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Nuclear factor kappa B (NF-κB)-mediated inflammatory responses play crucial roles in the pathogenesis of diarrhea caused by the Vibrio cholerae El Tor variant (EL), which is a major bacterial strain causing recent cholera outbreaks. Flufenamic acid (FFA) has previously been demonstrated to be a potent activator of AMP-activated protein kinase (AMPK), which is a negative regulator of NF-κB signaling. This study aimed to investigate the anti-diarrheal efficacy of FFA in a mouse model of EL infection and to investigate the mechanisms by which FFA activates AMPK in intestinal epithelial cells (IEC). In a mouse closed loop model of EL infection, FFA treatment (20mg/kg) significantly abrogated EL-induced intestinal fluid secretion and barrier disruption. In addition, FFA suppressed NF-κB nuclear translocation and expression of proinflammatory mediators and promoted AMPK phosphorylation in the EL-infected mouse intestine. In T84 cells, FFA induced AMPK activation. Furthermore, FFA promoted tight junction assembly and prevented interferon gamma (IFN-γ)-induced barrier disruption in an AMPK-dependent manner. Biochemical and molecular docking analyses indicated that FFA activates AMPK via a direct stimulation of calcium/calmodulin-dependent protein kinase kinase beta (CaMKKβ) activity. Collectively, our data indicate that FFA represents a class of existing drugs that may be of potential utility in the treatment of cholera caused by EL infection via AMPK-mediated suppression of NF-κB signaling in IEC., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H(2)O(2)/Cl(-) system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H(2)O(2)/Cl(-) system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC(50) = 1.1+/-0.3 microM) is the most efficient inhibitor of the MPO/H(2)O(2)/Cl(-) system and nimesulide (IC(50) = 2.1+/-0.3 microM) is more active than the other NSAIDs of the oxicam family (IC(50) = 8-12 microM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H(2)O(2)/Cl(-) system. Flufenamic acid has the best inhibiting activity towards the MPO/H(2)O(2)/Cl(-) system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.