The immunomodulation of dynorphin 1-17 and its biotransformation fragments on inflammatory signalling pathways

Abstract

Inflammation is a complex physiological process that involves host defense mechanisms in response to the intrusion of harmful external stimuli. Uncoordinated inflammatory responses, however, are the underlying pathophysiological cause of many chronic diseases. Inflammation is characterised by the burgeoning migration of leukocytes to the point of injury and subsequent release of pro-nociceptive mediators, generating inflammatory pain. Dynorphin 1-17 (DYN 1-17) is endogenously produced and released from leukocytes upon stimulation by local inflammatory factors in the inflamed area. This opioid peptide primarily binds to kappa-opioid receptor (KOR) to produce analgesia. Under inflammatory milieu, DYN 1-17 undergoes spontaneous degradation, yielding a variety of opioid and non-opioid fragments that may have significant implications in inflammation. The underlying cellular effects of these fragments remain unclear. This thesis seeks to explore potential mechanistic insights into selected major DYN 1-17 fragments, identified from a previous biotransformation study of DYN 1-17 in rodent inflamed tissue. This thesis examines the DYN 1-17 fragments modulation of intracellular signals associated with inflammation and thereby, gain an insight into novel therapeutic targets through exploring these endogenous mechanisms.Utilising THP-1 macrophages as an in vitro model of inflammation, this thesis begins with a semiquantitative assessment of nuclear factor-kappa B/p65 (NF-κB/p65) translocation, a major transcription factor that regulates genes responsible for immune and inflammatory responses. The findings presented in Chapter Three of this thesis demonstrated that DYN 1-17 and selected major fragments (DYN 1-6, 1-7, 1-9, 1-10, 1-11, 3-14, 6-12, 2-17 and 7-17) significantly attenuated NF-κB/p65 nuclear translocation induced by lipopolysaccharide (LPS), with the greatest reduction observed with DYN 1-7 at 10 nM. A selective KOR antagonist, ML-190, was used to examine KOR involvement in this inhibitory action. ML-190 significantly reversed the inhibition of NF-κB/p65 translocation produced by DYN 1-17, DYN 1-6, DYN 1-7 and DYN 1-9, but not DYN 1-10 and DYN 1-11.Cytokine production is linked as a downstream process to NF-κB activation; hence it is necessary to evaluate the ability of DYN 1-17 and selected biotransformation fragments in the modulation of IL-1β and TNF-α release in differentiated THP-1 cells, as presented in Chapter Four, to gain an insight into the effects on major pro-inflammatory cytokines. DYN 1-17 and the fragments, demonstrated differential modulatory effects on LPS-induced release of IL-1β and TNF-α. DYN 1-7 and DYN 1-6, inhibited and elevated the secretion of both cytokines, respectively, in a concentration-dependent manner (10 -11 to 10 -7 M). DYN 1-17, however, only inhibited IL-1β release from differentiated iii THP-1 cells and had no effect on TNF-α secretion. Intriguingly, DYN 3-14 at 10 -17 to 10 -11 M demonstrated significant inhibition on IL-1β release and paradoxically increased TNF-α lev...