The effects of reference monoterpene compounds and Gentiana lutea root extract on apoptosis and necroptosis in human peripheral blood mononuclear cells

Gentiana lutea je lekovita biljka koja se vekovima koristi u narodnoj medicini, a njena primena je u mnogim aspektima potkrepljena naučnim podacima. S obzirom da je zapaženo da u zavisnosti od eksperimentalnih uslova osim pozitivnih može ispoljavati i citotoksični i genotoksični uticaj, cilj ove doktorske disertacije je bila procena odgovora neizmenjenih mononuklearnih ćelija periferne krvi čoveka (PBMC) i stimulisanih na deobu fitohemaglutininom (PHA-PBMC) na tretman referentnim monoterpenskim komponentama (MRK) i ekstraktom korena Gentiana lutea (EKG), identifikacija ćelijske smrti tipa apoptoze i nekroptoze izazvane uticajem tretmana i procena selektivne aktivacije ispitivanih tipova ćelijske smrti. Rezultati ove doktorske disertacije pokazali su da citotoksični i genotoksični potencijal EKG zavise od koncentracije i dužine izlaganja, kao i od karakteristika ćelijske kulture. EKG sadrži značajne količine MRK, a posebno genciopikrozid (Gp), koje pojedinačno imaju veći genotoksični potencijal od samog EKG, stoga je uočeni citotoksični i genotoksični uticaj EKG verovatno uslovljen odnosima MRK i drugih sekundarnih metabolita dobijenih ekstrakcijom. Citotoksične koncentracije MRK aktiviraju kaspazno-zavisnu apoptozu usled indukovanog oštećenja molekula DNK, tako da je aktivacija apoptoze neselektivna i može biti praćena drugim tipovima ćelijske smrti. Dodatno, Gp i svertiamarin (Sm) tretmani u ćelijama mogu aktivirati i ćelijsku smrt tipa nekroptoze. Ovim saznanjima se ne osporava blagotvorno dejstvo EKG primenjenog u manjim dozama i u kraćem vremenskom periodu, već se ukazuje da neadekvatna priprema i primena lako dostupnog biljnog materijala može imati i štetne posledice.Gentiana lutea is a medicinal plant that has been used in folk medicine for centuries, while its application is, in many aspects, scientifically proven. Since its effects vary from positive to cytotoxic and genotoxic depending on the experimental conditions, the aim of this doctoral dissertation was to evaluate the response of primary human peripheral blood mononuclear cells (PBMC) and those stimulated with phytohemagglutinin (PHA-PBMC) to reference monoterpene components (MRK) and Gentiana lutea root extract (EKG) treatment; to identify the type of treatment-provoked cell death (apoptosis or necroptosis) and to assess the selectivity of investigated cell death types’ activation. The obtained results indicate that the cytotoxic and genotoxic potential of EKG depends on concentration and time of exposure to EKG, as well as on the characteristics of cell culture. EKG contains significant amounts of MRK, especially gentiopicroside (Gp), which individually have a higher genotoxic potential than the EKG, so the observed cytotoxic and genotoxic effects of the EKG are probably due to the ratios of MRK and other secondary metabolites acquired after extraction. Cytotoxic concentrations of MRK activate caspase-dependent apoptosis due to treatment-induced DNA damage, indicating that activation of apoptosis is nonselective and may be accompanied by the other types of cell death. Additionally, Gp and swertiamarin (Sm) treatments can activate necroptosis-type cell death. The findings of this study do not dispute the beneficial effects of EKG applied in smaller doses and over a shorter period of time, but they do emphasize the necessity of proper preparation and application of easily available herbal material that otherwise may have an adverse impact

Lomljivost telomera u bolničkih radnika profesionalno izloženih niskim dozama ionizirajućega zračenja

AbstractBiološki učinci ionizirajućega zračenja (IZ) pripisuju se oštećenjima DNA i indirektnim učincima kroz povećanu proizvodnju reaktivnih vrsta kisika. Iako se telomere rabe kao pokazatelji radioosjetljivosti, o njihovu ponašanju kao odgovoru na ionizirajuće zračenje u uvjetima profesionalne izloženosti i dalje se raspravlja. U ovom radu željeli smo istražiti duljinu i strukturu telomera u bolničkih radnika koji su profesionalno izloženi ionizirajućem zračenju te povezati te nalaze s oksidacijskim biomolekulama i kromosomskim aberacijama. Uzorci krvi izloženih ispitanika i zdravih kontrola uzeti su za analizu tijekom rutinskoga godišnjeg zdravstvenog pregleda. Osim kromosomskih aberacija, u uzorcima plazme izmjereni su i parametri oksidacijskoga stresa [prooksidacijska/antioksidacijska ravnoteža (PAB), lipidna peroksidacija i 8-okso-dG], a procjena duljine i strukture telomera provedena je metodom Q-FISH na metafaznim kromosomima. Analiza kromosomskih aberacija pokazala je da od 34 ispitanika njih 14 ima kromosomske aberacije (skupina 1), a 20 nije imalo aberacije (skupina 2). Nije bilo značajne razlike u spolu ili dobi ni u duljini telomera između skupina. Međutim, incidencija lomljivih telomera bila je značajno veća u objema skupinama ispitanika izloženih IZ-u u usporedbi s kontrolnim ispitanicima. Produkti peroksidacije lipida i 8-okso-dG također su bili značajno viši u objema skupinama. Učestalost lomljivih telomera u pozitivnoj je korelaciji (statistički značajna) s razinama 8-okso-dG

Optimizacija metode za izolaciju epitelnih stanica iz nežljezdanog dijela želuca štakora za protočnu citometriju

Traditional methods in cell proliferation studies are based on immunohistochemical detection of proliferating cells in the target tissue. Since they are time consuming, optimization of novel, more efficient methods is important for large scale proliferation studies. In this study, we aimed to optimize the isolation of single epithelial rat forestomach cells for flow cytometry. As a marker of cellular proliferation we used the Ki-67 antibody to detect this nuclear protein expressed in proliferating cells. We also performed immunohistochemical detection of Ki-67 positive cells and propidium iodide staining to validate the results. 3-tert- butyl -4-hydroxyanisole was used as the positive control to ensure cellular proliferation. The results showed that isolation of epithelial cells with collagenase, trypsin and cell strainer ensures great cell viability (>95%) and the purity of the samples. Flow cytometry and immunostaining with the Ki-67 antibody indicated that 3-tert- butyl-4-hydroxyanisole treatment leads to a significant increase in proliferation. A significant positive correlation was observed between the results obtained by immunohistochemistry and flow cytometry, but the flow cytometric data had a smaller measurement error, suggesting the equal sensitivity and greater accuracy of this method. Propidium iodide staining showed that the percentage of cells in the G2+S phase of the cell cycle correlated positively with the percentage of Ki-67 positive cells assessed by flow cytometry, indicating that Ki-67 positive cells reflect an active dividing cell pool. We conclude that the isolation of forestomach epithelial cells described is a simple and reliable method for obtaining viable cells for use in flow cytometry. Compared to immunohistochemistry, flow cytometric detection of the Ki-67 antigen is equally sensitive, but much faster and provides more accurate results.Tradicionalne metode u ispitivanju stanične proliferacije temelje se na imunohistokemijskom otkrivanju proliferacijskih stanica u ciljanom tkivu. Kako su dugotrajne, optimizacija novih i učinkovitijih metoda važna je za velika istraživanja o proliferaciji. U ovom smo radu željeli optimizirati izolaciju epitelnih stanica prednjeg želuca štakora za protočnu citometriju. Kao marker stanične proliferacije koristili smo Ki-67 protutijelo za otkrivanje ovoga nuklearnog proteina izraženog u proliferacijskim stanicama. Također smo učinili imunohistokemijsku detekciju Ki- 67 pozitivnih stanica i bojenje propidij-jodidom kako bismo potvrdili rezultate. Butil-hidroksianizol korišten je kao pozitivna kontrola da se osigura stanična proliferacija. Rezultati su pokazali da izolacija epitelnih stanica s kolagenazom, tripsinom i staničnim cjedilom osigurava veliku vijabilnost stanica (> 95 %) i čistoću uzoraka. Protočna citometrija i Ki-67 bojenje pokazali su da tretman butil-hidroksianizolom dovodi do znakovitog porasta proliferacije. Primijećena je znakovita pozitivna korelacija između rezultata dobivenih imunohistokemijom i protočnom citometrijom, dok su protočni citometrijski podaci imali manju pogrešku mjerenja, što upućuje na jednaku osjetljivost i veću točnost ove metode. Bojenje propidij-jodidom pokazalo je da postotak stanica u G2+S fazi staničnog ciklusa pozitivno korelira s postotkom Ki-67 pozitivnih stanica procijenjenih protočnom citometrijom, što upućuje na to da Ki-67 oslikava stanice u aktivnoj diobi. Zaključujemo da je opisana izolacija epitelnih stanica prednjeg želuca štakora jednostavna i pouzdana metoda za dobivanje održivih stanica za upotrebu u protočnoj citometriji. U usporedbi s imunohistokemijom, protočna citometrijska detekcija antigena Ki-67 jednako je osjetljiva, ali mnogo brža i daje točnije rezultate

Prolonged exposure to mesoporous silica decrease cell viability in vitro

„Contemporary Materials 2021“ : XIV International Scientific Conference : programme and book of abstracts : September 9-10, 2021; Banja Luka, Bosnia and Herzegovin

Synthesis and characterisation of SBA-15 with spherical particles

„Contemporary Materials 2021“ : XIV International Scientific Conference : programme and book of abstracts : September 9-10, 2021; Banja Luka, Bosnia and Herzegovin

Vodeni ekstrakt kaduljina lišća umanjuje upalu i oksidacijsku genotoksičnost u ljudskim mononuklearnim stanicama periferne krvi

Traditional medicine has used sage (Salvia officinalis L.) preparations for centuries to prevent and treat various inflammatory and oxidative stress-induced conditions. The aim of this in vitro study was to determine the bioactive properties of a sage leave extract obtained with environmentally friendly aqueous extraction and lyophilisation in primary human peripheral blood cells. To that end we measured the total phenolic and flavonoid content (TPC and TFC, respectively) with gas chromatography-mass spectrometry (GC-MS). Non-cytotoxic concentrations determined with the trypan blue assay were used to assess the antioxidant (DPPH, ABTS, and PAB assay), antigenotoxic (CBMN assay), immunomodulatory (IL-1β and TNF-α), and neuroprotective effects (AChE inhibition). The extract contained high TPC (162 mg GAE/g of dry extract) and TFC (39.47 mg QE/g of dry extract) concentrations, while β-thujone content was unexpectedly low (below 0.9 %). Strong radical-scavenging activity combined with glutathione reductase activation led to a decrease in basal and H2O2-induced oxidative stress and DNA damage. A decrease in TNF-α and increase in IL-1β levels suggest complex immunomodulatory response that could contribute to antioxidant and, together with mild AChE inhibition, neuroprotective effects. Overall, this study has demonstrated that aqueous sage leave extract reduces the levels of thujone, 1,8-cineole, pinene, and terpene ketones that could be toxic in high concentrations, while maintaining high concentrations of biologically active protective compounds which have a potential to prevent and/or treat inflammatory and oxidative stress-related conditions.Salvia officinalis L. stoljećima se koristi u tradicionalnoj medicini za prevenciju i liječenje raznih upalnih i oksidacijskim stresom izazvanih poremećaja. U ovoj studiji željeli smo ekstrahirati kaduljino lišće korištenjem ekološki prihvatljivog, “zelenog” pristupa vodenom ekstrakcijom i liofilizacijom te odrediti njegova bioaktivna svojstva u primarnim ljudskim perifernim krvnim stanicama. Ukupni sadržaj fenola i flavonoida i GC-MS korišteni su za karakterizaciju ekstrakta. Necitotoksične koncentracije, određene metodom bojenja s bojom tripan plavo, analizirane su za procjenu antioksidacijskih (DPPH, ABTS i PAB test), antigenotoksičnih (CBMN test), imunomodulacijskih (IL-1β i TNF-α) i neuroprotektivnih učinaka (AChE inhibicija). Ekstrakt je sadržavao visoku koncentraciju ukupnih fenola (162 mg GAE/g liofilizata) i flavonoida (39,47 mg QE/g liofilizata), dok je sadržaj β-tujona bio neočekivano nizak (niži od 0,9 %). Snažna aktivnost hvatanja radikala u kombinaciji s aktivacijom glutation reduktaze dovela je do smanjenja bazalnog i H2O2 induciranog oksidacijskog stresa i oštećenja DNA. Smanjenje TNF-α i povišenje razine IL-1β sugeriraju kompleksan imunomodulatorni odgovor koji bi mogao pridonijeti antioksidacijskim i, zajedno s blagom inhibicijom AChE, neuroprotektivnim učincima. Sveukupno, ova je studija pokazala da vodena ekstrakcija kaduljina lišća smanjuje toksične spojeve kao što su tujon, 1,8-cineol, pinen i terpenski ketoni, a održava visoku koncentraciju biološki aktivnih zaštitnih spojeva u ekstraktu, što bi moglo imati potencijal za prevenciju i/ili liječenje oksidacijskih i upalnih poremećaja

Influence of synthesis conditions on adsorption capacity of SBA-15/alginate adsorbent for removal ions of nickel(II) and lead(II) from aqueous solution

„Contemporary Materials 2021“ : XIV International Scientific Conference : programme and book of abstracts : September 9-10, 2021; Banja Luka, Bosnia and Herzegovin

Nanotechnology approach for diminishing quercetin toxicity toward peripheral blood mononuclear cells

Quercetin (Q) is one of the most common and well researched antioxidant flavonoids, which usually occurs in plant-based foods, and medicinal plants. It was shown that quercetin exerts many beneficial effects on human health, including prevention of cancer and heart diseases. Quercetin was found to be toxic toward various types of cancer cell, still, at higher concentrations it was also shown toxic toward normal human cells [1,2]. One of the approaches to overcome this shortcoming offers nanotechnology which enables the novel perspective of phytochemical usage in contemporary medicine [3]. The strategy of binding quercetin to the gold nanoparticles during their synthesis was used, which resulted in quercetin capped gold nanoparticles (NPQ) [4]. Trypan blue exclusion test [5] was used to evaluate peripheral blood mononuclear cells (PBMC) viability after their exposure to either NPQ or free Q during 24, 48 and 72 h, at 37 °C, in the range of quercetin concentrations from 5 to 50 μg/mL. A significant reduction in the cell count was observed in PBMC cultures treated with 10, 20, and 50 μg/mL of free Q, for all exposure times. The treatments of increasing concentrations and exposure times lowered the cells viability, resulting in 63% of the viable cells, following 72 h of the treatment with 50 μg/mL of free Q. Although NPQ treatments affected the cells viability in a concentration- and time-dependent manner the treatment with 50 μg/mL of NPQ for 72 h, had a milder effect on PBMC cultures than free Q, resulting in 81% of the viable cells (Figure 1). According to the obtained results, NPQ were shown less toxic toward PBMC than free Q.1-EuSPMF - 1st European Symposium on Phytochemicals in Medicine and Food; 7-9 September, 2022; Belgrad

Sweroside displays a cytotoxic effect by activating apoptosis in human peripheral blood mononuclear cells

Sweroside (Sw) is a secondary metabolite commonly found in plants belonging to the Gentianaceae family [1]. This iridoid compound is well-known for its anti-inflammatory [2], antidiabetic [3] and antitumor properties [4], which have been studied in pathological model systems. In transformed cell lines, Sw displays an antitumor effect by apoptosis activation [5]. Since healthy cells are also exposed to cancer therapy applied in vivo, our goal was to determine Sw’s cytotoxic concentration in primary human peripheral blood mononuclear cells (PBMCs) after 48 h of treatment with a range of concentration from 20 μM to 130 μM Sw in vitro, and to analyze whether the cytotoxic effect was due to activated apoptosis. According to the obtained results of the trypan blue dye exclusion test, 48 h of treatment with 50 μM Sw and higher concentrations led to a significant decrease in cell number. The DNA fragmentation assay indicated that following 50 μM Sw treatment, cells are dying in an apoptosis-like manner since the level of DNA fragments was 3.5 times higher than in the untreated control. The type of cell death was confirmed by immunoblot analysis of apoptosis- specific protein markers, which revealed the elevation of cleaved caspase-3 and PARP1 89 kDa fragments. Our findings showed that like in transformed cell lines, Sw in healthy cells can also activate apoptosis. A potential difference in sensitivity to Sw treatment between healthy and transformed cells could justify Sw treatment in anticancer therapy.1-EuSPMF - 1st European Symposium on Phytochemicals in Medicine and Food; 7-9 September, 2022; Belgrad

The influence of hydrochloric acid on the features of SBA-15 particles

The template method synthesis of both SBA-15 materials with elongated and spherical particles was performed using a surfactant Pluronic P123. The HCl (p.a.) was used to synthesize material with elongated particles connected in chain structures grouped into shapes resembling sheaves of wheat. In the synthesis of spherical SBA-15 with diameters ranging from 0.5 to 2 μm, a spent HCl solution which was obtained after chemical treatment of clay was used where the dominant presence of the spheres was confirmed by the SEM method. In addition to the methods mentioned above, XRD, EDS and FTIR methods characterize SBA-15 materials