Escherichia coli Nissle 1917 Protects Intestinal Barrier Function by Inhibiting NF-κB-Mediated Activation of the MLCK-P-MLC Signaling Pathway

Figure 6

EcN^sup suppressed the activation of NF-κB. (a) The nuclear protein of monolayers was harvested after incubation for 48 h in the absence or presence of 10 ng/mL TNF-α and 10 ng/mL IFN-γ with or without 100 ng/mL EcN^sup followed by western blot assays. (b) Caco-2 monolayers were stained for NF-κB p65 by immunofluorescence. Cotreatment with 100 ng/mL EcN^sup significantly inhibited the nuclear translocation of NF-κB p65 elicited by TNF-α/IFN-γ. A: The control group; B: Caco-2 monolayers were exposed to 100 ng/mL EcN^sup for 30 min; C: Caco-2 monolayers were incubated with 10 ng/mL TNF-α and 10 ng/mL IFN-γ for 30 min; and D: Caco-2 monolayers were incubated with 100 ng/mL EcN^sup for 30 min in the presence of 10 ng/mL TNF-α and 10 ng/mL IFN-γ. (c) Schematic illustration of human MLCK gene promoter containing two putative κB sites (κB-1 and κB-2) at -136 and -1451 as indicated. The PCR primer locations were also indicated. (d) Caco-2 cells were pretreated for 4 h with or without 100 ng/mL EcN^sup in the absence or presence of 10 ng/mL TNF-α and 10 ng/mL IFN-γ. ChIP assays were performed with anti-p65 antibodies or IgG. The results for κB-1 and κB-2 sites are shown. Similar results were seen for 10 h treatment. Results were expressed as the (). vs. control. ^# vs. TNF-α/IFN-γ.

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