Inducible nitric oxide synthase (iNOS)-induced nitric oxide (NO) and its reactive metabolite peroxynitrite (ONOO־) has been implicated as an important mediator in the pathogenesis of inflammatory bowel disease (IBD). Immune cells macrophages produce NO and ONOO־ to provide the host defence and play important roles in the regulation of inflammatory response. Several in vitro and In vivo studies have reported the role of probiotics and probiotic-released metabolites in the treatment and prevention of IBD. The study was therefore started with the hypothesis that the metabolites released by probiotics present in the formulation VSL#3 may have the effect on the pro-inflammatory mediator NO and its regulatory enzyme iNOS in the immune cells.Murine macrophage J774 cells were used in the study as these cells are readily induced by bacterial lipopolysaccharide (LPS) and produce pro-inflammatory mediators nitric oxide (NO) and prostaglandin E2 (PGE2) by the expression of enzymes iNOS and cyclooxygenase-2 (COX-2) respectively through the Nuclear Factor-κappa B (NF-B) signalling pathway. The cells were exposed to VSL#3-conditioned medium (VSL#3-CM) in the absence and presence of LPS and the inhibitors of a number of enzymes and signalling molecules. To exclude the possibility of endotoxin contamination in VSL#3-CM, experiments were also carried out in the absence and presence of an endotoxin neutralising compound polymyxin B (PmB).VSL#3-CM increased the basal NO production through the induction of iNOS on a concentration dependent manner. On the other hand, it suppressed the LPS-induced NO production but caused slight inhibition on the LPS-induced iNOS expression. PmB suppressed the LPS-induced NO production but not that caused by VSL#3-CM suggests the effects of the later were not LPS mediated. Similarly, VSL#3-CM at a higher concentration induced the basal COX-2. However, it caused slight inhibition on the LPS-induced COX-2 expression. Dexamethasone has partially inhibited the VSL#3-CM-induced NO and iNOS expression. Bisindolylmaleimide (BIM) and SB203580, inhibitor of protein kinase C (PKC) and p38 MAPK, a member of the mitogen activated protein kinases (MAPKs) family respectively, have markedly inhibited VSL#3-CM-induced iNOS expression and NO production. Slight inhibition was made by LY2904002 and CAY10470, inhibitors of Phosphatidylinocitol 3-kinase (PI3K) and NF-B respectively. Akt inhibitor XIII and MG132 the inhibitors of the protein kinase B (Akt) and proteasome showed no inhibition on VSL#3-CM-induced iNOS and NO production.A balanced expression of iNOS and NO production is necessary for the regulation of normal cellular functions. In this study, it has been shown that VSL#3 has the ability to regulate the function of iNOS and thus the over production of NO in presence of LPS. This action may explain, in part, the proposed anti-inflammatory effects of VSL#3 and thus its potential benefits in IBD. Interestingly, VSL#3 may also induce iNOS expression and NO production through the activation of PKC, p38 MAPK and PI3K pathways but only when applied alone under control conditions. Whether this is specific to macrophages or a common effect of VSL#3 is currently not clear. It would not be considered normal however to recommend VSL#3 for routine use in healthy individuals. The ability of VSL#3 to downregulate the expression and function of iNOS induced by endotoxin however, provides a good rational for its use in inflammatory disease states including IBD.
|Date of Award||1 Jun 2014|
|Supervisor||Janey Henderson (Supervisor)|