-1 and AGS human gastric epithelial cells for 24 h,and specimens were collected.Western blotting was used to measure expression of the CagA protein and levels of the mitochondrial fusion fission proteins Mfn1,Mfn2,Fis1,and Drp1 in each group of cells.After addition of a JC-1 fluorescent probe,fluorescence microscopy was used to observe changes in the mitochondrial membrane potential in each group of cells.An ATP detection kit was used to measure changes in levels of ATP production in each group of cells.Results Western blotting indicated that CagA was expressed in the wild-type 1004 H.pylori strain,but CagA was not expressed in 1004 H.pylori△cagA.The relative levels of expression of the proteins Mfn1,Mfn2,Drp1,and Fis1 were 4.23±0.25,1.03±0.19,1.33±0.33,and 1.82±0.21 in AGS cells infected with 1004 H.pylori△cagA.The relative levels of expression of the proteins Mfn1,Mfn2,Drp1,and Fis1 were 1.88±0.73,1.21±0.21,1.47±0.51,and 2.01±0.42 in GES-1 cells infected with 1004 H.pylori△cagA.The relative levels of expression of the proteins Mfn1,Mfn2,Drp1,and Fis1 were 3.23±0.42,0.73±0.12,1.78±0.37,and 2.43±0.22 in AGS cells infected with 1004 H.pylori.The relative levels of expression of the proteins Mfn1,Mfn2,Drp1,and Fis1 were 1.37±0.78,0.83±0.14,2.44±0.32,and 3.29±0.14 in GES-1 cells infected with 1004 H.pylori.Expression of the mitochondrial fusion proteins Mfn1 and Mfn2 decreased in cells infected with 1004 H.pylori compared to cells infected with 1004 H.pylori△cagA(t values:5.425,2.866,7.645,and 5.440;P<0.05),expression of the mitochondrial fission proteins Fis1 and Drp1 increased(t values:20.60,7.117,10.21,and 6.044;P<0.05),and the mitochondrial membrane potential decreased.Results for GES-1 cells agreed with those for AGS cells.The relative level of ATP production was 0.30±0.03 in AGS cells and 0.64±0.04 in GES-1 cells infected with 1004 H.pylori and 0.64±0.01 in AGS cells and 1.23±0.19 in GES-1 cells infected with 1004 H.pylori△cagA.The level of ATP production in AGS cells infected with 1004 H.pylori decreased by 53.1% compared to that in cells infected with 1004 H.pylori△cagA.The level of ATP production in GES-1 cells infected with 1004 H.pylori was 48.0% lower than that in GES-1 cells infected with 1004 H.pylori△cagA(P<0.05).Conclusion H.pylori cagA affects the dynamic balance of mitochondria and induces dysfunction,which may be one of the reasons for the pathological changes in the stomach caused by H.pylori.