Uticaj purinskih nukleozidnih analoga, ribavirina i tiazofurina, na aktivaciju mikroglije u uslovima inflamacije
The effect of purine nucleoside, analogues, ribavirin and tiazofurin, on microglial activation under inflammatory conditions
Doctoral thesis (Published version)
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Activation of microglia is the hallmark of experimental autoimmune encephalomyelitis (EAE) and other neurodegenerative disorders associated with chronic neuroinflammation. The present study addressed the potency of purine nucleoside analogues ribavirin (RBV) and tiazofurin (TR) to modulate the microglial inflammatory response in vivo and in vitro. In this study, we induced animal model of multiple sclerosis – experimental autoimmune encephalomyelitis (EAE) by subcutaneous injection of encephalitogenic emulsion in the hind paw of Dark Agouti rats and showed that therapeutic treatment of EAE animals with combination of these two nucleoside analogues reduced disease severity. In addition, histological analysis indicated that RBV and TR also decreased the number and activation status of microglial cells. Since the combined treatment with RBV and TR leads to suppression of EAE symptoms and reduction of reactive microgliosis, we proposed hypothesis that this treatment, in addition to previously described effects in immune and vascular system, may act in central nervous system targeting primarily microglial cells. Therefore, in further experiments we examined effect of RBV, TR and their combination (RBV+TR) on primary microglial cell culture. Activation of microglial cells with bacterial lipopolysaccharide (LPS) triggers typical microglial responses to inflammation. Anti-inflammatory potential of RBV, TR and RBV+TR was tested in terms of influencing microglial morphological changes, NO production, nuclear factor-κB translocation and proinflammatory cytokines release, after LPS stimulation. The cytotoxic effects of RBV (1 – 20 μM), TR (1 – 20 μM) and their combination (10 μM RBV + 5 μM TR) are determined, as well as the dosages that achieved half-maximal inhibitory effect (EC50). RBV, TR and combination treatment lowered NO levels in LPS stimulated cultures as a result of decreased cell number. RBV, TR and RBV+TR induced shrinkage of average cell surface of LPS activated amoeboid microglial cells. Treatment with RBV, TR and RBV+TR of activated microglia strongly reduced the nuclear translocation of NF-κB/p65 and suppressed proinflammatory immune response genes. Applied treatments also exerted suppression of TNF-α and IL-6 release in activated microglia manly by reduction of cell number. At the same time, RBV and RBV + TR treatment additionally increased LPS-induced IL-1β gene expression and release. In primary microglial cell culture, gained results confirmed gene and protein expression of equilibrative nucleoside transporters (ENTs), proteins that are responsible for the putative entry of RBV and TR into cells. Results obtained in this study indicate that RBV and TR, alone or in a combination, attenuate neuroinflammatory response, probably by exerting cytotoxicity on activated microglial cells, reduction of NO levels, attenuation NF-κB signaling and decrease of proinflammatory cytokine release.
Keywords:Central nervous system; Inflammation; Microglia; Experimental autoimmune encephalomyelitis; Cell culture
Source:University of Belgrade, Faculty of Biology, 2012, 1-108
- Cellular and molecular basis of neuroinflamation: potential targets for translational medicine and therapy (RS-41014)