acidophilus determined by the percentage of LDH leakage (in triplicate) and non-stained trypan blue (single) Bacteria and MOI Cytotoxicitya (% LDH) Viable cell count (× 106) Cell only for 4 and 8 hours 18.0, 18.0
1.36 H. pylori for 4 hours MOI 100 18.1 selleck kinase inhibitor 1.00 Lactobacillus for 8 hours MOI 1 18.4 1.00 MOI 10 18.0 1.11 MOI 100 18.7 1.24 MOI 1000 24.2 0.77 aAll cytotoxicity data were presented with mean value of three tests H. pylori stimulated IL-8 and TNF-α but not TGF-β1 production in vitro In MKN45 cells incubated with H. pylori (MOI 100) at various time periods, the IL-8 level increased from the 4th to the 8th hour after co-incubation, as determined by ELISA (MCC950 in vivo Figure 1A). For TNF-α, the post-incubation level rose after the 4th hour and maintained a plateau until the 8th hour (Figure 1B). However, the TGF-β1 level did not increase after H. pylori incubation for 4 hours (data not shown). Figure 1 (A) IL-8 and (B) TNF-α concentrations in the supernatant of MKN45 cells culture after variable duration of H. pylori and L. acidophilus
infection (MOI = 100). Data were expressed as means ± standard deviation (SD) (in triplicate). HDAC inhibitor In contrast, L. acidophilus did not induce IL-8, TNF-α, and TGF-β1 expressions of MKN45 at least within the 8-hour co-incubation period. Pre-treatment of L. acidophilus attenuated H. pylori-induced IL-8 Because the IL-8 level of MKN45 cells could be induced by H. pylori challenge for 4 hours, the time- and dose-dependent effects of probiotics in reducing pro-inflammatory cytokines and TGF-β1 on the 4th hour were
studied. The IL-8 and TGF-β1 concentrations were PD184352 (CI-1040) shown for MKN cells challenged by H. pylori and with variable doses of L. acidophilus pretreatment for 8 hours (Figure 2). Compared to the control group, L. acidophilus pre-treatment with higher bacterial colony count (MOI 100) reduced H. pylori-induced IL-8 expressions in MKN45 cells (P < 0.05). The TGF-β1 level did not change (P > 0.05). Figure 2 The concentrations of IL-8 (blank column) and TGF-β1 (black column) in the supernatant of MKN45 cells pre-treated with different MOI (0: control; 1: 1 × 10 6 c.f.u.; 10: 1 × 10 7 c.f.u.; 100: 1 × 10 8 c.f.u.) of L. acidophilus. The cells were washed thrice with PBS to remove the L. acidophilus and then infected with H. pylori (MOI = 100) for 4 hours. Data are expressed as means ± SD (in triplicate). Statistical analysis was performed in each measurement with comparisons to the controls (cells treated H. pylori only; IL-8 2034 ± 865 pg/ml and TGF-β1 587.2 ± 39.8 pg/ml) (*P < 0.05). L. acidophilus reduced H. pylori-induced NF-κB by increasing IκBα The study determined that MKN45 cells (MOI 100) incubated with H. pylori led to a peak increase of nuclear NF-κB production within one hour. Thus, nuclear NF-κB levels of MKN45 cells co-incubated with H. pylori, after prior pre-treatments by various MOIs (1-100) of L.