20.500.12556/RUL-177874 Vplivi kanabidiola na fetalne humane astrocite Influences of cannabidiol on fetal human astrocytes Kanabidiol (CBD) je fitokanabinoid, naravno prisoten v rastlinah rodu Cannabis (konoplja). Kljub njegovi naraščujoči priljubljenosti, molekularni mehanizmi njegovega delovanja še niso v celoti raziskani. Toksični učinki ∆9-tetrahidrokanabinola (THC) so znani že nekaj časa, vpliv CBD na razvijajoče se možgane pa ostaja nepojasnjen. V okviru magistrske naloge smo želeli preučiti vplive CBD na fetalne humane astrocite iz možganske skorje v kulturi. Predvidevali smo, da CBD deluje citotoksično, znižuje celični metabolizem astrocitov in vstopa v interakcije z endogenim kanabinoidnim sistemom (EKS) humanih fetalnih astrocitov. Celice smo izpostavili 0,5; 1 ali 5 µM CBD v različnih časovnih intervalih (1 do 24 ur). Citotoksičnost smo preverjali s testom viabilnosti AlamarBlue®, z merjenjem aktivnosti laktat dehidrogenaze (LDH) zunaj celic in merjenjem aktivnosti kaspaze-3/7, določili pa smo tudi vsebnost proteinov v celicah. CBD je v vseh preučevanih koncentracijah povzročil znižanje viabilnosti celic. Aktivnost LDH je bila povišana pri višjih koncentracijah (1 in 5 µM), iz česar sklepamo, da je bila motena integriteta celične membrane, kar je značilno za nekroptozo in nekrozo. Pri istih koncentracijah je CBD povzročil upad vsebnosti proteinov in povišano aktivnost kaspaze-3/7, ki ima osrednjo vlogo v procesu apoptoze. Vpliv na celični metabolizem smo preučevali z merjenjem znotrajcelične koncentracije ATP in merjenjem sprememb mitohondrijskega membranskega potenciala (∆Ψm), pri čemer smo ugotovili, da je CBD povzročil hiter upad količine ATP ter upad ∆Ψm po 24-urni izpostavitvi. Da bi preverili, ali CBD vstopa v interakcije z EKS, smo preučili vpliv različnih koncentracij CBD na izražanje genov komponent EKS. CBD je znižal ekspresijo genov CNR1 in CNR2 za receptorje CB1 in CB2, ter zvišal ekspresijo genov TRPV1 in MGLL, ki kodirata receptor TRPV1 in encim MAGL. Zaključili smo, da CBD deluje citotoksično in znižuje presnovno aktivnost celic ter vpliva na funkcijo EKS fetalnih humanih astrocitov v kulturi. Cannabidiol (CBD) is a phytocannabinoid that is naturally present in plants from genus Cannabis. Despite its growing popularity, CBD's molecular mechanisms of action are not fully understood yet. Toxic effects of ∆9-tetrahydrocannabinol (THC) have been known for a while now, meanwhile possible effects of CBD on the developing brain remain unexplained. The aim of our study was to investigate possible influences of cannabidiol on cortical fetal human astrocytes in culture. We hypothesized that CBD exerts cytotoxic effects, decreases cell metabolism and interacts with endocannabinoid system (ECS) of astrocytes. Cells were incubated with 0,5; 1 or 5 µM CBD over different time intervals (1 to 24 h). To evaluate potential cytotoxic effects, we employed a cell viability test AlamarBlue®, lactate dehydrogenase (LDH) activity assay and caspase-3/7 activity assay, as well as measurments of protein content in cells. All CBD concentrations caused a decrease in cell viability. LDH activity was increased at higher concentrations of CBD (1 and 5 µM), suggesting the cell membrane integrity was compromised, which is a sign of necroptosis or necrosis. Furthermore, the same CBD concentrations caused a decrease of cellular protein content and an increase of caspase-3/7 activity, pointing to apoptosis. In order to assess the influences of CBD on cell metabolism, we measured intracellular ATP content and changes in mitochondrial membrane potential (∆Ψm). CBD induced a rapid ATP depletion and a decrease in ∆Ψm after 24 h of exposure. To determine whether CBD interacts with ECS, we concluded a gene expression analysis. Different concentrations of CBD downregulated the expression of CBN1 and CBN2, which encode CB1 and CB2 receptors, upregulated the expression of TRPV1 and MGLL, which encode TRPV1 receptor and MAGL enzyme, while having no significant influence on the expression of PPARG, DAGLA, NAPEPLD and FAAH. Based on our results, we conclude that CBD has cytotoxic effects, lowers cell metabolism and affects the function of ECS in fetal human astrocytes. astrociti kanabidiol fitokanabinoidi endokanabinoidni sistem citotoksičnost celični metabolizem astrocytes cannabidiol phytocannabinoids endocannabinoid system cytotoxicity cell metabolism true false false [V. Z. Štok] Slovenski jezik Angleški jezik Magistrsko delo/naloga 2026-01-11 07:15:07 2026-01-11 07:15:32 2026-01-12 08:13:46 0000-00-00 00:00:00 2025 0 0 0000-00-00 NiDoloceno NiDoloceno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 1970-01-01 576.34:57.083.36(043.2) 278527 264244995 55604.pdf 55604.pdf 1 292D0BB9B3FDC1BC2C5CEF293A2EA46A fa33cccc40b0c2dfd22f86c0e05de0c9d0db094b1136eb5462015f5bfb129b62 ea239294-eeb4-11f0-bf92-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=225263 Biotehniška fakulteta 0 0 0