Cytotoxicity Of Hydrogen Peroxide Research Articles

Pharmacological effects of methysticin and L-sulforaphane through the Nrf2/ARE signaling pathway in MLO-Y4 osteocytes: in vitro study

BackgroundOxidative stress plays a crucial role in the pathogenesis of many skeletal diseases by inducing osteocyte death. The transcription factor nuclear factor erythroid 2-related factor 2 (Nrf2) is a master regulator of various antioxidant gene expressions through antioxidant response element (ARE) against cellular oxidative stress and can be induced by various stimulants, including the phytochemicals methysticin (MET) and L-sulforaphane (SFN). This study aimed to establish an osteocyte in vitro model to investigate the pharmacological effects of MET and SFN on the Nrf2/ARE pathway. MethodsMLO-Y4 murine osteocytes and the stably transduced MLO-Y4-SIN-lenti-ARE reporter gene cell line were used. MET and SFN were used as Nrf2 inducers. The cytotoxicity of MET, SFN, and hydrogen peroxide (H2O2) was evaluated using the CytoTox-Glo™ Assay. Time- and dose-dependent ARE induction was examined by Monoluciferase Assay. The mRNA and protein expressions of Nrf2 target markers, such as heme-oxygenase 1 (Ho-1), NADPH quinone dehydrogenase 1 (Nqo1), and thioredoxin reductase 1 (Txnrd1), were detected by RT-qPCR, Western Blot, and immunofluorescence staining, respectively. Osteogenesis markers, osteopontin, and osteocalcin were compared with and without treatment by immunofluorescence staining. ResultsThe experimental data showed that MET and SFN induced ARE activity in a time- and dose-dependent manner and increased the mRNA and protein expression of antioxidant markers compared to vehicle-treated controls. The protein expression of osteopontin and osteocalcin in the samples treated with SFN were significantly higher than without treatment, and the number of cell death treated with SFN was significantly lower than without treatment under H2O2-induced stress conditions. ConclusionsNrf2 inducers MET and SFN increased the mRNA expression of antioxidant genes through the Nrf2/ARE pathway in osteocytes. Notably, SFN increased the protein expression of osteocyte-associated osteogenic markers and suppressed cell death under H2O2-induced stress condition. Thus, Nrf2 stimulators can exert stress-relieving and osteogenic effects on osteocytes.

Melatonin alleviates oxidative stress-induced injury to nucleus pulposus-derived mesenchymal stem cells through activating PI3K/Akt pathway

BackgroundThe changes in the microenvironment of degenerative intervertebral discs cause oxidative stress injury and excessive apoptosis of intervertebral disc endogenous stem cells. The purpose of this study was to explore the possible mechanism of the protective effect of melatonin on oxidative stress injury in NPMSCs induced by H2O2. MethodsThe Cell Counting Kit-8 assay was used to evaluate the cytotoxicity of hydrogen peroxide and the protective effects of melatonin. ROS content was detected by 2′7′-dichlorofluorescin diacetate (DCFH-DA). Mitochondrial membrane potential (MMP) was detected by the JC-1assay. Transferase mediated d-UTP Nick end labeling (TUNEL) and Annexin V/PI double staining were used to determine the apoptosis rate. Additionally, apoptosis-associated proteins and PI3K/Akt signaling pathway-related proteins were evaluated by immunofluorescence, immunoblotting and PCR. ECMs were evaluated by RT‒PCR and immunofluorescence. In vivo, X-ray, Magnetic resonance imaging (MRI) and Histological analyses were used to evaluate the protective effect of melatonin. ResultsMelatonin had an obvious protective effect on NPMSCs treated with 0–10 μM melatonin for 24 h. In addition, melatonin also had obvious protective effects on mitochondrial dysfunction, decreased membrane potential and cell senescence induced by H2O2. More importantly, melatonin could significantly reduce the apoptosis of nucleus pulposus mesenchymal stem cells induced by H2O2 by regulating the expression of apoptosis-related proteins and decreasing the rate of apoptosis. After treatment with melatonin, the PI3K/Akt pathway was significantly activated in nucleus pulposus mesenchymal stem cells, while the protective effect was significantly weakened after PI3K-IN-1 treatment. In vivo, the results of X-ray, MRI and histological analyses showed that therapy with melatonin could partially reduce the degree of intervertebral disc degeneration. ConclusionOur research demonstrated that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of nucleus pulposus mesenchymal stem cells induced by oxidative stress via the PI3K/Akt pathway, which provides a novel idea for the therapy of intervertebral disc degeneration. The translational potential of this articleThis study indicates that melatonin can effectively alleviate the excessive apoptosis and mitochondrial dysfunction of NPMSCs through activating the PI3K/Akt pathway. Melatonin might serve as a promising candidate for the prevention and treatment of Intervertebral disc degeneration disease (IVDD) in the future.

Hydrogen Peroxide Diffusion through Dental Tissues-In Vitro Study.

Whitening products commonly utilize hydrogen peroxide (HP) as an active principle, which can penetrate dental tissues with potential side effects due to its low molecular weight. This study aimed to evaluate the HP diffusion of two in-office whitening products, namely 6% VivaStyle Paint On Plus (VS) and Opalescence Boost 40% (OP), in different tooth types. Additionally, the influence of the area of exposure, dental tissue thickness and pulp chamber volume was assessed. Each group consisted of eighteen intact anterior (A), premolar (PM) and molar (M) human teeth, and a positive pulpal pressure model was employed. The samples were analyzed using spectrophotometry, and results were expressed as the mean and 95% confidence interval. Statistical tests and linear regression models were appropriately applied at α = 5%. The total HP (µg) retrieved was as follows: VS-A, 1.333 [1.214, 1.452]; OP-A, 1.538 [1.457, 1.620]; VS-PM, 1.208 [1.123, 1.291]; OP-PM, 3.628 [3.401, 3.855]; VS-M, 2.560 [2.297, 2.823]; and OP-M, 4.197 [3.997, 4.396], with statistically significant differences in diffusion kinetics between whitening products for PM and M. Several HP concentrations attained a minimum cytotoxicity value of 2.22 µg/mL. The regression model shows that OP exposed the pulp chamber to 1.421 µg of HP more than that of VS. Different whitening products can cause cytotoxic HP concentrations in the pulp chamber, with a higher risk observed in molars.

Antioxidant activity of fruit jellies enriched with phytochemicals from Pinus sylvestris L.

The aim of this study was to examine the antioxidant properties of fruit jellies made from concentrated fruit juice with the addition of bioactive compounds acquired from young pine shoots. The scope of work included: determination of the optimal extraction temperature of bioactive compounds with the use of concentrated apple juice as an osmotic agent, preparation of jellies from obtained extract, as well as analysis of the phytochemical and antioxidant activity of jellies using S. cerevisiae as a model organism. The study showed that concentrated apple juice can be a good extractant of bioactive ingredients contained in young pine shoots. However, it has been demonstrated that the recovery efficiency of these components is dependent on the process temperature. The most efficient extraction of phytochemicals can be achieved by conducting the process at 60 °C. The jellies made from the extract showed high antioxidant activity which was closely correlated with the amount of raw material used. In addition, the composition of bioactive compounds presented in the jellies reduced the level of oxidative stress in yeast cells treated with H2O2, which was demonstrated by lower intracellular ROS and lower SOD activity. The result was reduced cytotoxicity of hydrogen peroxide and increased cell viability.

Targeting Hydroxybenzoic Acids to Mitochondria as a Strategy to Delay Skin Ageing: An In Vitro Approach.

Targeting antioxidants to mitochondria is considered a promising strategy to prevent cellular senescence and skin ageing. In this study, we investigate whether four hydroxybenzoic acid-based mitochondria-targeted antioxidants (MitoBENs, MB1-4) could be used as potential active ingredients to prevent senescence in skin cells. Firstly, we evaluated the chemical stability, cytotoxicity, genotoxicity and mitochondrial toxicity of all compounds. We followed this by testing the antioxidant protective capacity of the two less toxic compounds on human skin fibroblasts. We then assessed the effects of the best hit on senescence, inflammation and mitochondrial remodeling on a 3D skin cell model, while also testing its mutagenic potential. Cytotoxicity and mitochondrial toxicity rankings were produced: MB3 < MB4 ≃ MB1 < MB2 and MB3 < MB1 < MB4 < MB2, respectively. These results suggest that pyrogallol-based compounds (MB2 and MB4) have lower cytotoxicity. The pyrogallol derivative, MB2, containing a 6-carbon spacer, showed a more potent antioxidant protective activity against hydrogen peroxide cytotoxicity. In a 3D skin cell model, MB2 also decreased transcripts related to senescence. In sum, MB2’s biological safety profile, good chemical stability and lack of mutagenicity, combined with its anti-senescence effect, converts MB2 into a good candidate for further development as an active ingredient for skin anti-ageing products.

Importance of Locations of Iron Ions to Elicit Cytotoxicity Induced by a Fenton-Type Reaction.

Simple SummaryThe Fenton reaction generates the hydroxyl radical (•OH), which is the most reactive and toxic reactive oxygen species and widely recognized as a key player in oxidative stress. To clarify whether this highly reactive molecule travels to its biological target, the effects of the site of the Fenton reaction on cytotoxicity were investigated. Cytotoxicity induced by the Fenton reaction was affected by the distribution of iron ions surrounding and/or being incorporated into cells. Cytotoxicity was enhanced when the Fenton reaction occurred inside cells. Instead of enhancing cytotoxicity, extracellular iron ions exerted protective effects against the cytotoxicity of extracellular hydrogen peroxide in an ion concentration-dependent manner. Distance had a negative impact on the reactivity of extracellular •OH and biologically effective targets. Furthermore, an assessment of plasmid DNA breakage showed that the Fenton reaction system did not effectively induce DNA breakage.The impact of the site of the Fenton reaction, i.e., hydroxyl radical (•OH) generation, on cytotoxicity was investigated by estimating cell lethality in rat thymocytes. Cells were incubated with ferrous sulfate (FeSO4) and hydrogen peroxide (H2O2), or pre-incubated with FeSO4 and then H2O2 was added after medium was replaced to remove iron ions or after the medium was not replaced. Cell lethality in rat thymocytes was estimated by measuring cell sizes using flow cytometry. High extracellular concentrations of FeSO4 exerted protective effects against H2O2-induced cell death instead of enhancing cell lethality. The pre-incubation of cells with FeSO4 enhanced cell lethality induced by H2O2, whereas a pre-incubation with a high concentration of FeSO4 exerted protective effects. FeSO4 distributed extracellularly or on the surface of cells neutralized H2O2 outside cells. Cytotoxicity was only enhanced when the Fenton reaction, i.e., the generation of •OH, occurred inside cells. An assessment of plasmid DNA breakage showed that •OH induced by the Fenton reaction system did not break DNA. Therefore, the main target of intracellularly generated •OH does not appear to be DNA.

Apoptosis Inducing Activity of Rhinacanthin-C in Doxorubicin-Resistant Breast Cancer MCF-7 Cells.

Rhinacanthin-C is a natural bioactive naphthoquinone ester with potential chemotherapeutic value in cancer treatment. In this study, we investigated its apoptotic induction ability and the involved mechanisms through the mitogen-activated protein kinases (MAPK) and protein kinase B/glycogen synthase kinase-3β/nuclear factor erythroid 2-related factor 2 (Akt/GSK-3β/Nrf2) signaling pathways in doxorubicin-resistant breast cancer MCF-7 (MCF-7/DOX) cells. Our 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay showed that rhinacanthin-C (3-28 µM) significantly decreased the viability of MCF-7/DOX cells and potentiated hydrogen peroxide cytotoxicity. This naphthoquinone was able to increase intracellular reactive oxygen species (ROS), as measured by the 2',7'-dichlorofluorescein diacetate (DCFH-DA) assay. This compound increased the number of apoptotic cells by elevating the ratio of apoptotic checkpoint proteins Bax/Bcl-2 and by decreasing the expression of poly(ADP-ribose) polymerase (PARP) protein. Furthermore, Western blotting analyses showed that treatment with rhinacanthin-C (3-28 µM) for 24 h significantly decreased the expression levels of the phosphorylated forms of MAPK proteins (i.e., extracellular signal regulated protein kinase 1/2 (ERK1/2), c-Jun N-terminal kinases (JNK) and p38), Akt, GSK-3β and Nrf2 proteins in MCF-7/DOX cells. Inhibition of the Akt/GSK-3β/Nrf2 pathway led to a significant reduction in heme oxygenase-1 (HO-1) and reduced nicotinamide adenine dinucleotide phosphate (NADP)(H): quinone oxidoreductase 1 (NQO1) proteins. These findings suggested that rhinacanthin-C was able to induce apoptosis in MCF-7/DOX cells through increased ROS production and suppression of the cell survival systems mediated by the MAPKs and Akt/GSK-3β/Nrf2 signaling pathways.

PRDX4 Improved Aging-Related Delayed Wound Healing in Mice

Aging-related delayed wound healing is an issue of concern worldwide. Oxidative stress is involved in wound healing. Antioxidative enzymes have various roles in this process. PRDX4, a member of the PRDX family, is upregulated after injury. To investigate the effects of PRDX4 on aging-related wound healing, we subjected C57BL/6J (wild-type), human Prdx4‒transgenic (i.e., hPrdx4+/+), Prdx4-knockout (i.e., Prdx4-/y) mice of three age groups (young, adult, and aged) to skin wound formation. The overexpression of PRDX4 accelerated wound healing in adult and aged mice but not in young mice. Aged hPrdx4+/+ mice showed reduced oxidative stress and inflammation, lower numbers of neutrophils, increased macrophage infiltration, increased angiogenesis, and increased GF levels. The granulation tissue of adult and aged hPrdx4+/+ mice was richer in fibroblasts than that in the matched wild-type mice. PRDX4 deficiency was associated with mortality in adult and aged mice. Invitro, the overexpression of PRDX4 promoted the proliferation and migration of fibroblasts derived from adult or aged mice and made fibroblasts more resistant to the cytotoxicity of hydrogen peroxide. PRDX4 is essential for wound healing and can improve the healing process from multiple aspects, suggesting that it may be very beneficial to wound treatment, especially for the elderly.

Детекция окислительного стресса в артериальных эндотелиальных клетках человека при воздействии кальций-фосфатных бионов

Введение. Кальций-фосфатные бионы (КФБ) формируются в организме человека при перенасыщении сыворотки ионами кальция и фосфора и вызывают дисфункцию эндотелия, однако молекулярные механизмы нарушения функционирования эндотелия при воздействии КФБ не ясны. Цель исследования - выяснение роли кальций-фосфатных бионов различной формы в развитии окислительного стресса в артериальных эндотелиальных клетках (ЭК) человека. Методика. Для детекции окислительного стресса к конфлюэнтным культурам первичных ЭК коронарной и внутренней грудной артерии человека добавляли равные концентрации КФБ сферической или игольчатой формы (СКФБ и ИКФБ соответственно) с последующим культивированием в течение 1 и 4 ч, добавлением флюоресцентных индикаторов окислительного стресса MitoSOX Red и CellROX Green и конфокальной микроскопией. Измеряли концентрацию продуктов перекисного окисления липидов в культуральной жидкости через 24 ч экспозиции эндотелиальных клеток КФБ. Анализ нейтрализации цитотоксических эффектов перекисного окисления липидов проводили путем добавления к ЭК супероксиддисмутазы и каталазы на 4 или 24 ч (одновременно с КФБ). Для сравнения механизмов клеточной гибели при воздействии СКФБ и ИКФБ анализировали цитотоксичность обоих типов бионов при одновременном воздействии лизосомального ингибитора бафиломицина А1. Результаты. Значимого увеличения генерации активных форм кислорода (АФК) в результате экспозиции СКФБ (независимо от линии ЭК и продолжительности экспозиции) не было выявлено. В то же время наблюдалось повышение генерации супероксида через 4 ч, а иных свободных радикалов через 1 ч после добавления ИКФБ к ЭК. Предварительная нейтрализация АФК супероксиддисмутазой и каталазой частично защищала ЭК от индуцируемой ИКФБ гибели. При этом добавление бафиломицина А1 к ЭК частично защищало их от гибели только при воздействии СКФБ, но не ИКФБ. Заключение. Гибель ЭК при воздействии СКФБ происходит в результате первичного повреждения лизосом, а при воздействии ИКФБ - в первую очередь вследствие окислительного стресса. Background. Calcium phosphate bions (CPB) form in the human blood upon its supersaturation with calcium and phosphate and provoke endothelial dysfunction; however, the molecular mechanisms of these pathological processes remain unclear. Aim. To elucidate the role of differently shaped CPBs in induction of oxidative stress in human arterial endothelial cells (Ecs). Methods. For detection of oxidative stress, equal concentrations of spherical CPB (CPB-S) or needle-shaped CPB (CPB-N) were added to confluent cultures of primary human coronary artery and internal thoracic artery ECs for 1 and 4 h; this was followed by MitoSOX Red and CellROX Green staining and subsequent confocal microscopy. Concentration of thiobarbituric acid-reactive substances was measured in the EC culture supernatant at 24 h of the CPB exposure. The lipid peroxidation cytotoxicity was neutralized by adding superoxide dismutase and catalase to ECs for 4 or 24 h. To compare cell death subroutines induced by CPB-S and CPB-N, the effect of bafilomycin A1, a lysosomal inhibitor, on CRB cytotoxicity was studied. Results. No increase in reactive oxygen species generation was observed in the CPB-S exposure, regardless of the EC line and exposure duration. However, addition of CPB-N to ECs increased the production of superoxide and other free radicals after four- and one-hour exposure, respectively. Prior neutralization of reactive oxygen species with superoxide dismutase and catalase partially protected ECs from CPB-N- but not CPB-S-induced death while bafilomycin A1, vice versa, protected ECs from CPB-S- but not CPB-N-induced death. Conclusion. CPB-S cause cell death due to primary damage of lysosomes whereas CPB-N induce apoptosis due to oxidative stress.

Homocysteine-induced decrease in HUVEC cells\u2019 resistance to oxidative stress is mediated by Akt-dependent changes in iron metabolism

PurposeHyperhomocysteinemia is an independent risk factor for cardiovascular diseases and also promotes neuronal death in various neurodegenerative diseases. There is evidence that iron can mediate homocysteine (Hcy) toxicity. Thus, the aim of this study was to investigate the effect of Hcy on iron metabolism in HUVEC and SH-SY5Y cells.MethodsHUVEC and SH-SY5Y cells were treated with 3 mM Hcy for a defined time.ResultsWe demonstrate that Hcy induced the upregulation of ferritins type L and H in HUVEC cells in a time-dependent manner and had no effect on the ferritins in SH-SY5Y cells. The change in ferritin expression was preceded by a significant decrease in the cellular level of the active form of Akt kinase in HUVEC but not in SH-SY5Y cells. An increase in ferritin L and H protein levels was observed in the Akt1, Akt2, Akt3 siRNA transfected cells, while in the cells transfected with FOXO3a siRNA, a decrease in both ferritins levels was noticed. Moreover, in the HUVEC cells treated with Hcy for 6 days, the active form of kinase Akt returned to the control level and it was accompanied by a drop in ferritin L and H protein levels. Cytotoxicity of hydrogen peroxide significantly increased in HUVEC cells pre-treated with Hcy for 24 h.ConclusionsThese data indicate that Hcy induces an increase in cellular ferritin level, and the process is mediated by alterations in Akt-FOXO3a signaling pathway.

Correlation between Nucleotide Substitutions in Glycerol-3-Phosphate Oxidase Gene, the Level of Hydrogen Peroxide Production, and Cytotoxicity of Mycoplasma pneumoniae

Hydrogen peroxide, one of the main factors of Mycoplasma pneumoniae pathogenicity, is released in the reaction catalyzed by glycerol-3-phosphate (G3P) oxidase enzyme (EC 1.1.3.21). During the study, nucleotide substitutions in the glycerol-3-phosphate oxidase gene of Mycoplasma pneumoniae clinical isolates, the presence of which was accompanied by different levels of hydrogen peroxide production and cytotoxicity of Mycoplasma pneumoniae isolates regarding respiratory epithelial cells, were detected.

Addition of hydrogen peroxide to methylene blue conjugated to β-cyclodextrin in photodynamic antimicrobial chemotherapy in S. mutans biofilm.

This study evaluated the effect of hydrogen peroxide addition on β-cyclodextrin-conjugated methylene blue in antimicrobial photodynamic therapy(a-PDT) in S. mutans biofilm model using laser or light emitting diode (LED) (λ = 660 nm). A preliminary assay was performed to evaluate the cytotoxicity of hydrogen peroxide in oral fibroblasts by the colorimetric method (MTT). Afterwards, groups were divided into (n = 3, in triplicate): C (negative control), CX - chlorhexidine 0.2% (positive control), P (methylene blue/β-cyclodextrin), H (Hydrogen Peroxide at 40 μM), PH, L (Laser), LP, LH (Laser+Hydrogen Peroxide), LPH, LED, LEDP, LEDH, and LEDPH. The biofilm was formed in 24 h with BHI + 1% sucrose (w/v). Light irradiations were conducted with laser, 9 J, 323 J/cm2, 113 s or with LED, 8.1 J, 8.1 J/cm2 for 90 s. Microbial reduction was evaluated by counting the viable microorganisms of the biofilm after the respective treatments, in a selective culture medium, and laser confocal microscopy evaluation. LP, LH, LPH, LEDP, LEDH, and LEDPH groups statistically reduced the counts of S.mutans compared with the C group and the log reductions were of 1.87, 1.94, 2.19, 0.91, 0.92, and 1.33, respectively; the addition of hydrogen peroxide did not potentiate the microbial reductions (LPH and LEDPH) compared with the LP and LEDP groups. The association of hydrogen peroxide with the conjugated β-cyclodextrin nanoparticle as photosensitizer did not result in an enhanced effect of a-PDT; hydrogen peroxide behaved as a photosensitizer, since it reduced the number of S. mutans when associated with laser light.

Cell-Type-Specific Modulation of Hydrogen Peroxide Cytotoxicity and 4-Hydroxynonenal Binding to Human Cellular Proteins In Vitro by Antioxidant Aloe vera Extract.

Although Aloe vera contains numerous bioactive components, the activity principles of widely used A. vera extracts are uncertain. Therefore, we analyzed the effects of genuine A. vera aqueous extract (AV) on human cells with respect to the effects of hydrogen peroxide (H2O2) and 4-hydroxynonenal (HNE). Fully developed A. vera leaves were harvested and analyzed for vitamin C, carotenoids, total soluble phenolic content, and antioxidant capacity. Furthermore, human cervical cancer (HeLa), human microvascular endothelial cells (HMEC), human keratinocytes (HaCat), and human osteosarcoma (HOS) cell cultures were treated with AV extract for one hour after treatment with H2O2 or HNE. The cell number and viability were determined using Trypan Blue, and endogenous reactive oxygen species (ROS) production was determined by fluorescence, while intracellular HNE–protein adducts were measured for the first time ever by genuine cell-based HNE–His ELISA. The AV extract expressed strong antioxidant capacities (1.1 mmol of Trolox eq/g fresh weight) and cell-type-specific influence on the cytotoxicity of H2O2, as well as on endogenous production of ROS and HNE–protein adducts induced by HNE treatment, while AV itself did not induce production of ROS or HNE–protein adducts at all. This study, for the first time, revealed the importance of HNE for the activity principles of AV. Since HMEC cells were the most sensitive to AV, the effects of AV on microvascular endothelia could be of particular importance for the activity principles of Aloe vera extracts.

Captan-induced increase in the concentrations of intracellular Ca2+ and Zn2+ and its correlation with oxidative stress in rat thymic lymphocytes

Captan, a phthalimide fungicide, is considered to be relatively nontoxic to mammals. There is a possibility that captan affects membrane and cellular parameters of mammalian cells, resulting in adverse effects, because of high residue levels. To test the possibility, we examined the effects of captan on rat thymic lymphocytes using flow-cytometry with appropriate fluorescent probes. Treatment with 10 and 30 μM captan induced apoptotic and necrotic cell death. Before cell death occurred, captan elevated the intracellular concentrations of Ca2+ and Zn2+ and decreased the concentration of cellular thiol compounds. These captan-induced phenomena are shown to cause cell death and are similar to those caused by oxidative stress. Captan also elevated the cytotoxicity of hydrogen peroxide. Results indicate that 10 and 30 μM captan cause cytotoxic effects on mammalian cells. Despite no report on the significant environmental toxicity hazard of captan in humans, it may exhibit adverse effects, described above, on wild organisms.

Human Lymphocyte-Protective Effects of an Ethanol Extract from Detarium microcarpum Guill. and Perr. (Caesalpiniaceae) Fruit Pulp.

The current study aimed to evaluate, in vitro, the antioxidant capacity and the human lymphocyte-protective effect of the ethanolic extract from Detarium microcarpum fruit pulp against oxidative stress damage. Human lymphocytes were incubated with different concentrations of extract, followed by the addition of hydrogen peroxide or tert-butyl hydroperoxide. Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Furthermore, the antioxidant property of the extract was evaluated in vitro using hydrogen peroxide and nitric oxide radical-scavenging assays. Compared to the vehicle, the fruit pulp ethanol extract did not exhibit a cytotoxic effect on human lymphocytes. Furthermore, the cytotoxicity of hydrogen peroxide and tert-butyl hydroperoxide to human lymphocytes was significantly reduced by fruit extract pretreatment. The extract and ascorbic acid exhibited similar cytoprotective activity (p > 0.05). The fruit pulp extract showed more antioxidant activity than gallic acid in the hydrogen peroxide-scavenging model, while in the nitric oxide-quenching model, the fruit extract and gallic acid showed similar activity. The fruit pulp of D. microcarpum contains potent antioxidant and cell-protective compounds. The use of the fruit pulp of D. microcarpum as a food supplement could rescue cellular oxidative damage responsible for numerous pathologies.

Antiaging and antioxidant effects of topical autophagy activator: A randomized, placebo-controlled, double-blinded study.

Recently, potential roles of autophagy in skin homeostasis received many interests. But, little has been reported for the potential antiaging effects of autophagy activator. With the newly synthesized autophagy activator, heptasodium hexacarboxymethyl dipeptide-12 (Aquatide™) invitro and clinical efficacy of the topical autophagy activator as an antiaging cosmeceutical ingredient was evaluated. Antioxidant effect of Aquatide™ was evaluated by radical scavenging assay. In vitro effect was assessed by measuring the cytotoxicity of hydrogen peroxide in cultured normal human epidermal keratinocytes. Clinical evaluation was performed by a randomized, placebo-controlled, double-blinded study. Antioxidant efficacy was observed by measuring the carbonylated proteins in stratum corneum (SC) by fluorescein-5-thiosemicarbazide (FTZ) staining. Radical scavenging effects of Aquatide were observed with the ABTS assay, and significant reduction in hydrogen peroxide-induced cytotoxicity was observed in Aquatide™-treated cells. Autophagy inhibitor treatment abrogated cytoprotective effects of Aquatide™. In a clinical study, statistically significant increase in skin elasticity was observed after 4 and 8weeks. Quantitative analysis of carbonylated proteins in SC also showed significant reduction in Aquatide™-treated group, which is consistent with the invitro data. These results suggest that autophagy plays important roles in antioxidant system and aging process in skin, and topical autophagy activators can be potential cosmeceutical ingredients for skin antiaging.

Synergistic Degradation of a Hyperuricemia-Causing Metabolite Using One-Pot Enzyme-Nanozyme Cascade Reactions

Multi-enzyme cascade reactions are frequently found in living organisms, in particular when an intermediate should be eliminated. Recently, enzyme-mimic nanomaterials (nanozymes) received much attention for various applications, because they are usually more stable and cost-effective than enzymes. However, enzyme-nanozyme cascade reations have not been yet extensively exploited. Therefore, in this study, we investigated one-pot enzyme-nanozyme cascade reactions using urate oxidase (UOX) and catalase-mimic gold nanoparticle nanozyme (AuNP) with the ultimate goal of treatment of hyperuricemia. UOX degrades hyperuricemia-causing uric acid, but also generates hydrogen peroxide raising several health concerns. We successfully demonstrated that one-pot UOX-AuNP cascade systems degrade uric acid more than five times faster than UOX alone, by eliminating potentially cytotoxic hydrogen peroxide, similar to enzyme-enzyme reactions.

A novel AhR ligand, 2AI, protects the retina from environmental stress.

Various retinal degenerative diseases including dry and neovascular age-related macular degeneration (AMD), retinitis pigmentosa, and diabetic retinopathy are associated with the degeneration of the retinal pigmented epithelial (RPE) layer of the retina. This consequently results in the death of rod and cone photoreceptors that they support, structurally and functionally leading to legal or complete blindness. Therefore, developing therapeutic strategies to preserve cellular homeostasis in the RPE would be a favorable asset in the clinic. The aryl hydrocarbon receptor (AhR) is a conserved, environmental ligand-dependent, per ARNT-sim (PAS) domain containing bHLH transcription factor that mediates adaptive response to stress via its downstream transcriptional targets. Using in silico, in vitro and in vivo assays, we identified 2,2′-aminophenyl indole (2AI) as a potent synthetic ligand of AhR that protects RPE cells in vitro from lipid peroxidation cytotoxicity mediated by 4-hydroxynonenal (4HNE) as well as the retina in vivo from light-damage. Additionally, metabolic characterization of this molecule by LC-MS suggests that 2AI alters the lipid metabolism of RPE cells, enhancing the intracellular levels of palmitoleic acid. Finally, we show that, as a downstream effector of 2AI-mediated AhR activation, palmitoleic acid protects RPE cells from 4HNE-mediated stress, and light mediated retinal degeneration in mice.

Insights on the virulence of swine respiratory tract mycoplasmas through genome-scale metabolic modeling.

BackgroundThe respiratory tract of swine is colonized by several bacteria among which are three Mycoplasma species: Mycoplasma flocculare, Mycoplasma hyopneumoniae and Mycoplasma hyorhinis. While colonization by M. flocculare is virtually asymptomatic, M. hyopneumoniae is the causative agent of enzootic pneumonia and M. hyorhinis is present in cases of pneumonia, polyserositis and arthritis. The genomic resemblance among these three Mycoplasma species combined with their different levels of pathogenicity is an indication that they have unknown mechanisms of virulence and differential expression, as for most mycoplasmas.MethodsIn this work, we performed whole-genome metabolic network reconstructions for these three mycoplasmas. Cultivation tests and metabolomic experiments through nuclear magnetic resonance spectroscopy (NMR) were also performed to acquire experimental data and further refine the models reconstructed in silico.ResultsEven though the refined models have similar metabolic capabilities, interesting differences include a wider range of carbohydrate uptake in M. hyorhinis, which in turn may also explain why this species is a widely contaminant in cell cultures. In addition, the myo-inositol catabolism is exclusive to M. hyopneumoniae and may be an important trait for virulence. However, the most important difference seems to be related to glycerol conversion to dihydroxyacetone-phosphate, which produces toxic hydrogen peroxide. This activity, missing only in M. flocculare, may be directly involved in cytotoxicity, as already described for two lung pathogenic mycoplasmas, namely Mycoplasma pneumoniae in human and Mycoplasma mycoides subsp. mycoides in ruminants. Metabolomic data suggest that even though these mycoplasmas are extremely similar in terms of genome and metabolism, distinct products and reaction rates may be the result of differential expression throughout the species.ConclusionsWe were able to infer from the reconstructed networks that the lack of pathogenicity of M. flocculare if compared to the highly pathogenic M. hyopneumoniae may be related to its incapacity to produce cytotoxic hydrogen peroxide. Moreover, the ability of M. hyorhinis to grow in diverse sites and even in different hosts may be a reflection of its enhanced and wider carbohydrate uptake. Altogether, the metabolic differences highlighted in silico and in vitro provide important insights to the different levels of pathogenicity observed in each of the studied species.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2644-z) contains supplementary material, which is available to authorized users.

Control of oxygen release from peroxides using polymers.

An important limitation in cell therapy for the regeneration of tissue is the initial lack of oxygen. After implantation of large 3D cell-seeded structures, cells die rather than contribute to tissue regenerating. Here we've tested oxygen-releasing materials to improve cell survival and growth after implantation. Calcium peroxide (CaO2) in a polymer matrix was used as source of oxygen. Two polymers were tested in order to slow down and extend the period of oxygen release, poly(D,L-lactic acid) and poly(lactic-co-glycolic acid). Compared to CaO2 particles, both releasing systems showed an initially higher and shorter oxygen release. Human mesenchymal stromal cells cultured on casted films of these oxygen-releasing composites required catalase to proliferate, indicating the production of cytotoxic hydrogen peroxide as intermediate. Poly(D,L-lactic acid) and poly(lactic-co-glycolic acid) are less suited for slowly oxygen-releasing materials. Catalase was able to reduce the cytotoxic effect of H2O2.