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Reactive Species Detection in Biology || UV–Vis Absorption and Chemiluminescence Techniques
摘要： Spectrophotometric techniques involving ultraviolet and colorimetric detection offer a convenient way of detecting reactive species (RS) formation due to the prevalence of UV-Vis spectrophotometer in research laboratories. Conventional spectrophotometric measurement of RS exploits their unique chemical reactivity with a small organic molecule and/or enzyme that has specificity to these RS where formation (or disappearance) of absorption peak/s at a particular wavelength is observed at the UV to the visible region of the electromagnetic spectrum. Direct detection of O2 from xanthine oxidase in the past involves rapid-freeze technique using electron paramagnetic resonance (EPR) spectroscopy. At the same time, spectrophotometric detection of O2 generated from xanthine oxidase/xanthine system was also employed for the investigation of the enzymatic property of superoxide dismutase (SOD) where the O2 levels were measured through reduction of ferricytochrome c, tetranitromethane, or oxidation of epinephrine to adenochrome. Several approaches have been developed since then to improve sensitivity with the use of submicromolar probe concentration thus allowing minimal interferences of the probe on the biological process being investigated. Specificity has also been improved to increase the reaction rate of probes to certain RS through synthesis of new and innovative analogues that exploit the unique chemistry between the probe and RS. Chromophore stability was also achieved through improved molecular design, optimized experimental conditions, or addition of supramolecular reagents since chromophores impart inherent thermodynamic stability as a function of its chemical structure, solvent polarity, pH, temperature, or due to presence of oxido-reductants or other reactive substances. Improved sample preparation and high-throughput analysis were also developed in order to maximize efficiency in the measurement markers of oxidative stress and determination of antioxidant capacity (AOC) of known molecules, food, biological fluid, or tissue. Therefore, spectrophotometric techniques for RS measurement have found broad application in the fields of biomedical research, clinical chemistry, plant biology, food chemistry, environmental chemistry, radiation chemistry, pharmaceuticals, toxicology, or material science to name a few, or just simply for the investigation of RS production in simple chemical systems. However, unlike the fluorescence probes, spectrophotometric as well as most applications of chemiluminescence probes do not provide spectrospatial image of the RS localization in cells, hence, one cannot deduce the site of radical production unless multiple probes and/or inhibitors are used with varying compartmentalization property (i.e., extracellular or intracellular). For example, in the investigation of radical production in cellular NADPH oxidase, several methods had been suggested such as measurement of O2 consumption, use of SOD-ferricytochrome c and horseradish peroxidase (HRP)/inhibitable probe such as Amplex Red for extracellular O2 and H2O2, respectively, and the HPLC analysis of the 2-OH-E marker for the quantification of intracellular O2.
关键词： Clinical Chemistry,Oxidative Stress,Spectrophotometric Techniques,Environmental Chemistry,Reactive Species,Antioxidant Capacity,Biomedical Research,Chemiluminescence,Superoxide Dismutase,UV-Vis Absorption
更新于2025-09-04 15:30:14
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Knockdown of FOXO6 inhibits high glucose–induced oxidative stress and apoptosis in retinal pigment epithelial cells
摘要： Oxidative stress and apoptosis in retinal pigment epithelium cells are involved in the pathogenesis of diabetic retinopathy (DR). Forkhead box class O 6 (FOXO6) is a member of the FOXO family that can regulate diabetes‐induced oxidative stress. However, the role of FOXO6 in DR has not been clarified. The aim of the present study was to investigate the effects of FOXO6 on high glucose (HG)‐induced oxidative stress and apoptosis in ARPE‐19 cells. The results showed that FOXO6 was overexpressed in clinical vitreous samples from DR patients and in HG‐induced ARPE‐19 cells. Knockdown of FOXO6 by small interfeing RNA targeting FOXO6 (si‐FOXO6) mitigated the HG‐induced the production of reactive oxygen species and malondialdehyde, as well as the inhibition of superoxide dismutase activity. Knockdown of FOXO6 reduced the rate of cell apoptosis in HG‐induced ARPE‐19 cells. The increase in bax expression and decrease in bcl‐2 expression caused by HG stimulation were reversed by si‐FOXO6 transfection. Furthermore, knockdown of FOXO6 enhanced the activation of Akt/Nrf2 pathway in HG‐stimulated ARPE‐19 cells. Taken together, suppression of FOXO6 protects ARPE‐19 cells from HG‐induced oxidative stress and apoptosis, which is in part mediated by the activation of Akt/Nrf2 pathway.
关键词： Akt/Nrf2 pathway,oxidative stress,retinal pigment epithelium cells,forkhead box class O 6,diabetic retinopathy,apoptosis
更新于2025-09-04 15:30:14
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The Neuroprotective Effect of Maltol against Oxidative Stress on Rat Retinal Neuronal Cells
摘要： Purpose: Maltol (3-hydroxy-2-methyl-4-pyrone), formed by the thermal degradation of starch, is found in coffee, caramelized foods, and Korean ginseng root. This study investigated whether maltol could rescue neuroretinal cells from oxidative injury in vitro. Methods: R28 cells, which are rat embryonic precursor neuroretinal cells, were exposed to hydrogen peroxide (H2O2, 0.0 to 1.5 mM) as an oxidative stress with or without maltol (0.0 to 1.0 mM). Cell viability was monitored with the lactate dehydrogenase assay and apoptosis was examined by the terminal deoxynucleotide transferase-mediated terminal uridine deoxynucleotidyl transferase nick end-labeling (TUNEL) method. To investigate the neuroprotective mechanism of maltol, the expression and phosphorylation of nuclear factor-kappa B (NF-κB), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 were evaluated by Western immunoblot analysis. Results: R28 cells exposed to H2O2 were found to have decreased viability in a dose- and time-dependent manner. However, H2O2-induced cytotoxicity was decreased with the addition of maltol. When R28 cells were exposed to 1.0 mM H2O2 for 24 hours, the cytotoxicity was 60.69 ± 5.71%. However, the cytotoxicity was reduced in the presence of 1.0 mM maltol. This H2O2-induced cytotoxicity caused apoptosis of R28 cells, characterized by DNA fragmentation. Apoptosis of oxidatively-stressed R28 cells with 1.0 mM H2O2 was decreased with 1.0 mM maltol, as determined by the TUNEL method. Western blot analysis showed that treatment with maltol reduced phosphorylation of NF-κB, ERK, and JNK, but not p38. The neuroprotective effects of maltol seemed to be related to attenuated expression of NF-κB, ERK, and JNK. Conclusions: Maltol not only increased cell viability but also attenuated DNA fragmentation. The results obtained here show that maltol has neuroprotective effects against hypoxia-induced neuroretinal cell damage in R28 cells, and its effects may act through the NF-κB and mitogen-activated protein kinase signaling pathways.
关键词： Oxidative stress,Neuroprotection,Maltol,Rat retinal neuronal cell
更新于2025-09-04 15:30:14
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Photobiomodulation at Multiple Wavelengths Differentially Modulates Oxidative Stress <i>In Vitro</i> and <i>In Vivo</i>
摘要： Photobiomodulation (PBM) is emerging as an effective strategy for the management of multiple inflammatory conditions, including oral mucositis (OM) in cancer patients who receive chemotherapy or radiotherapy. Still, the poor understanding of the mechanisms by which the light interacts with biological tissues and the heterogeneity of light sources and protocols employed worldwide significantly limits its applicability. Reactive oxygen species (ROS) are massively generated during the early phases of OM and play a major role in the pathogenesis of inflammation in general. Here, we report the results of a clinical and experimental study, aimed at evaluating the effect of laser light at different wavelengths on oxidative stress in vivo in oncologic patients suffering from OM and in vitro in two cell types abundantly present within the inflamed oral mucosa, neutrophil polymorphonuclear (PMN) granulocytes, and keratinocytes. In addition to standard ROS detection methods, we exploited a roGFP2-Orp1 genetically encoded sensor, allowing specific, quantitative, and dynamic imaging of redox events in living cells in response to oxidative stress and PBM. We found that the various wavelengths differentially modulate ROS production. In particular, the 660 nm laser light increases ROS production when applied either before or after an oxidative stimulus. In contrast, the 970 nm laser light exerted a moderate antioxidant activity both in the saliva of OM patients and in both cell types. The most marked reduction in the levels of ROS was detected in cells exposed either to the 800 nm laser light or to the combination of the three wavelengths. Overall, our study demonstrates that PBM exerts different effects on the redox state of both PMNs and keratinocytes depending on the used wavelength and prompts the validation of a multiwavelength protocol in the clinical settings.
关键词： Reactive oxygen species,Laser therapy,Photobiomodulation,Oxidative stress,Oral mucositis
更新于2025-09-04 15:30:14
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Refluxed Esterification of Fullerenes Conjugated P25 TiO2 Promotes Free Radical Scavenging Capacity and Facilitates Anti-aging Potentials in Human Cells
摘要： Titanium dioxide nanomaterials have good capability to prevent human cells from damage under UV irradiation. However, some studies indicated that the nanoscale of titanium dioxide could potentially cause harmful effects such as free radicals generation under UV irradiation and thereby accelerate the progress of cell aging. Fullerenes can scavenge large amounts of free radicals due to the fact that fullerenes contain enormous amount of π electrons with low lying lowest unoccupied molecular orbital (LUMO), but its adverse properties, such as the poor solubility in water, restricted the applicability. In this study, we employed water-soluble carboxylic acid fullerenes (C60-COOH and C70-COOH) as the free radical scavenger and modify onto the surface of titanium dioxide by refluxed esterification (P25/C60-COOH or C70-COOH) technique. The conformation and properties of theses nanomaterials were characterized by techniques and equipments such as X-ray diffraction (XRD), energy dispersive spectroscopy analysis (EDS), scanning electron microscopy (SEM), thermal gravimetric analysis (TGA), high-resolution transmission electron microscopy (HRTEM), and Fourier transform infrared spectroscopy (FT-IR). We also introduced methylene blue (MB) and rhodamine B (Rh B) as indicator to evaluate and demonstrate the scavenging capacity of these nanomaterials. Moreover, we examined the biocompatibility and UV protection capacity of our P25/fullerene composites in human 293T cells, and applied luciferase activity assay to investigate the possible underlying cell protection mechanisms exhibited by these nanomaterials. Our data indicate that both P25/C60-COOH and P25/C70-COOH could protect human cells against UV exposure. P25/C70-COOH exhibits great anti-inflammation capacity, while as P25/C60-COOH exhibits great anti-oxidative stress and anti-DNA damage capacity. Our results suggest that most of our P25/fullerenes composite materials have the ability to reduce free radicals and exhibit high biomedical potential in anti-inflammation, anti-oxidant, and anti-aging applications.
关键词： refluxed esterification,anti-oxidative stress,UV protection,TiO2/fullerene composites,anti-inflammation
更新于2025-09-04 15:30:14