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The effect of purine nucleoside, analogues, ribavirin and tiazofurin, on microglial activation under inflammatory conditions

dc.contributor.advisorPeković, Sanja
dc.contributor.otherNedeljković, Nadežda
dc.contributor.otherLavrnja, Irena
dc.contributor.otherBožić, Biljana
dc.creatorSavić, Danijela
dc.date.accessioned2016-01-05T11:45:45Z
dc.date.available2016-01-05T11:45:45Z
dc.date.available2020-07-03T08:14:05Z
dc.date.issued2012-10-01
dc.identifier.urihttp://eteze.bg.ac.rs/application/showtheses?thesesId=29
dc.identifier.urihttps://nardus.mpn.gov.rs/handle/123456789/2043
dc.identifier.urihttps://fedorabg.bg.ac.rs/fedora/get/o:2285/bdef:Content/download
dc.identifier.urihttp://vbs.rs/scripts/cobiss?command=DISPLAY&base=70036&RID=41536015
dc.description.abstractAktivacija mikroglije jedno je od glavnih obeležja eksperimentalnog autoimunskog encefalomijelitisa (EAE) i drugih neuroloških poremećaja koje odlikuje hronična inflamacija. Cilj ove studije bio je da se proceni sposobnost dva analoga purinskih nukleozida, ribavirina (RBV) i tiazofurina (TR), da modulišu inflamatorni odgovor aktivirane mikroglije, in vivo i in vitro. U in vivo studiji, koja je izvedena na EAE modelu indukovanom ubrzgavanjem encefalitogene emulzije u šapu Dark Agouti pacova, pokazano je da tretman životinja kombinacijom oba analoga, počev od pojave prvih simptoma pa tokom sledećih 15 dana, dovodi do smanjenja težine bolesti, a na histološkom planu do smanjenja broja i stepena aktivacije mikroglije. Budući da kombinovani tretman RBV i TR dovodi do supresije simptoma EAE i do redukcije reaktivne mikroglioze, formulisana je hipoteza da ovaj tretman, pored ranije opisanih sistemskih efekata u imunskom i vaskularnom sistemu, ostvaruju centralni efekat i to delujući prvenstveno na ćelije mikroglije. Uticaj RBV i TR, kao i kombinovane primene oba analoga detaljnije je ispitan u primarnoj kulturi mikroglijskih ćelija. Aktivacija mikroglije, indukovana lipopolisaharidom bakterijskog zida gram negativnih bakterija, (LPS), izazvala je tipičan odgovor reaktivne mikroglioze. Antiinflamatorni potencijal RBV, TR i RBV+TR ispitan je u pogledu citotoksičnosti, indukcije morfoloških promena, produkcije azot monoksida (NO), oslobađanja proinflamatornih citokina i nuklearne translokacije NF-κB. Ispitan je citototoksični potencijal RBV (1 – 20 μM), TR (1 – 20 μM) i kombinovanog tretmana (10 μM RBV + 5 μM TR) i određene su EC50 doze pri kojima se postiže polovina maksimalnog citotoksičnog efekta. RBV, TR i kombinovani tretman smanjuju ukupnu produkciju NO u kulturi stimulisanoj LPS-om, a taj efekat se pripisuje smanjenju ukupnog broja ćelija u kulturi. RBV, TR i RBV+TR menjaju ameboidnu morfologiju aktivirane mikroglije, tako što smanjuju prosečnu površinu ćelija u kulturi. Tretman RBV, TR i RBV+TR smanjuju translokaciju NF-κB/p65 u jedro. Primenjeni tretmani smanjuju ukupno oslobađanje TNF-α i IL-6, što je verovatno posledica smanjenja ukupnog broja aktiviranih ćelija, a RBV i kombinovani tretman povećavaju ekspresiju IL-1β. U primarnoj kulturi mikroglijskih ćelija potvrđena je genska i proteinska ekspresija uravnotežujućih nukleozidnih transportera 1 i 2 (ENT1, 2), koji učestvuju u transportu RBV i TR u ćelije. Ukupno gledano, rezultati dobijeni ovom studijom ukazuju da RBV i TR, pojedinačno ili u kombinaciji, deluju citotoksično na aktiviranu mikrogliju i menjaju njenu morfologiju, a ispoljavaju i imunomodulatorno dejstvo tako što snižavaju produkciju NO, smanjuju signalizaciju posredovanu NF-κB/p65 i redukuju produkciju proinflamatornih citokina.sr
dc.description.abstractActivation 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.en
dc.formatapplication/pdf
dc.languagesr
dc.publisherУниверзитет у Београду, Биолошки факултетsr
dc.relationinfo:eu-repo/grantAgreement/MESTD/Integrated and Interdisciplinary Research (IIR or III)/41014/RS//
dc.rightsopenAccessen
dc.rights.urihttps://creativecommons.org/licenses/by-nc-nd/4.0/
dc.sourceУниверзитет у Београдуsr
dc.subjectCentralni nervni sistemsr
dc.subjectCentral nervous systemen
dc.subjectinflamacijasr
dc.subjectmikroglijasr
dc.subjecteksperimentalni autoimunski encefalomijelitissr
dc.subjectćelijska kulturasr
dc.subjectinflammationen
dc.subjectmicrogliaen
dc.subjectexperimental autoimmune encephalomyelitisen
dc.subjectcell cultureen
dc.titleUticaj purinskih nukleozidnih analoga, ribavirina i tiazofurina, na aktivaciju mikroglije u uslovima inflamacijesr
dc.titleThe effect of purine nucleoside, analogues, ribavirin and tiazofurin, on microglial activation under inflammatory conditionsen
dc.typedoctoralThesisen
dc.rights.licenseBY-NC-ND
dcterms.abstractПековић, Сања; Божић, Биљана; Недељковић, Надежда; Лаврња, Ирена; Савић, Данијела; Утицај пуринских нуклеозидних аналога, рибавирина и тиазофурина, на активацију микроглије у условима инфламације; Утицај пуринских нуклеозидних аналога, рибавирина и тиазофурина, на активацију микроглије у условима инфламације;
dc.identifier.fulltexthttps://nardus.mpn.gov.rs/bitstream/id/3279/Disertacija.pdf
dc.identifier.fulltexthttp://nardus.mpn.gov.rs/bitstream/id/3279/Disertacija.pdf
dc.identifier.doi10.2298/bg20121001savic
dc.identifier.rcubhttps://hdl.handle.net/21.15107/rcub_nardus_2043


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