Flufenamic Acid Prevents Vibrio cholerae El Tor Variant‐Induced Diarrhea via AMPK Activation and Suppression of NF‐κB‐Mediated Inflammation

Abstract

Pathogenesis of cholera diarrhea caused by infection with Vibrio cholerae El Tor variant (EL), a current epidemic strain of Vibrio cholerae that causes severe diarrhea in several regions, involves the nuclear factor kappa B (NF‐κB)‐mediated inflammatory responses including cyclooxygenase‐2 (COX‐2) upregulation and prostaglandin E2 (PGE2)‐induced intestinal fluid secretion and barrier disruption. The aim of this study was to evaluate the anti‐diarrheal efficacy of flufenamic acid (FFA), an non‐steroidal anti‐inflammatory drug (NSAID) that has previously been reported to stimulate AMP‐activated protein kinase (AMPK), a negative regulator of NF‐κB. We found that intraperitoneal injection of FFA (20 mg/kg) effectively reduced EL‐induced intestinal fluid secretion and intestinal barrier leakage in a mouse closed‐loop model of EL infection. In addition, FFA activated AMPK and suppressed p65 NF‐κB nuclear translocation, mRNA expression of proinflammatory cytokines and protein expressions of COX‐2 and inducible nitric oxide synthase (iNOS) in the mouse intestine infected with EL. In human intestinal epithelial cells (T84 cells), FFA induced tight junction assembly via AMPK activation. Mechanistic studies using immunoblot analysis and molecular docking prediction demonstrated that FFA activated AMPK in T84 cells by directly stimulating Ca(2+)/calmodulin dependent kinase kinase beta (CaMKKβ). Taken together, our results indicate that FFA and related compounds may be repurposed for the treatment of cholera caused by EL infection.Support or Funding InformationThis work was supported by grants BRG5980008 from the Thailand Research Fund and Mahidol University and by the Faculty of Science, Mahidol University and Faculty of graduate studies, Mahidol University.