Human immunity involves both innate and adaptive defence mechanisms, with inflammation playing a central role in responding to cellular injury, pathogenic infections, and allergic stimuli. Reactive oxygen species (ROS) are closely associated with the onset and progression of inflammation. While moderate ROS levels function as crucial signalling molecules, excessive ROS can damage cellular components. This study aimed to evaluate the anti-inflammatory and antioxidant potential of plant-derived bioactive compounds including chlorogenic acid, oleuropein, tomatine, and tyrosol using human monocytic cell models (U-937 and THP-1). Differentiation of U-937 and THP-1 cells was induced prior to treatment with the selected bioactive compounds. Cell morphology and integrity were examined utilizing confocal microscopy. Gene expression stability was evaluated using reference genes β-actin and glyceraldehyde 3-phosphate dehydrogenase (GAPDH). Protein expression levels of key inflammatory markers were determined by Western blot analysis. In addition, molecular docking studies were conducted to assess the binding affinity of the compounds to human target proteins [Interleukin-4 (IL-4), 5-Lipoxygenase (LOX-5), Myeloperoxidase (MPO), and Tumor necrosis factor-alpha ( TNF-α)]. No cytotoxic effects were observed in treated cells, and GAPDH was confirmed as a stable reference gene under all experimental conditions. In U-937 cells, treatment with the bioactive compounds led to increased expression of the anti-inflammatory cytokine IL-4 and decreased expression of MPO. Notably, exposure to chlorogenic acid and tyrosol reduced MPO activity. Oleuropein and tyrosol demonstrated a strong suppressive effect on the expression of LOX-5, an enzyme responsible for leukotriene production. All tested bioactive compounds significantly reduced the phorbol 12-myristate 13-acetate (PMA) induced increase in LOX-5 activity. Molecular docking supported the potential of these compounds to interact with key inflammatory proteins, contributing to reduced oxidative stress. The plant-derived compounds, particularly oleuropein and tyrosol from olives, exhibit promising anti-inflammatory and antioxidant effects by modulating ROS-associated signalling pathways and downregulating inflammatory markers. These findings support the therapeutic potential of agricultural waste-derived bioactive in inflammation management and oxidative stress regulation.

• Klíčová slova
• Antioxidants, Bioactive compound, Free radicals, Inflammation, Interleukin-4, LOX-5, Macrophages, Monocytes, Reactive oxygen species,
• MeSH
• antiflogistika * farmakologie   chemie   MeSH
• antioxidancia farmakologie   MeSH
• arachidonát-5-lipoxygenasa metabolismus   MeSH
• fenethylalkohol analogy a deriváty   farmakologie   MeSH
• iridoidní glukosidy farmakologie   MeSH
• iridoidy farmakologie   MeSH
• kyselina chlorogenová farmakologie   MeSH
• lidé MeSH
• makrofágy * účinky léků   metabolismus   MeSH
• monocyty * účinky léků   metabolismus   MeSH
• oxidační stres * účinky léků   MeSH
• peroxidasa metabolismus   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• simulace molekulového dockingu MeSH
• THP-1 buňky MeSH
• TNF-alfa metabolismus   MeSH
• U937 buňky MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• 4-hydroxyphenylethanol MeSH Prohlížeč
• antiflogistika * MeSH
• antioxidancia MeSH
• arachidonát-5-lipoxygenasa MeSH
• fenethylalkohol MeSH
• iridoidní glukosidy MeSH
• iridoidy MeSH
• kyselina chlorogenová MeSH
• oleuropein MeSH Prohlížeč
• peroxidasa MeSH
• reaktivní formy kyslíku MeSH
• TNF-alfa MeSH

In this study, we investigated the properties of ascorbic acid (vitamin C), which is a naturally occurring water-soluble vitamin. Our goal is to evaluate its pro-oxidative and/or antioxidant capabilities. To do this, we initially used a confocal laser scanning microscope (CLSM) to visualize the differentiation pattern in U-937 cells under the treatment of variable concentrations of ascorbic acid. Prior to induction, U-937 cells showed a spherical morphology. After treatment, significant morphological changes were observed in the form of prominent pseudopodia and amoeboid structures. Interestingly, pseudopodia incidences increased with an increase in ascorbic acid concentrations. In addition, our analysis of protein modification using anti-malondialdehyde antibodies showed changes in more than one protein. The findings reveal the link between the differentiation of U-937 cells into macrophages and the protein modifications triggered by the production of reactive oxygen species when U-937 cells are exposed to ascorbic acid. Furthermore, the transformation of ascorbic acid from a pro-oxidative to an antioxidant property is also demonstrated.

• Klíčová slova
• Antioxidants, Human cells, Pro-oxidant, Reactive oxygen species, Vitamin C,
• Publikační typ
• časopisecké články MeSH

N-acetylcysteine (NAC), often used as an antioxidant-scavenging reactive oxygen species (ROS) in vitro, was recently shown to increase the cytotoxicity of other compounds through ROS-dependent and ROS-independent mechanisms. In this study, NAC itself was found to induce extensive ROS production in human leukemia HL-60 and U937 cells. The cytotoxicity depends on ROS-modulating enzyme expression. In HL-60 cells, NAC activated NOX2 to produce superoxide (O2•-). Its subsequent conversion into H2O2 by superoxide dismutase 1 and 3 (SOD1, SOD3) and production of ClO- from H2O2 by myeloperoxidase (MPO) was necessary for cell death induction. While the addition of extracellular SOD potentiated NAC-induced cell death, extracellular catalase (CAT) prevented cell death in HL-60 cells. The MPO inhibitor partially reduced the number of dying HL-60 cells. In U937 cells, the weak cytotoxicity of NAC is probably caused by lower expression of NOX2, SOD1, SOD3, and by the absence of MOP expression. However, even here, the addition of extracellular SOD induced cell death in U937 cells, and this effect could be reversed by extracellular CAT. NAC-induced cell death exhibited predominantly apoptotic features in both cell lines. Conclusions: NAC itself can induce extensive production of O2•- in HL-60 and U937 cell lines. The fate of the cells then depends on the expression of enzymes that control the formation and conversion of ROS: NOX, SOD, and MPO. The mode of cell death in response to NAC treatment bears apoptotic and apoptotic-like features in both cell lines.

• Klíčová slova
• HL-60 cells, MPO, N-acetylcysteine, NOX, SOD, U937 cells, oxidative stress,
• MeSH
• acetylcystein farmakologie   MeSH
• HL-60 buňky MeSH
• katalasa genetika   MeSH
• leukemie farmakoterapie   genetika   metabolismus   MeSH
• lidé MeSH
• NADPH-oxidasa 2 genetika   MeSH
• oxidační stres účinky léků   MeSH
• peroxidasa genetika   MeSH
• proliferace buněk účinky léků   MeSH
• reaktivní formy kyslíku metabolismus   MeSH
• regulace genové exprese u nádorů účinky léků   MeSH
• stanovení celkové genové exprese MeSH
• superoxiddismutasa genetika   MeSH
• U937 buňky MeSH
• viabilita buněk účinky léků   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetylcystein MeSH
• CYBB protein, human MeSH Prohlížeč
• katalasa MeSH
• MPO protein, human MeSH Prohlížeč
• NADPH-oxidasa 2 MeSH
• peroxidasa MeSH
• reaktivní formy kyslíku MeSH
• superoxiddismutasa MeSH

Free radical-mediated activation of inflammatory macrophages remains ambiguous with its limitation to study within biological systems. U-937 and HL-60 cell lines serve as a well-defined model system known to differentiate into either macrophages or dendritic cells in response to various chemical stimuli linked with reactive oxygen species (ROS) production. Our present work utilizes phorbol 12-myristate-13-acetate (PMA) as a stimulant, and factors such as concentration and incubation time were considered to achieve optimized differentiation conditions. ROS formation likely hydroxyl radical (HO●) was confirmed by electron paramagnetic resonance (EPR) spectroscopy combined with confocal laser scanning microscopy (CLSM). In particular, U-937 cells were utilized further to identify proteins undergoing oxidation by ROS using anti-DMPO (5,5-dimethyl-1-pyrroline N-oxide) antibodies. Additionally, the expression pattern of NADPH Oxidase 4 (NOX4) in relation to induction with PMA was monitored to correlate the pattern of ROS generated. Utilizing macrophages as a model system, findings from the present study provide a valuable source for expanding the knowledge of differentiation and protein expression dynamics.

• Klíčová slova
• HL-60 cells, NADPH oxidase, NOX4, U-937 cells, macrophages, phorbol 12-myristate 13-acetate, protein-centered radicals,
• MeSH
• acetofenony farmakologie   MeSH
• barvení a značení MeSH
• buněčná diferenciace * účinky léků   MeSH
• elektronová paramagnetická rezonance MeSH
• HL-60 buňky MeSH
• hydroxylový radikál MeSH
• lidé MeSH
• monocyty cytologie   účinky léků   metabolismus   MeSH
• NADP metabolismus   MeSH
• proliferace buněk účinky léků   MeSH
• proteiny metabolismus   MeSH
• tetradekanoylforbolacetát farmakologie   MeSH
• tvar buňky účinky léků   MeSH
• U937 buňky MeSH
• viabilita buněk účinky léků   MeSH
• volné radikály metabolismus   MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• acetofenony MeSH
• acetovanillone MeSH Prohlížeč
• hydroxylový radikál MeSH
• NADP MeSH
• proteiny MeSH
• tetradekanoylforbolacetát MeSH
• volné radikály MeSH