TDK conference 2011

The oxidative stress via causing cell and tissue damage can lead to formation of acute and chronic intestinal inflammation. The goal of our experimental work was the development of in vitro system mimicking intestinal epithelium, where oxidative stimuli can be introduced by hydrogen-peroxide administration and the protective effect of probiotics can be tested.

Porcine intestinal epithelial cells, IPEC-J2 cell line on membrane insert was used to investigate the oxidative stress, probiotics and their interaction on intestinal eithelium. The prerequisite for finding the optimal concentration of hydrogen-peroxide was the maintenance of constant cell layer permeability whereas the changes in relative gene expression level of inflammatory cytokines could indicate the acute phase of inflammatory processes.

IL-6 gene expression remained unchanged during the exposure time and a day long follow-up period. Expression of IL-8 and TNF-α, however, increased significantly, and the peak level was reached for both cytokines when 1 mM H2O2 was added for 1 hour without time for recovery. The transepithelial electrical resistance (TER) did not decrease and the expression of proteins in junctional complexes did not show changes in cell membranes after 1 mM peroxide addition.

We investigated the effects of the spent culture supernatants (SCSs) of 5 bacterial strains (Lactobacillus plantarum 2142, Lactobacillus casei Shirota, Bifidobacterium animalis subsp. Lactis Bb-12, Bacillus amyloliquefaciens, Enterococcus faecium) on the upregulation of IL-8 and TNF- α. Only the Lactobacillus plantarum 2142 had significant lowering effect on inflammatory cytokine level.

Dianisidine-based spectrophotometric method was used for the quantitative determination of spontaneous decomposition of hydrogen-peroxide and for estimation if chemical interaction exists between SCSs of probiotics and hydrogen-peroxide. We demonstrated that apart from Bacillus amyloliquefaciens, no bacterial SCSs entered chemical reaction with hydrogen-peroxide, which excludes the possibility that the antiinflammatory effect of Lactobacillus plantarum 2142 SCS could be attributed to its direct peroxide-decomposing activity.

Further research designed to isolate and characterize potential bioactive components of lactobacilli SCSs and to trace the mode of action behind probiotic protection against acute oxidative-stress induced cellular damage is still ongoing.