Influence of lactic acid bacteria and their spent culture supernatant on hydrogen peroxide-induced interleukin-8 synthesis and necrosis of Caco-2 cells

Figures & data

Figure 1.  Influence of pre-incubation with L. plantarum 2142 on hydrogen peroxide-induced IL-8 synthesis of Caco-2 cells. Caco-2 cells were pre-treated with L. plantarum 2142 for 1 h. After removing lactobacilli by washing with plain DMEM, Caco-2 cells were exposed to 1 mM hydrogen peroxide for 1 h. Caco-2 cells exposed to 1 mM hydrogen peroxide for 1 h without pre-treatment served as positive control. After hydrogen peroxide was removed by washing, cells were allowed to recover in plain DMEM medium for up to 24 h. In the supernatants IL-8 was determined. Mean and SD of triplicates are given. There were no significant differences (p<0.05) between the IL-8 levels in the lactobacilli pre-treated cells and non-pre-treated cells.

Figure 2.  Effect of simultaneous application of Lactobacillus plantarum 2142 and 1 mM H₂O₂ on IL-8 production of Caco-2 cells. Caco-2 cells were treated for 1 h either with the combination of L. plantarum 2142 and 1 mM hydrogen peroxide or with 1 mM hydrogen peroxide alone. After removing lactobacilli and hydrogen peroxide by washing with plain DMEM, Caco-2 cells were allowed to recover in plain DMEM medium for up to 24 h. In the supernatants IL-8 was determined. Mean and SD of triplicates are given. There were no significant differences (p<0.05) between the IL-8 levels in supernatants of Caco-2 cells exposed either to lactobacilli and hydrogen peroxide concomitantly or to hydrogen peroxide alone.

Figure 3.  Effect of SCS of L. plantarum 2142 on hydrogen peroxide-induced IL-8 production by Caco-2 cells. Non-filter grown Caco-2 cells were treated for 1 h with 1 mM hydrogen peroxide and the combinations of 150 µl MRS broth or SCS with 850 µl of hydrogen peroxide. After the treatment period, cells were washed with plain DMEM, and allowed to recover for 24 h. In the supernatants, IL-8 was determined. Linear equations were calculated by R program. H₂O₂: −27.178 + 175.293*log(time + 1); H₂O₂+MRS: −62.682 + 171.092*log(time + 1); H₂O₂+SCS:−5.454 + 21.791*log(time + 1). There was no significant difference between the linear equations of treatment with H₂O₂ and H₂O₂+MRS. H₂O₂+SCS was significantly different from H₂O₂ or H₂O₂+MRS.

Figure 4.  Effect of hydrogen peroxide and L. plantarum 2142 on TER and necrosis in filter grown Caco-2 cells. Caco-2 cells were exposed to various concentrations of hydrogen peroxide (A) or simultaneously to hydrogen peroxide and L. plantarum 2142 (B) for 1 h. Furthermore, Caco-2 cells were pre-treated with L. plantarum 2142 for 1 h, and after removing the bacteria by washing with plain DMEM, the cells were exposed to the hydrogen peroxide concentrations for 1 h (C). At the end of the treatment periods, TER was measured. After washing, cells were stained with DAPI. TER is expressed as relative TER, necrosis as percentage of DAPI-stained nuclei. Means and SD of triplicates are given.

Figure 5.  Protective effect of L. plantarum 2142 against 30 mM hydrogen peroxide-induced necrosis in filter grown Caco-2 cells. Caco-2 cells were exposed to 30 mM hydrogen peroxide (A) or a combination of 30 mM hydrogen peroxide with L. plantarum 2142 (B) for 1 h. Furthermore, Caco-2 cells were pre-treated with L. plantarum 2142 for 1 h followed by incubation with 30 mM hydrogen peroxide for 1 h (C). After the treatments, cells were stained with DAPI.