ABSTRACT
Selective microbes used as probiotics can enhance epithelial cell protection. We have previously shown that a Lactobacillus plantarum strain 299v (Lp299v) has the ability to induce mucin genes. In the current study, we utilized a cytokine model of inflammation in cell culture to study the modulation of apoptosis by this probiotic. HT-29 cells were pre-incubated with the Lp299v or L. plantarum strain adh- (Lpadh-), a non-adherent derivative of Lp299v. Cells were challenged with a mixture of cytokines (TNF-α, IFN-γ, and IL-1a) to imitate conditions of inflammation. To assess for cell death, we evaluated TUNEL, multi-caspase, and caspase-3 and caspase-7 activity assays. There was a marked decrease in apoptosis as measured by TUNEL(+) cells in samples pre-treated with Lp299v (18.7 ± 4.1%, p < 0.01) and Lpadh- (16.6 ± 3.2%, p < 0.05) prior to cytokine exposure when compared to cells (43.6 ± 6.2%) exposed to the cytokine mixture. Lp299v pre-incubation with HT-29 cells reduced caspase(+) cells in the multi-caspase activity assay (3.6 ± 0.6%, p < 0.05) compared to cells exposed to cytokines (68.9 ± 5.1%) whereas Lpadh- did not (46.8 ± 17.5%, p > 0.05). Similarly, caspase-3, caspase-7 activity was also reduced by Lp299v. Selected probiotics may confer an exogenous protective effect at the mucosal-luminal interface for intestinal epithelial cells via alteration of caspase-dependent apoptotic pathways.
ABSTRACT
Upon ingestion, probiotics may act to protect the host through a number of protective mechanisms including modulation of genes involved in intestinal innate mucosal defense such as epithelial cell-derived mucin glycoproteins and inhibitor of apoptosis proteins. To determine the specificity of effect and sustainability of response in vivo, Lactobacillus plantarum 299v (Lp299v), Lactobacillus rhamnosus R0011 (LrR0011), and Bifidobacterium bifidum R0071 (BbR0071) were added repeatedly or intermittently to the drinking water of Sprague-Dawley rats. After killing the rats via CO2 suffocation, Muc2, Muc3, neuronal apoptosis inhibitor protein (NAIP), human inhibitor of apoptosis protein 1/cellular inhibitor of apoptosis 2 (HIAP1/cIAP2), and human inhibitor of apoptosis protein 2/cellular inhibitor of apoptosis 1 (HIAP2/cIAP1) mRNA and protein levels were analyzed via RT-PCR and immunohistochemistry. Live Lp299v, BbR0071, and LrR0011 increased Muc3 protein and mRNA expression in jejunum and ileum. Heat-killed and a nonadherent derivative of Lp299v failed to induce Muc3 expression. Lp299v did induce expression of HIAP2/cIAP1 and NAIP expression. Muc3 mucin expression was elevated for 5 d after oral administration of Lp299v; however, this effect was not sustained despite ongoing daily ingestion of a probiotic. Intermittent pulse ingestion of probiotics, however, was found to repeatedly increase Muc3 expression. We conclude that selected probiotics can induce protective genes of mucosal intestinal epithelial cells, an effect that is reproducible with pulse probiotic administration.
