ABSTRACT
Context The effect of 6-gingerol (6G), the bioactive component of Zingiber officinale Roscoe (Zingiberaceae), in the reduction of Vibrio cholerae (Vibrionaceae)-induced inflammation has not yet been reported. Materials and methods Cell viability assay was performed to determine the working concentration of 6G. Elisa and RT-PCR were performed with Int 407 cells treated with 50 µM 6G and 100 multiplicity of infection (MOI) V. cholerae for 0, 2, 3, 3.5, 6 and 8 h to determine the concentration of IL-8, IL-6, IL-1α and IL-1ß in both protein and RNA levels. Furthermore, the effect of 50 µM 6G on upstream MAP-kinases and NF-κB signalling pathways was evaluated at 0, 10, 15, 30, 60 and 90 min. Results The effective dose (ED50) value of 6G was found to be 50 µM as determined by cell viability assay. Pre-treatment with 50 µM 6G reduced V. cholerae infection-triggered levels of IL-8, IL-6, IL-1α and IL-1ß by 3.2-fold in the protein level and two-fold in the RNA level at 3.5 h. The levels of MAP-kinases signalling molecules like p38 and ERK1/2 were also reduced by two- and three-fold, respectively, after 30 min of treatment. Additionally, there was an increase in phosphorylated IκBα and down-regulation of p65 resulting in down-regulation of NF-κB pathway. Conclusion Our results showed that 6G could modulate the anti-inflammatory responses triggered by V. cholerae-induced infection in intestinal epithelial cells by modulating NF-κB pathway.
Subject(s)
Anti-Inflammatory Agents/pharmacology , Catechols/pharmacology , Cytokines/metabolism , Epithelial Cells/drug effects , Fatty Alcohols/pharmacology , Inflammation Mediators/metabolism , Intestines/drug effects , NF-kappa B/metabolism , Vibrio cholerae/immunology , Cytokines/genetics , Cytokines/immunology , Down-Regulation , Enzyme Activation , Epithelial Cells/immunology , Epithelial Cells/metabolism , Epithelial Cells/microbiology , Hep G2 Cells , Host-Pathogen Interactions , Humans , Inflammation Mediators/immunology , Intestinal Mucosa/metabolism , Intestines/immunology , Intestines/microbiology , Mitogen-Activated Protein Kinases/metabolism , NF-KappaB Inhibitor alpha/metabolism , NF-kappa B/immunology , Phosphorylation , Signal Transduction/drug effects , Time Factors , Transcription Factor RelA/metabolism , Vibrio cholerae/pathogenicityABSTRACT
Vibrio cholerae is one of the major bacterial pathogens responsible for the devastating diarrheal disease called cholera. Chemotherapy is often used against V. cholerae infections; however, the emergence of V. cholerae with multidrug resistance (MDR) toward the chemotherapeutic agents is a serious clinical problem. This scenario has provided us with the impetus to look into herbal remediation, especially toward blocking the action of cholera toxin (CT). Our studies were undertaken to determine the antidiarrheal potential of 6-gingerol (6G) on the basis of its effect on CT, the virulence factor secreted by V. cholerae. We report here that 6G binds to CT, hindering its interaction with the GM1 receptor present on the intestinal epithelial cells. The 50% inhibitory concentration (IC50) was determined to be 10 µg/ml. The detailed mechanistic study was conducted by enzyme-linked immunosorbent assay (ELISA), fluorescence spectroscopy, and isoelectric focusing. These results were validated with in vitro studies performed with the CHO, HeLa, and HT-29 cell lines, whereas a rabbit ileal loop assay was done to estimate the in vivo action, which confirms the efficacy of 6G in remediation of the choleragenic effects of CT. Thus, 6G can be an effective adjunctive therapy with oral rehydration solution for severe CT-mediated diarrhea.
