Secreted factors from Bifidobacterium animalis subsp. lactis inhibit NF-κB-mediated interleukin-8 gene expression in Caco-2 cells - PubMed (original) (raw)

Secreted factors from Bifidobacterium animalis subsp. lactis inhibit NF-κB-mediated interleukin-8 gene expression in Caco-2 cells

Zhonggui Wang et al. Appl Environ Microbiol. 2011 Nov.

Abstract

The objective of the present study was to evaluate the anti-inflammatory effects of Bifidobacterium animalis subsp. lactis strain BB12 in stimulated Caco-2 cells and to characterize the factors responsible for these anti-inflammatory effects. Characterization and purification studies indicate that BB12's anti-inflammatory factors might include a 50-kDa proteinaceous compound that is stable under a variety of heat and pH conditions.

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Figures

Fig. 1.

Fig. 1.

Inhibitory effect of BB12 on TNF-α-induced IL-8 expression is mediated through suppression of NF-κB activation in Caco-2 cells. (A) Total RNA was extracted from BB12-treated Caco-2 cells after TNF-α stimulation, and IL-8 mRNA expression was quantified by real-time PCR. Data are presented as means and standard deviations. *, P < 0.01 compared with TNF-α-stimulated cells without the BB12 pretreatment. (B) IL-8 protein secretion in culture supernatants was quantified with an ELISA kit. Data are presented as means and standard deviations. *, P < 0.01 compared with TNF-α-stimulated cells without the BB12 pretreatment. (C) Caco-2 cells were pretreated with BB12 (1 × 109 CFU/ml) for 12 h and then stimulated with TNF-α (10 ng/ml) for 15 min. Proteins were extracted from the cells. Nuclear extracts were analyzed for NF-κB (p65) using anti-human NF-κB antibody. Cytosolic extracts were analyzed with anti-human IκBα and phospho-IκBα (pIκBα) antibodies. (D) Caco-2 cells were transfected with a NF-κB luciferase (firefly) construct together with pRL-TK (Renilla luciferase) as a transfection control using the GeneJammer transfection reagent. After transfection, the cells were stimulated with TNF-α in the presence of BB12. Luciferase activities were determined using a dual-luciferase reporter assay kit and normalized to the control values. Data are means and standard deviations from three independent experiments. *, P < 0.01 compared with TNF-α-stimulated cells without the BB12 pretreatment.

Fig. 2.

Fig. 2.

Secreted factors from BB12 inhibit TNF-α-induced IL-8 production. (A) Caco-2 cells were incubated for 12 h with the nontreated (NT) control, viable (live) or heat-killed (HK) BB12, fresh conditioned medium (CM), or heat-inactivated CM of BB12 (HI CM) in the presence or absence of TNF-α. (B) Caco-2 cells were incubated for 12 h with the NT control, CM, BB12 culture supernatant (CS), or HI CS in the presence or absence of TNF-α. IL-8 production in the supernatant of Caco-2 cells was estimated by ELISA. Data are presented as means and standard deviations. *, P < 0.05 compared with TNF-α-stimulated cells in the absence of BB12. (C) Caco-2 cells were treated with the nontreated (NT) control or the component extracts from CS either alone or together with TNF-α (10 ng/ml). The IL-8 protein level in the supernatant of Caco-2 cells was estimated by ELISA. Data are presented as means and standard deviations. *, P < 0.05 compared with TNF-α-stimulated cells in the absence of BB12. (D) Caco-2 cells were treated with the nontreated (NT) control or CS from different pH conditions either alone or together with TNF-α (10 ng/ml). The IL-8 protein level in the supernatant of Caco-2 cells was estimated by ELISA. Data are presented as means and standard deviations. *, P < 0.05 compared with TNF-α-stimulated cells in the absence of BB12.

Fig. 3.

Fig. 3.

Purification of the active IL-8 inhibitory components in CS. (A) Eluted proteins were separated by SDS-PAGE and stained with a colloidal blue stain kit (lane 3). Proteins present in fractions of broth and BB12 CS and concentrated using a 5-kilodalton cutoff filter are shown (lanes 1 and 2). (B) Caco-2 cells were pretreated with purified p50 at the indicated concentrations for 12 h and then stimulated with TNF-α (10 ng/ml). IL-8 protein secretion in culture supernatants was quantified with an ELISA kit.

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