Search Results (34)

Floridoside (2-O-D-glycerol-α-D-galactopyranoside) is a natural product typically found in red algae. It serves as the algae’s carbon reserve and is produced as a protective response against osmotic and heat stress. Both floridoside and its acylated derivatives have been associated with modulating redox homeostasis and inflammatory responses. Therefore, we aimed to evaluate whether the newly synthesized floridoside phosphotriesters (1b–1d, 1f–1h) and acylated floridoside derivative (1e) can modulate the oxidative burst in stimulated human neutrophils. Synthetic strategies included the glycosylation of the thioglycoside donor with glycerol derivatives, having NIS/TfOH as the promoter. Phosphorylation was achieved with POCl₃ in the presence of pyridine. The compounds were analysed for their cytotoxicity, with 1b and 1h being cytotoxic at 50 μM, while the others showed no cytotoxicity in the tested concentrations. The detection of the neutrophils’ oxidative burst was performed using multiple probes [luminol, aminophenyl fluorescein (APF), and Amplex Red (AR)] to evaluate reactive species levels. Compound 1e prevented the oxidative burst in activated human neutrophils (IC₅₀ = 83 ± 7 μM). All the other tested compounds were ineffective in inhibiting APF and AR oxidation under the present experimental conditions. These findings highlight the potential of floridoside-based derivatives as candidates for targeting inflammatory pathways. Full article

Background/Objectives: Oxidized low-density lipoprotein (ox-LDL) is a proinflammatory particle associated with various diseases and affects cell proliferation and viability in multiple cell types. However, its impact on intestinal epithelial cells remains underexplored. This study investigates the effect of ox-LDL on colonic epithelial cell proliferation and viability, as well as the underlying mechanisms involved. Methods: The expression levels of ox-LDL receptors in human colonoids were analyzed at baseline and in response to proinflammatory signals by qRT-PCR. The effect of ox-LDL on organoid proliferation was analyzed using morphometric measurements, viability assays, and the incorporation of a thymidine analog into DNA. The generation of reactive oxygen species (ROS) was determined by Amplex Red assays. Additionally, ox-LDL-induced ROS-dependent organoid proliferation was studied by exposing colonoids to an antioxidant or ROS inhibitors. Results: Colonic epithelial cells express ox-LDL receptors. Ox-LDL significantly induces the proliferation of colonic epithelial cells, which are dependent on ROS generation. Notably, ROS scavengers and NADPH inhibitors reduced ox-LDL-induced proliferation, highlighting the crucial role of oxidative stress in this process. Conclusions: This study demonstrates for the first time that ox-LDL stimulates CEC proliferation mediated by ROS production and validates that the colonic organoid model enables the analysis of potential pharmacological strategies for intestinal diseases characterized by oxidative stress and inflammation. Full article

Pulmonary artery endothelial cells (PAECs) are a major contributor to hypoxic pulmonary hypertension (PH) due to the possible roles of reactive oxygen species (ROS). However, the molecular mechanisms and functional roles of ROS in PAECs are not well established. In this study, we first used Amplex UltraRed reagent to assess hydrogen peroxide (H₂O₂) generation. The result indicated that hypoxic exposure resulted in a significant increase in Amplex UltraRed-derived fluorescence (i.e., H₂O₂ production) in human PAECs. To complement this result, we employed lucigenin as a probe to detect superoxide (O₂⁻) production. Our assays showed that hypoxia largely increased O₂⁻ production. Hypoxia also enhanced H₂O₂ production in the mitochondria from PAECs. Using the genetically encoded H₂O₂ sensor HyPer, we further revealed the hypoxic ROS production in PAECs, which was fully blocked by the mitochondrial inhibitor rotenone or myxothiazol. Interestingly, hypoxia caused an increase in the migration of PAECs, determined by scratch wound assay. In contrast, nicotine, a major cigarette or e-cigarette component, had no effect. Moreover, hypoxia and nicotine co-exposure further increased migration. Transfection of lentiviral shRNAs specific for the mitochondrial Rieske iron–sulfur protein (RISP), which knocked down its expression and associated ROS generation, inhibited the hypoxic migration of PAECs. Hypoxia largely increased the proliferation of PAECs, determined using Ki67 staining and direct cell number accounting. Similarly, nicotine caused a large increase in proliferation. Moreover, hypoxia/nicotine co-exposure elicited a further increase in cell proliferation. RISP knockdown inhibited the proliferation of PAECs following hypoxia, nicotine exposure, and hypoxia/nicotine co-exposure. Taken together, our data demonstrate that hypoxia increases RISP-mediated mitochondrial ROS production, migration, and proliferation in human PAECs; nicotine has no effect on migration, increases proliferation, and promotes hypoxic proliferation; the effects of nicotine are largely mediated by RISP-dependent mitochondrial ROS signaling. Conceivably, PAECs may contribute to PH via the RISP-mediated mitochondrial ROS. Full article

Chikungunya virus (CHIKV) infection is characterized by febrile illness, severe joint pain, myalgia, and cardiovascular complications. Given that CHIKV stimulates reactive oxygen species (ROS) and pro- and anti-inflammatory cytokines, events that disrupt vascular homeostasis, we hypothesized that CHIKV induces arterial dysfunction by directly impacting redox-related mechanisms in vascular cells. Wild-type (WT) and iNOS knockout (iNOS^−/−) mice were administered either CHIKV (1.0 × 10⁶ PFU/µL) or Mock vehicle via the intracaudal route. In vivo, CHIKV infection induced vascular dysfunction (assessed by a wire myograph), decreased systolic blood pressure (tail-cuff plethysmography), increased IL-6 and IFN-γ, but not TNF-α levels (determined by ELISA), and increased protein content by Western blot. Marked contractile hyporesponsiveness to phenylephrine was observed 48 h post-infection, which was restored by endothelium removal. L-NAME, 1400W, Tiron, and iNOS gene deletion prevented phenylephrine hyporesponsiveness. CHIKV infection increased vascular nitrite concentration (Griess reaction) and superoxide anion (O₂^•−) generation (lucigenin chemiluminescence), and decreased hydrogen peroxide (H₂O₂, by Amplex Red) levels 48 h post-infection, alongside increased TBARS levels. In vitro, CHIKV infected endothelial cells (EA.hy926) and upregulated ICAM-1 and iNOS protein expression (determined by Western blot). These data support the conclusion that CHIKV-induced alterations in vascular ROS/NF-kB/iNOS/NO signaling potentially contribute to cardiovascular events associated with Chikungunya infection. Full article

Inhibitors of the serine protease furin have been widely studied as antimicrobial agents due to their ability to block the cleavage and activation of certain viral surface proteins and bacterial toxins. In this study, the antipseudomonal effects and safety profiles of the furin inhibitors MI-1851 and MI-2415 were assessed. Fluorescence quenching studies suggested no relevant binding of the compounds to human serum albumin and α₁-acid glycoprotein. Both inhibitors demonstrated significant antipseudomonal activity in Madin–Darby canine kidney cells, especially compound MI-1851 at very low concentrations (0.5 µM). Using non-tumorigenic porcine IPEC-J2 cells, neither of the two furin inhibitors induced cytotoxicity (CCK-8 assay) or altered significantly the intracellular (Amplex Red assay) or extracellular (DCFH-DA assay) redox status even at a concentration of 100 µM. The same assays with MI-2415 conducted on primary human hepatocytes also resulted in no changes in cell viability and oxidative stress at up to 100 µM. Microsomal and hepatocyte-based CYP3A4 activity assays showed that both inhibitors exhibited a concentration-dependent inhibition of the isoenzyme at high concentrations. In conclusion, this study indicates a good safety profile of the furin inhibitors MI-1851 and MI-2415, suggesting their applicability as antimicrobials for further in vivo investigations, despite some inhibitory effects on CYP3A4. Full article

The increasing prevalence of antimicrobial resistance against zoonotic bacteria, including Streptococcus (S.) suis, highlights the need for new therapeutical strategies, including the repositioning of drugs. In this study, susceptibilities of bacterial isolates were tested toward ten different 3-amidinophenyalanine (Phe(3-Am)) derivatives via determination of minimum inhibitory concentration (MIC) values. Some of these protease inhibitors, like compounds MI-432, MI-471, and MI-476, showed excellent antibacterial effects against S. suis. Their drug interaction potential was investigated using human liver microsomal cytochrome P450 (CYP450) measurements. In our work, non-tumorigenic IPEC-J2 cells and primary porcine hepatocytes were infected with S. suis, and the putative beneficial impact of these inhibitors was investigated on cell viability (Neutral red assay), on interleukin (IL)-6 levels (ELISA technique), and on redox balance (Amplex red method). The antibacterial inhibitors prevented S. suis-induced cell death (except MI-432) and decreased proinflammatory IL-6 levels. It was also found that MI-432 and MI-476 had antioxidant effects in an intestinal cell model upon S. suis infection. Concentration-dependent suppression of CYP3A4 function was found via application of all three inhibitors. In conclusion, our study suggests that the potential antiviral Phe(3-Am) derivatives with 2′,4′ dichloro-biphenyl moieties can be considered as effective drug candidates against S. suis infection due to their antibacterial effects. Full article

The neuroblastoma cell lines SH-SY5Y and Neuro2A are commonly utilized models in neurobiological research. DMEM supplemented with different nutrients and 5–10% Fetal Calf Serum (FCS) is typically used for culturing these cell lines. During special treatments, a reduced FCS content is often deployed to reduce cellular proliferation or the content of bioactive compounds. The impact of the reduction of FCS in culture media on the metabolic profile of SH-SY5Y and Neuro2A cells is currently unknown. Using an Amplex Red Assay, this study showed that the consumption of L-glutamine decreased after FCS reduction. Glucose and pyruvate consumption increased in both cell lines after the reduction of FCS. Thus, lactate production also increased with reduced FCS concentration. The reduction of FCS in the cell culture medium resulted in a reduced aerobic ATP production for SH-SY5Y cells and a complete shut down of aerobic ATP production for Neuro2A cells, measured using the Seahorse XF Real-Time ATP Rate Assay. Utilizing the Seahorse XF Glutamine Oxidation Stress Test, Neuro2A cells showed an increased utilization of L-glutamine oxidation after reduction of FCS. These results indicate that changes in FCS concentration in culture media have an impact on the different energy production strategies of SH-SY5Y and Neuro2A cells which must be considered when planning special treatments. Full article

Ultrasound contrast agents are clinically used for diagnosis of internal organs, but ultrasound contrast agents are rarely applied clinically in musculoskeletal disorders. Our study aims to comparatively analyze the differences between ultrasonographic images through peri-injury injection of the clinically used microbubble and researched nanoparticle contrast agents in various muscular injury models. To compare contrast-enhanced images in different muscle injury models, we prepared groups of rats with sham, laceration, punch, contusion, and toxin injection injuries. We measured H₂O₂ levels using the Amplex Red assay by extracting tissue from the damaged area. As comparative contrast agents, SonoVue^®, a commercially available microbubble contrast agent, and poly(vanillinoxalte) (PVO) nanoparticles, which are H₂O₂-responsive nanoparticles, were used. The difference in contrast between the two contrast agents was recorded as an ultrasound movie, and J-image software 1.53p was used to quantify and analyze the maximum and minimum echogenicity values of the images after contrast enhancement. In the Amplex red assay for the highest H₂O₂ level in each muscle injury model, the maximum level showed 24 h after the modeling. In the sham rats, PVO injection showed no increased echogenicity except at the needle insertion site, but SonoVue^® injection showed increased echo signal throughout the injected muscle immediately after injection. One day after the preparation of the lesion, PVO and SonoVue^® were injected into the lesion site and ultrasound was performed on the lesion site. After the injection of PVO nanoparticles, contrast enhancement was observed at the lesion site immediately. SonoVue^® injections, on the other hand, showed a widespread pattern of echo signals and an increase in echo retention only at the lesion site over time, but this was not clear. There were statistically significant differences between the highest and lowest echogenicity in PVO and SonoVue^® contrast-enhanced images in all models. Contrast enhancement lasted more than 3 h in the PVO injection, but disappeared within 3 h in the SonoVue^® injection. PVO nanoparticles showed the possibility of physiologic contrast by CO₂ generated by conjugation with H₂O₂ generated by muscle injuries, and SonoVue^® injection observed the possibility of microbubble contrast as a contrast agent with a pooling effect that lasts longer on the lesion. Further research is needed to investigate the use of various ultrasound contrast agents, including nanoparticles, in musculoskeletal disorders, as well as the potential for further utilities of microbubble contrast agents. Full article

Liver cancer is a prevalent form of cancer worldwide. While research has shown that increasing sphingomyelin (SM) hydrolysis by activating the cell surface membrane-associated neutral sphingomyelinase 2 (nSMase2) can control cell proliferation and apoptosis, the role of total glutathione depletion in inducing tumor cell apoptosis via nSMase2 activation is still under investigation. Conversely, glutathione-mediated inhibition of reactive oxygen species (ROS) accumulation is necessary for the enzymatic activity of nSMase1 and nSMase3, increased ceramide levels, and cell apoptosis. This study evaluated the effects of depleting total glutathione in HepG2 cells using buthionine sulfoximine (BSO). The study assessed nSMases RNA levels and activities, intracellular ceramide levels, and cell proliferation using RT-qPCR, Amplex red neutral sphingomyelinase fluorescence assay, and colorimetric assays, respectively. The results indicated a lack of nSMase2 mRNA expression in treated and untreated HepG2 cells. Depletion of total glutathione resulted in a significant increase in mRNA levels but a dramatic reduction in the enzymatic activity of nSMase1 and nSMase3, a rise in ROS levels, a decrease in intracellular levels of ceramide, and an increase in cell proliferation. These findings suggest that total glutathione depletion may exacerbate liver cancer (HCC) and not support using total glutathione-depleting agents in HCC management. It is important to note that these results are limited to HepG2 cells, and further studies are necessary to determine if these effects will also occur in other cell lines. Additional research is necessary to explore the role of total glutathione depletion in inducing tumor cell apoptosis. Full article

Malaria is an infectious disease caused by a Plasmodium genus parasite that remains the most widespread parasitosis. The spread of Plasmodium clones that are increasingly resistant to antimalarial molecules is a serious public health problem for underdeveloped countries. Therefore, the search for new therapeutic approaches is necessary. For example, one strategy could consist of studying the redox process involved in the development of the parasite. Regarding potential drug candidates, ellagic acid is widely studied due to its antioxidant and parasite-inhibiting properties. However, its low oral bioavailability remains a concern and has led to pharmacomodulation and the synthesis of new polyphenolic compounds to improve antimalarial activity. This work aimed at investigating the modulatory effect of ellagic acid and its analogues on the redox activity of neutrophils and myeloperoxidase involved in malaria. Overall, the compounds show an inhibitory effect on free radicals as well as on the enzyme horseradish peroxidase- and myeloperoxidase (HRP/MPO)-catalyzed oxidation of substrates (L-012 and Amplex Red). Similar results are obtained with reactive oxygen species (ROS) produced by phorbol 12-mystate acetate (PMA)-activated neutrophils. The efficiency of ellagic acid analogues will be discussed in terms of structure–activity relationships. Full article

(1) Background: Ionic transport in Trypanosoma cruzi is the object of intense studies. T. cruzi expresses a Fe-reductase (TcFR) and a Fe transporter (TcIT). We investigated the effect of Fe depletion and Fe supplementation on different structures and functions of T. cruzi epimastigotes in culture. (2) Methods: We investigated growth and metacyclogenesis, variations of intracellular Fe, endocytosis of transferrin, hemoglobin, and albumin by cell cytometry, structural changes of organelles by transmission electron microscopy, O₂ consumption by oximetry, mitochondrial membrane potential measuring JC-1 fluorescence at different wavelengths, intracellular ATP by bioluminescence, succinate-cytochrome c oxidoreductase following reduction of ferricytochrome c, production of H₂O₂ following oxidation of the Amplex^® red probe, superoxide dismutase (SOD) activity following the reduction of nitroblue tetrazolium, expression of SOD, elements of the protein kinase A (PKA) signaling, TcFR and TcIT by quantitative PCR, PKA activity by luminescence, glyceraldehyde-3-phosphate dehydrogenase abundance and activity by Western blotting and NAD⁺ reduction, and glucokinase activity recording NADP⁺ reduction. (3) Results: Fe depletion increased oxidative stress, inhibited mitochondrial function and ATP formation, increased lipid accumulation in the reservosomes, and inhibited differentiation toward trypomastigotes, with the simultaneous metabolic shift from respiration to glycolysis. (4) Conclusion: The processes modulated for ionic Fe provide energy for the T. cruzi life cycle and the propagation of Chagas disease. Full article

Horseradish peroxidase (HRP) combined with its fluorescence substrates is attracting increasing attention for biochemical analysis. Amplex red is the most widely used fluorescence substrate to HRP; however, it suffers from some drawbacks, such as nonspecific responsiveness toward carboxylesterases. Discovering a new small molecular fluorescence substrate with improved sensitivity and selectivity for HRP is thus desired. Herein, three dihydrofluorescein derivatives (DCFHs) are presented to serve as HRP substrates through fluorescence turn-on methods. The most promising one, 2,7-dichloro-9-(2-(hydroxymethyl)phenyl)-9H-xanthene-3,6-diol (DCFH-1), exhibited excellent sensitivity in the detection of HRP. Moreover, DCFH-1 does not respond to carboxylesterase, thus holding advantages over Amplex red. In the further study, the detection reagent in the commercial ELISA kits was replaced with DCFH-1 to establish a new fluorescence ELISA, which works very well in the quantification of inflammatory cytokine biomarkers from in vitro models. Full article

Cationic antimicrobial peptides (CAMPs) have gained attention as promising antimicrobial therapeutics causing lower or no bacterial resistance. Considerable achievements have been made in designing new CAMPs that are highly active as antimicrobials. However, there is a lack of research on their interaction with biologically important proteins. This study focused on CAMPs’ effects on myeloperoxidase (MPO), an enzyme which is microbicidal and concomitantly damaging to host biomolecules and cells due to its ability to produce reactive oxygen and halogen species (ROS/RHS). Four CAMPs designed by us were employed. MPO catalytic activity was assessed by an absorbance spectra analysis and by measuring enzymatic activity using Amplex Red- and Celestine Blue B-based assays. The peptide Hm-AMP2 accelerated MPO turnover. Pept_1545 and Hm-AMP8 inhibited both the MPO chlorinating and peroxidase activities, with components of different inhibition types. Hm-AMP8 was a stronger inhibitor. Its K_i towards H₂O₂ and Cl^– was 0.3–0.4 μM vs. 11–20 μM for pept_1545. Peptide tyrosine and cysteine residues were involved in the mechanisms of the observed effects. The results propose a possible dual role of CAMPs as both antimicrobial agents and agents that downregulate MPO activation, and suggest CAMPs as prototypes for the development of antioxidant compounds to prevent MPO-mediated ROS/RHS overproduction. Full article

Ochratoxin A (OTA) and lipopolysaccharide (LPS) intake can cause gastrointestinal disorders. Polyphenolic chrysin (CHR) and luteolin (LUT) display anti-inflammatory and antioxidant properties. Porcine intestinal epithelial (jejunal) IPEC-J2 cells were treated with OTA (1 µM, 5 µM and 20 µM), E. coli LPS (10 µg/mL), CHR (1 µM) and LUT (8.7 µM) alone and in their combinations. Cell viabilities (MTS assay) and extracellular (EC) hydrogen-peroxide (H₂O₂) production (Amplex red method) were evaluated. Intracellular (IC) reactive oxygen species (ROS) were assessed using a 2′-7′dichlorodihydrofluorescein diacetate (DCFH-DA) procedure. ELISA assay was used to evaluate IL-6 and IL-8 secretion. OTA decreased cell viabilities (p < 0.001) which could not be alleviated by LUT or CHR (p > 0.05); however, EC H₂O₂ production was successfully suppressed by LUT in IPEC-J2 cells (p < 0.001). OTA with LPS elevated the IC ROS which was counteracted by CHR and LUT (p < 0.001). IL-6 and IL-8 secretion was elevated by LPS + OTA (p < 0.001) which could be inhibited by LUT (p < 0.01 for IL-6; p < 0.001 for IL-8). Based on our results, CHR and LUT exerted beneficial effects on IC ROS levels and on cytokine secretion (LUT) in vitro; thus, they might be used as dietary and feed supplements to avoid OTA- and LPS-related health risks. Full article

Streptococcus mutans (S. mutans) and Candida albicans (C. albicans) are prominent microbes associated with rapid and aggressive caries. In the present study, we investigated the antimicrobial efficacy, cytotoxicity, and mechanism of toluidine blue O (TBO)-mediated antimicrobial photodynamic therapy (aPDT) and potassium iodide (KI). The dependence of KI concentration, TBO concentration and light dose on the antimicrobial effect of aPDT plus KI was determined. The cytotoxicity of TBO-mediated aPDT plus KI was analyzed by cell counting kit-8 (CCK-8) assay. A singlet oxygen (¹O₂) probe test, time-resolved ¹O₂ detection, and a ¹O₂ quencher experiment were performed to evaluate the role of ¹O₂ during aPDT plus KI. The generation of iodine and hydrogen peroxide (H₂O₂) were analyzed by an iodine starch test and Amplex red assay. The anti-biofilm effect of TBO-mediated aPDT plus KI was also evaluated by counting forming unit (CFU) assay. KI could potentiate TBO-mediated aPDT against S. mutans and C. albicans in planktonic and biofilm states, which was safe for human dental pulp cells. ¹O₂ measurement showed that KI could quench ¹O₂ signals, implicating that ¹O₂ may act as a principal mediator to oxidize excess iodide ions to form iodine and H₂O₂. KI could highly potentiate TBO-mediated aPDT in eradicating S. mutans and C. albicans due to the synergistic effect of molecular iodine and H₂O₂. Full article