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Resveratrol je rastlinski fitoestrogen, ki je obetavno terapevtsko sredstvo za preprečevanje in zdravljenje nevrodegenerativnih obolenj. Prehaja krvno-možgansko pregrado, uravnava delovanje živčnih celic in je pomemben modulator funkcije astrocitov. Nevrotrofični dejavnik nevrotrofin-3 (NT-3) omogoča preživetje in diferenciacijo živčnih celic različnih fenotipov in sodeluje pri procesih sinaptične plastičnosti. Astrociti predstavljajo pomemben lokalni vir NT-3, kjer njegovo sintezo uravnavajo različni dejavniki. Vpliv resveratrola na sintezo NT-3 ni poznan, zato smo v pričujoči nalogi proučili možno aktivno vlogo tega fitoestrogena pri spodbujanju sinteze in sproščanja NT-3 iz astrocitov. Študija je potekala na primarnih celičnih kulturah astrocitov možganske skorje podgane. Celice smo izpostavili vplivu resveratrola, agonistom in antagonistom estrogenskih receptorjev ter različnim zaviralcem znotrajceličnih signalnih poti. S specifično encimsko-imunsko metodo za kvantitativno določanje NT-3 proteina smo določili znotrajcelično vsebnost in sproščanje NT-3 iz astrocitov. Spodbudni vpliv resveratrola na sintezo NT-3 je hiter in prehoden ter odvisen od koncentracije in časa inkubacije celic v prisotnosti te snovi. Najučinkovitejši je pri koncentraciji 100 nM, ki po 12-urni inkubaciji povzroči 3-kratno povišanje znotrajcelične vsebnosti NT-3, 12 ur kasneje pa tudi 2,5-kratno povečanje sproščanja NT-3 v primerjavi s kontrolnimi celicami. Z uporabo agonistov (17ß-estradiol, PPT in DPN) in antagonistov (ICI 182,780, MPP, PHTPP) estrogenskih receptorjev smo ugotovili, da je sinteza astrocitnega NT-3 pod vplivom resveratrola receptorski proces, kjer sta aktivno udeležena oba podtipa estrogenskih receptorjev, ER in ERβ. K vplivu resveratrola pomembno prispeva dvig znotrajceličnega Ca2+ in aktivacija PKC in CaMKII, aktivacija encimaPI3K in serin/treonin protein kinaze Akt ter aktivacija MAP kinazne kaskade, ki predstavlja konvergenčno točko omenjenih znotrajceličnih poti. Na podlagi dobljenih rezultatov zaključujemo, da resveratrol preko specifičnih estrogenskih receptorskih mehanizmov učinkovito spodbuja sintezo in sproščanje NT-3 v astrocitih možganske skorje podgane. Resveratrol torej lahko preko spodbujanja nevrotrofične aktivnosti astrocitov pomembno pripomore k procesom sinaptične plastičnosti in ohranjanja funkcije osrednjega živčevja. Resveratrol, a plant phytoestrogen, is a prospective therapeutic agent for prevention and treatment of neurodegenerative diseases. It passes blood-brain barrier, modulates nerve cells and importantly contributes to the astrocyte function. Neurotrophic factor neurotrophin-3 (NT-3) takes part in survival and differentiation of various nerve cell phenotypes, and contributes to synaptic plasticity. Astrocytes are important local source of NT-3, which synthesis is mediated by several factors. The influence of resveratrol on NT-3 synthesis is not known. Therefore in our work, we wanted to test the phytoestrogen's influence on astrocytic NT-3 synthesis and release. In our study we used primary cell cultures of rat cortical astrocytes. The cells were exposed to the influence of resveratrol, oestrogen receptor agonists and antagonists and several intracellular protein inhibitors. By specific enzyme immunoassay for quantitative NT-3 determination, we determined intracellular NT-3 content and release from astrocytes. Positive influence of resveratrol on NT-3 synthesis is fast but temporal, and dependent from incubation time and concentration of the substance. Most effective is at the 100 nM concentration, which causes a 3-fold increase in intracellular levels of NT-3 after 12-hour of incubation and 2.5-fold increase in NT-3 secretion 12 hours later, in comparison with control cells. With the oestrogen receptor agonists, 17ß-estradiol, PPT or DPN and antagonists ICI 182,780, MPP or PHTPP, we discovered that astrocytic NT-3 synthesis is both, ERα and ERβ, oestrogen receptor subtypes dependent process. Resveratrol causes intracellular Ca2+ elevation and further PKC and CaMKII activation. It also activates PI3K and serin/threonin Akt kinasis and MAP kinase downstream cascade, which is the convergent point of the above mentioned protein cascades. Based on results obtained from our study we conclude that resveratrol effectively promotes synthesis and release of NT-3 in rat cortical astrocytes through specific oestrogen receptor mechanisms. Through astrocytic neurotrophic activity support resveratrol can significantly contribute to synaptic plasticity and central nervous system activity. |
