探讨屎肠球菌的VanA调控人正常结直肠黏膜细胞FHC凋亡的机制

Mechanism of VanA of Enterococcus faecium regulating FHC apoptosis in human normal colorectal mucosa cells

目的探讨屎肠球菌的万古霉素替考拉宁A型抗性蛋白/D-丙氨酸-D-丙氨酸连接酶(Vancomycin Teicoplanin A-type resistance protein D-alanine-D-alanine ligase,VanA)调控人正常结直肠黏膜细胞FHC凋亡的机制。方法在人正常结直肠黏膜细胞FHC中使用屎肠球菌感染,Annxin-V染色检测细胞凋亡情况。使用屎肠球菌的VanA蛋白刺激,检测FHC细胞凋亡情况、ROS水平以及ROS标志蛋白MDA、GSH和SOD的表达水平。ROS抑制Acetylcysteine处理VanA刺激的FHC细胞后,检测细胞凋亡相关蛋白的表达水平。结果屎肠球菌与人正常结直肠黏膜细胞FHC共培养后,人正常结直肠黏膜细胞FHC的凋亡水平明显升高(t=2.876,P=0.0452),并且VanA蛋白能促进FHC凋亡水平(t=5.579,P=0.0051),同时细胞凋亡相关蛋白CLEAVED-CAS9、BAK的表达量上升,BCL-2的表达量下降。屎肠球菌的VanA蛋白刺激后,发现正常结直肠黏膜细胞FHC的ROS水平上升(t=10.190,P=0.0005),ROS标志蛋白MDA(t=4.315,P=0.0125)和SOD(t=5.751,P=0.0045)的表达水平上升,GSH(t=5.225,P=0.0064)的表达水平下降,但是,ROS抑制剂Acetylcysteine能够抑制这种现象。结论屎肠球菌的VanA通过提高细胞内ROS水平来促进人正常结直肠黏膜细胞FHC凋亡。

Objective To investigate the mechanism of vancomycin-induced regulation of FHC apoptosis by Enterococci in human normal colorectal mucosa cells.Methods Enterococcus faecium infection was induced in human normal colorectal mucosal cells FHC,and apoptosis was detected with Annexin-V staining.When stimulated with the VanA protein of Enterococcus faecium,the apoptosis of FHC cells,level of ROS,and the expression levels of ROS marker proteins MDA,GSH and SOD were detected.After ROS inhibiting the Acetylcysteine treatment of VanA-stimulated FHC cells,the expression level of apoptosis-related proteins was examined.Results After co-culturing of Enterococcus faecium with human normal colorectal mucosal cells FHC,the apoptosis level of FHC in human normal colorectal mucosa cells significantly increased(t=2.876,P=0.0452),and VanA protein promoted FHC apoptosis(t=5.579,P=0.0051).The expression levels of apoptosis-related proteins CLEAVED-CAS9 and BAK increased,while that of BCL-2 decreased.After stimulation with VanA protein of Enterococcus faecium,the ROS level of FHC in normal colorectal mucosa cells increased(t=10.190,P=0.0005),the expression levels of ROS marker protein MDA(t=4.315,P=0.0125)and SOD(t=5.751,P=0.0045)increased,and the expression level of GSH(t=5.225,P=0.0064)decreased;but the ROS inhibitor Acetylcysteine was able to inhibit this phenomenon.Conclusion The VanA of Enterococcus faecium promotes FHC apoptosis in human normal colorectal mucosa cells by increasing intracellular ROS levels.