Ispitivanje protektivnog dejstva grafenskih kvantnih tačaka u modelu neuroinflamacije kod pacova

Abstract

Eksperimentalni autoimunski encefalomijelitis (EAE) je jedan od najizučavanijih animalnih modela neuroinflamacije i najčešće je korišćen animalni model multiple skleroze (MS). Grafenske kvantne tačke (GKT) su tanki listići grafena (<100 nm), elipsastog oblika, sačinjeni iz 1-10 slojeva grafena. U biološkim sistemima, GKT mogu da ispoljavaju prooksidativno i antioksidativno dejstvo a u skorašnjim studijama je pokazano i njihovo imunomodulatorno dejstvo. S toga je cilj ovog istraživanja je bio da se ispita efekat primene GKT na klinički tok bolesti i inflamaciju centralnog nervnog sistema (CNS) u EAE modelu kao i celularne i molekularne mehanizme koji se nalaze u osnovi potencijalnog povoljnog efekta. Efekat GKT kao i mehanizmi njihovog delovanja su ispitivani primenom in vivo, in vitro i ex vivo pristupa. Za indukciju EAE korišćeni su pacovi Dark Agouti (DA) soja koji su imunizovani homogenatom kičmene moždine pacova u kompletnom Frojndovom adjuvansu. U cilju definisanja faze bolesti u kojoj GKT ostvaruju svoje potencijalno povoljno dejstvo, životinje su tretirane sa GKT (10 mg/kg tt, ip) tokom induktivne, efektorske ili obe faze bolesti. Ispitivanje lokalizacije GKT u tkivima životinja, analiza limfocitne infiltracije i apoptoze ćelija CNS i demijelinizacije vršeno je odgovarajućim histološkim metodama. Koristeći tkiva drenirajućeg limfnog čvora koji oslikava aktivnost induktivne faze bolesti i kičmene moždine (KM) koja oslikava aktivnost efektorske faze bolesti, metodom protočne citofluorimetrije je vršena fenotipska karakterizacija izolovanih mononuklearnih ćelija, qRT-PCR metodom ispitivana ekspresija gena za medijatore inflamacije a imunoblotom analizirana aktivacija proteina koji učestvuju u autofagiji i Akt/MAPK signalnim putevima. U in vitro modelu kokultivacije oligodendrocita i neurona sa stimulisanim limfocitima ispitan je protektivan efekat GKT tretmana, MTT testom...

Experimental autoimmune encephalomyelitis (EAE) is one of the most studied animal models of neuroinflammation and also, one of the most commonly used animal models of multiple sclerosis (MS). Graphene quantum dots (GQD) are thin ellipsoidal graphene sheets (<100 nm), made of 1-10-layer thick sheets of graphene. GQD exibit prooxidative and antioxidative effect in biological systems. Also, previous studies show its immunomodulatory effect. Therefore, the aim of this study was to investigate the effect of GQD on clinical course of a disease and central nervous system (CNS) inflammation in EAE model, as well as cellular and molecular mechanisms underlying its potential protective effect. The effect of GQD and its mechanisms of action were examined using in vivo, in vitro and ex vivo approaches. Dark Agouti (DA) rats were immunized with spinal cord homogenate in complete Freund's adjuvant, in order to induce EAE. GQD treatment (10 mg/kg bw, ip) was administrated during the inductive, effector and both phases of a disease. Using the appropriate histological approaches, the presence of lymphocyte infiltration, apoptosis and demyelination of CNS was investigated, as well as the ability of GQD to accumulate in cells of immune system and CNS. Flow cytometry analysis was used to determine phenotype characterization of mononuclear cells isolated from the draining lymph nodes (inductive phase) and spinal cord (SC) tissue (effector phase), while the level of mRNA for the inflammatory mediators was determined by qRT-PCR. Autophagy and Akt/MAPK signaling pathways in draining lymph nodes and SC tissue were investigated using immunoblot assay while the protective effect of GQD treatment on oligodendrocytes and neuronal cells that were co-cultivated with ConA stimulated lymphocytes in vitro, was verified by MTT test...