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11 February 2019 : Original article  

Escherichia Coli Outer Membrane Vesicles Induced DNA Double-Strand Breaks in Intestinal Epithelial Caco-2 Cells

Zhou Ling1A, Chen Dayong1A, Yu Denggao1A, Wang Yiting1A, Fang Liaoqiong1A*, Wang Zhibiao1A

DOI: 10.12659/MSMBR.913756

Med Sci Monit Basic Res 2019; 25:45-52

Abstract

BACKGROUND: Recent studies have shown that Escherichia coli induced digestive tract diseases may be related to outer membrane vesicles (OMVs) induced intestinal double-strand breaks (DSBs) in intestinal epithelial cells. This study aimed to compare the impact of OMVs forces on DSBs in intestinal epithelial Caco-2 cells, and provide a new treatment for digestive diseases caused by E. coli.

MATERIAL AND METHODS: E.coli OMVs were prepared and co-cultured with Caco-2 cells. The uptake of OMVs by Caco-2 cells was observed by confocal microscopy. The γ-H2AX protein was detected by western-blots. The DSBs caused by OMVs was detected by single cell gel electrophoresis.

RESULTS: The particle size analyzer showed that the average diameters of OMVs centrifuged at 20 000×g and 50 000×g were 217.5±7.29 nm and 186.3±6.59 nm (P<0.05), respectively. Transmission electron microscopy of the OMVs revealed a lipid bilayer structure with a variety of different sizes. Confocal fluorescence microscopy revealed that OMVs almost completely entered Caco-2 cells after 24 hours. The ratio of γ-H2AX protein band gray value normalized data in the OMVs centrifuged at 20 000×g and 50 000×g, and the control group (without OMVs) were 2.23±0.18, 1.58±0.20, 1±0.30 (P<0.05), respectively, while DNA levels of the comet tail (TailDNA%, TDNA%) were 72.21±14.61%, 23.11±4.98%, and 1.02±1.41% (P<0.05), respectively. The corresponding DNA damage was categorized as high (grade 3), moderate (grade 2), and no damage (grade 0).

CONCLUSIONS: Different sizes of OMVs induced different degrees of DNA damage in intestinal epithelial Caco-2 cells.

Keywords: Intestinal Epithelial Caco-2 Cells, DNA Damage, E. coli OMV, Bacterial Outer Membrane Proteins, Caco-2 Cells, DNA Breaks, Double-Stranded, Escherichia coli, Escherichia coli Proteins, Intestinal Mucosa

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Medical Science Monitor Basic Research eISSN: 2325-4416
Medical Science Monitor Basic Research eISSN: 2325-4416