butyricum and IL-10 production or IL-10 mRNA expression was dose-dependent. Figure 1 IL-10 mRNA expression and IL-10 protein secretion were stimulated by C. butyricum . The cells were exposed to 1 × 106, 1 × 107, 1 × 108 CFU ml−1 of C. butyricum for 2 h. (A) At the end of the incubation period, cell culture supernatants were collected to determine IL-10 protein concentration by sandwich ELISA. (B) The same cells were harvested for real-time quantitative PCR. Data represent the mean ± the

standard error of the mean for three experiments. *, P < 0.01 compared with learn more the control. C: levels of IL-10 in control HT-29 cells. Neutralization of IL-10 released by HT-29 cells enhances the effects of C. butyricum-induced NF-κB activation and IL-8 expression Our previous study demonstrated that C. butyricum could induce HT-29 cells to release low levels of pro-inflammatory cytokines, which is similar to other probiotics such as Lactobacilli[15]. We also found that C. butyricum could increase the expression of anti-inflammatory cytokines, which may be associated with the beneficial properties of C. butyricum. In the current study, we have shown that C. butyricum can induce HT-29 cells to secrete IL-10. To determine whether this IL-10 present in culture supernatant affects EPZ015938 the C. butyricum-induced immune response in HT-29 cells, an IL-10 antibody was utilized to treat

HT-29 cells. Neutralization of IL-10 using anti-IL-10 for 48 h resulted in a significant

degradation of cytoplasmic IκB protein and an increase in nuclear NF-κB and supernatant IL-8 levels (Figure 2). Therefore, it can be concluded that down-regulation of inflammatory cytokines and inhibition of excessive immunity ZD1839 in HT-29 cells induced by C. butyricum is probably mediated through IL-10. Figure 2 Activation of NF-κB and up-regulation of IL-8 expression in HT-29 cells by C. butyricum were enhanced in the presence of IL-10 antibody. (A) Immunoblot showing levels of NF-κB (p50/p105 subunits) and IκB in cells compared with the control. (B) IL-8 secretion in response to C. butyricum in control and anti-IL-10 treated cells. (C) IL-8 transcript levels as CRT0066101 measured using real-time PCR. Results are mean ± SE for three experiments. *, P < 0.01 compared to the control without IL-10 antibody treatment (C- vs. C + and T- vs. T+). C: levels of NF-κB, IκB or IL-8 in control HT-29 cells. T: levels of NF-κB, IκB or IL-8 in HT-29 cells treated with C. butyricum. Knockdown of IL-10 enhances the effects of C. butyricum-induced NF-κB activation and IL-8 expression To further confirm the effects of IL-10 on the activation of NF-κB and secretion of IL-8, NF-κB, IκB and IL-8 levels were measured after pre-treating HT-29 cells with siNEG (negative control-specific siRNA) or siIL-10 (IL-10 small interfering RNA) for 48 h, and then treating them with C. butyricum for 2 h.