Reduction Of Intracellular Reactive Oxygen Species Research Articles

Norcantharidin-Encapsulated C60-Modified Nanomicelles: A Potential Approach to Mitigate Cytotoxicity in Renal Cells and Simultaneously Enhance Anti-Tumor Activity in Hepatocellular Carcinoma Cells.

The objective of this study was to examine the preparation process of DSPE-PEG-C60/NCTD micelles and assess the impact of fullerenol (C60)-modified micelles on the nephrotoxicity and antitumor activity of NCTD. The micelles containing NCTD were prepared using the ultrasonic method and subsequently optimized and characterized. The cytotoxicity of micelles loaded with NCTD was assessed using the CCK-8 method on human hepatoma cell lines HepG2 and BEL-7402, as well as normal cell lines HK-2 and L02. Acridine orange/ethidium bromide (AO/EB) double staining and flow cytometry were employed to assess the impact of NCTD-loaded micelles on the apoptosis of the HK-2 cells and the HepG2 cells. Additionally, JC-1 fluorescence was utilized to quantify the alterations in mitochondrial membrane potential. The generation of reactive oxygen species (ROS) following micelle treatment was determined through 2',7'-dichlorofluorescein diacetate (DCFDA) staining. The particle size distribution of the DSPE-PEG-C60/NCTD micelles was determined to be 91.57 nm (PDI = 0.231). The zeta potential of the micelles was found to be -13.8 mV. The encapsulation efficiency was measured to be 91.9%. The in vitro release behavior of the micelles followed the Higuchi equation. Cellular experiments demonstrated a notable decrease in the toxicity of the C60-modified micelles against the HK-2 cells, accompanied by an augmented inhibitory effect on cancer cells. Compared to the free NCTD group, the DSPE-PEG-C60 micelles exhibited a decreased apoptosis rate (12%) for the HK-2 cell line, lower than the apoptosis rate observed in the NCTD group (36%) at an NCTD concentration of 75 μM. The rate of apoptosis in the HepG2 cells exhibited a significant increase (49%), surpassing the apoptosis rate observed in the NCTD group (24%) at a concentration of 150 μM NCTD. The HK-2 cells exhibited a reduction in intracellular ROS and an increase in mitochondrial membrane potential (ΔψM) upon exposure to C60-modified micelles compared to the NCTD group. The DSPE-PEG-C60/NCTD micelles, as prepared in this study, demonstrated the ability to decrease cytotoxicity and ROS levels in normal renal cells (HK-2) in vitro. Additionally, these micelles showed an enhanced antitumor activity against human hepatocellular carcinoma cells (HepG2, BEL-7402).

The effects of purslane (Portulaca oleracea) and fennel (Foeniculum vulgare Mill) hydroalcoholic extracts on the functional parameters of human spermatozoa after vitrification.

Reactive oxygen species (ROS) are produced during cryopreservation of human sperm and impair sperm function. Antioxidant compounds, such as fennel and purslane, reduce the damaging effects of ROS. This study aimed to evaluate motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), intracellular ROS, and DNA damage to determine the optimum concentrations of hydroalcoholic extracts of fennel and purslane for human spermatozoa cryopreservation. Twenty human sperm samples were used and divided into seven equal groups consisting of fennel hydroalcoholic extract (5, 10, and 15 mg/L), purslane hydroalcoholic extract (25, 50, and 100 mg/L), and no additive. Supplementation of 25 mg/L and 50 mg/L purslane extract and 10 mg/L fennel extract in cryopreservation extender significantly increased the motility and PMI of sperm with a significant reduction in intracellular ROS compared to control groups (p<0.05). A 50 mg/L concentration of purslane extract elevated progressive motility and MMP compared to the control group (p<0.05). No significant differences were seen for motion patterns and DNA damage of frozen-thawed human sperm in extender containing these extracts. The results showed that supplementation of 50 mg/L purslane extract and 10 mg/L fennel extract in semen cryopreservation extender has the potential to decrease intracellular ROS and subsequently elevate the motility and PMI of human sperm.

Antioxidant, Anti-Inflammatory and Attenuating Intracellular Reactive Oxygen Species Activities of Nicotiana tabacum var. Virginia Leaf Extract Phytosomes and Shape Memory Gel Formulation.

Oxidative stress is one of the major causes of skin aging. In this study, the shape memory gels containing phytosomes were developed as a delivery system for Nicotiana tabacum var. Virginia fresh (VFL) and dry (VDL) leaf extracts. The extracts were loaded in the phytosomes by a solvent displacement method. The physical and chemical characteristics and stability of phytosomes were evaluated by dynamic light scattering and phytochemistry, respectively. The in vitro antioxidant activity and intracellular reactive oxygen species reduction of phytosomes and/or extracts were investigated by the DPPH and ABTS radical scavenging assays, FRAP assay, and DCFH-DA fluorescent probe. The cytotoxicity and anti-inflammatory activity of VDL and VFL phytosomes were studied by an MTT and a nitric oxide assay, respectively. Here, we first reported the total phenolic content in the dry leaf extract of N. tabacum var. Virginia was significantly greater than that of the fresh leaf extract. The HPLC analysis results revealed that VDL and VFL extracts contained 4.94 ± 0.04 and 3.13 ± 0.01 µg/mL of chlorogenic acid and 0.89 ± 0.00 and 0.24 ± 0.00 µg/mL of rutin, respectively. The phytosomes of the VDL and VFL extracts displayed stable size, polydispersity index, zeta potential values, and good chemical stability. VDL and VDL phytosomes showed higher phenolic and flavonoid contents which showed stronger DPPH and ABTS radical scavenging effects and reduced the intracellular ROS. The results suggested that the phenolic compounds are the main factor in their antioxidant activity. Both VDL and VFL phytosomes inhibited nitric oxide production induced by LPS, suggesting the anti-inflammatory activity of the phytosomes. The shape memory gel containing VDL and VFL phytosomes had good physical stability in terms of pH and viscosity. The VDL and VFL phytosomes dispersed in the shape memory gels can be considered as a promising therapeutic delivery system for protecting the skin from oxidation and reactive oxygen species.

Participation of lipopolysaccharide in hyperplasic adipose expansion: Involvement of NADPH oxidase/ROS/p42/p44 MAPK-dependent Cyclooxygenase-2.

Obesity is a world‐wide problem, especially the child obesity, with the complication of various metabolic diseases. Child obesity can be developed as early as the age between 2 and 6. The expansion of fat mass in child age includes both hyperplasia and hypertrophy of adipose tissue, suggesting the importance of proliferation and adipogenesis of preadipocytes. The changed composition of gut microbiota is associated with obesity, revealing the roles of lipopolysaccharide (LPS) on manipulating adipose tissue development. Studies suggest that LPS enters the circulation and acts as a pro‐inflammatory regulator to facilitate pathologies. Nevertheless, the underlying mechanisms behind LPS‐modulated obesity are yet clearly elucidated. This study showed that LPS enhanced the expression of cyclooxygenase‐2 (COX‐2), an inflammatory regulator of obesity, in preadipocytes. Pretreating preadipocytes with the scavenger of reactive oxygen species (ROS) or the inhibitors of NADPH oxidase or p42/p44 MAPK markedly decreased LPS‐stimulated gene expression of COX‐2 together with the phosphorylation of p47phox and p42/p44 MAPK, separately. LPS activated p42/p44 MAPK via NADPH oxidase‐dependent ROS accumulation in preadipocytes. Reduction of intracellular ROS or attenuation of p42/p44 MAPK activation both reduced LPS‐mediated COX‐2 expression and preadipocyte proliferation. Moreover, LPS‐induced preadipocyte proliferation and adipogenesis were abolished by the inhibition of COX‐2 or PEG2 receptors. Taken together, our results suggested that LPS enhanced the proliferation and adipogenesis of preadipocytes via NADPH oxidase/ROS/p42/p44 MAPK‐dependent COX‐2 expression.

Effect of a Novel Hydroxybenzoic Acid Based Mitochondria Directed Antioxidant Molecule on Bovine Sperm Function and Embryo Production.

Simple SummaryGametes are particularly susceptible to oxidative stress that impairs the reproductive function. This study was conducted to study the effect of a mitochondria-targeted dietary antioxidant (AntiOxBEN2) on bovine sperm function. Different doses of this antioxidant were tested during spermatozoa capacitation and/or fertilization processes. Spermatozoa mitochondrial function was improved when AntiOxBEN2 was supplemented to the capacitation medium. Supplementation of both capacitation and fertilization media with AntiOxBEN2 (lowest dose) improved the fertilization process and embryo production. Our results showed that AntiOxBEN2 can be used for the prevention of oxidative stress in bovine spermatozoa and may constitute a putative novel therapeutic strategy to improve the outcomes of assisted reproductive techniques.Sperm cells are particularly vulnerable to reactive oxygen species (ROS), impairing their fertilizing ability. Our objective was to study the effect of a novel mitochondrial-directed antioxidant, AntiOxBEN2, on bovine sperm function. This antioxidant was added to the semen capacitation medium (CAP), during the swim-up process, and to the fertilization medium (FERT) during the co-incubation of matured oocytes and capacitated spermatozoa, in concentrations of 0 (control), 1, and 10 µM. After the swim-up, sperm motility (CASA and visual analysis), vitality (eosin-nigrosin), mitochondrial membrane potential (JC1), intracellular ROS, adenosine triphosphate (ATP) levels, and basal metabolism (Seahorse Xfe96) were evaluated. Embryo development and quality were also assessed. Higher cleavage rates were obtained when 1 µM AntiOxBEN2 were added to CAP and FERT media (compared to control, p < 0.04). A positive effect of AntiOxBEN2 on intracellular ROS reduction (p = 0.01), on the increment of mitochondrial membrane potential (p ≤ 0.003) and, consequently, on the sperm quality was identified. However, the highest dose impaired progressive motility, ATP production, and the number of produced embryos. The results demonstrate a beneficial effect of AntiOxBEN2 (1 µM) on sperm capacitation and fertilization processes, thus improving embryonic development. This may constitute a putative novel therapeutic strategy to improve the outcomes of assisted reproductive techniques (ART).

The antioxidant roles of L-carnitine and N-acetyl cysteine against oxidative stress on human sperm functional parameters during vitrification.

ObjectiveAmino acids can protect sperm structure in cryopreservation due to their antioxidant properties. Therefore, the present study aimed to investigate the protective effect of L-carnitine (LC) and N-acetyl cysteine (NAC) on motility parameters, plasma membrane integrity (PMI), mitochondrial membrane potential (MMP), DNA damage, and human sperm intracellular reactive oxygen species (ROS) during vitrification.MethodsTwenty normal human sperm samples were examined. Each sample was divided into six equal groups: LC (1 and 10 mM), NAC (5 and 10 mM), and cryopreserved and fresh control groups.ResultsThe groups treated with LC and NAC showed favorable findings in terms of motility parameters, DNA damage, and MMP. Significantly higher levels of intracellular ROS were observed in all cryopreserved groups than in the fresh group (p≤0.05). The presence of LC and NAC at both concentrations caused an increase in PMI, MMP, and progressive motility parameters, as well as a significant reduction in intracellular ROS compared to the control group (p≤0.05). The concentrations of the amino acids did not show any significant effect.ConclusionLC and NAC are promising as potential additives in sperm cryopreservation.

Redox nanomedicine ameliorates chronic kidney disease (CKD) by mitochondrial reconditioning in mice

Targeting reactive oxygen species (ROS) while maintaining cellular redox signaling is crucial in the development of redox medicine as the origin of several prevailing diseases including chronic kidney disease (CKD) is linked to ROS imbalance and associated mitochondrial dysfunction. Here, we have shown that a potential nanomedicine comprising of Mn3O4 nanoparticles duly functionalized with biocompatible ligand citrate (C-Mn3O4 NPs) can maintain cellular redox balance in an animal model of oxidative injury. We developed a cisplatin-induced CKD model in C57BL/6j mice with severe mitochondrial dysfunction and oxidative distress leading to the pathogenesis. Four weeks of treatment with C-Mn3O4 NPs restored renal function, preserved normal kidney architecture, ameliorated overexpression of pro-inflammatory cytokines, and arrested glomerulosclerosis and interstitial fibrosis. A detailed study involving human embryonic kidney (HEK 293) cells and isolated mitochondria from experimental animals revealed that the molecular mechanism behind the pharmacological action of the nanomedicine involves protection of structural and functional integrity of mitochondria from oxidative damage, subsequent reduction in intracellular ROS, and maintenance of cellular redox homeostasis. To the best of our knowledge, such studies that efficiently treated a multifaceted disease like CKD using a biocompatible redox nanomedicine are sparse in the literature. Successful clinical translation of this nanomedicine may open a new avenue in redox-mediated therapeutics of several other diseases (e.g., diabetic nephropathy, neurodegeneration, and cardiovascular disease) where oxidative distress plays a central role in pathogenesis.

Seasonality Modulates the Cellular Antioxidant Activity and Antiproliferative Effect of Sonoran Desert Propolis.

The main chemical composition and pharmacological potential of propolis from arid and semi-arid regions of the Sonoran Desert have been previously reported. Caborca propolis (CP), from an arid zone of the Sonoran Desert, has shown a polyphenolic profile that suggests a mixed plant origin, presenting poplar-type markers, as well as a 6-methoxylated flavonoid, xanthomicrol, characteristic of Asteraceae plants. In addition, CP has shown significant antioxidant properties and antiproliferative activity on cancer cells. In this study, we analyzed the influence of collection time on the chemical constitution, antiproliferative activity and protective capacity of CP against reactive oxygen species (ROS), by using HPLC–UV–diode array detection (DAD) analysis, 3-(4,5-dimethylthiazol-2-yl)-2,5-Dimethyltetrazoliumbromide (MTT) and 2,2-diphenyl-1-picryl-hydrazyl (DPPH) assays, as well as cellular antioxidant activity (CAA) assay on murine B-cell lymphoma M12.C3.F6 cells. HPLC–UV–DAD analyses of seasonally collected CP (one-year period) revealed quantitative differences among the most abundant CP constituents: pinocembrin, galangin, chrysin and pinobanksin-3-O-acetate. Though all seasonal samples of CP induced an antiproliferative effect in M12.C3.F6 cells, CP from autumn showed the highest inhibitory activity (IC50: 5.9 ± 0.6 µg/mL). The DPPH assay pointed out that CP collected in autumn presented the highest antioxidant potential (IC50: 58.8 ± 6.7 µg/mL), followed by winter (65.7 ± 12.2 µg/mL) and spring (67.0 ± 7.5 µg/mL); meanwhile, the summer sample showed a lesser antioxidant capacity (IC50: 98.7 ± 2.5 µg/mL). The CAA assay demonstrated that CP induced a significant protective effect against ROS production elicited by H2O2 in M12.C3.F6 cells. Pretreatment of M12.C3.F6 cells with CP from spring and autumn (25 and 50 µg/mL for 1 h) showed the highest reduction in intracellular ROS induced by H2O2 (1 and 5 mM). These results indicate that the antiproliferative effect and cellular antioxidant activity of CP are modulated by quantitative fluctuations in its polyphenolic profile due to its collection time.

Porous chitosan derivative scaffolds affect proliferation and osteogenesis of mesenchymal stem cell via reducing intracellular ROS

The proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) needed to be promoted for their use in stem cell-based therapy for large bone defects. This study aimed to prepare porous 2a-2 g scaffolds with antioxidant activity to reduce intracellular reactive oxygen species (ROS), which resulted in promoting the proliferation and osteogenic differentiation of MSCs. A series of novel chemically modified 2a-2 g scaffolds were fabricated by an acid-soluble/alkali-insoluble method. Besides, these 2a-2 g scaffolds had good biocompatibility, physicochemical properties and the ability to promote osteogenic differentiation of human adipose-derived stem cells (hADSCs). However, the proliferation ability of hADSCs on 2a-2f scaffolds was weakened. Interestingly, 2 g scaffold had a positive effect on hADSCs proliferation. These results indicated that the reduction of intracellular ROS was not conducive to hADSCs proliferation but beneficial to hADSCs osteogenic differentiation. Taken together, these significant results highlighted potential therapeutic benefit of 2 g scaffold in large bone defects.

DIBI, a novel 3-hydroxypyridin-4-one chelator iron-binding polymer, inhibits breast cancer cell growth and functions as a chemosensitizer by promoting S-phase DNA damage.

Breast cancer is a leading cause of cancer-related death in women; however, chemotherapy of breast cancer is often hindered by dose-limiting toxicities, demonstrating the need for less toxic approaches to treatment. Since the rapid growth and metabolism of breast cancer cells results in an increased requirement for iron, withdrawal of bioavailable iron using highly selective iron chelators has been suggested to represent a new approach to breast cancer treatment. Here we show that the recently developed iron-binding polymer DIBI inhibited the growth of five different breast cancer cell lines (SK-BR3, MDA-MB-468, MDA-MB-231, MCF-7, and T47D). In cultures of MDA-MB-468 breast cancer cells, which were most sensitive to DIBI-mediated growth inhibition, iron withdrawal was associated with increased expression of transferrin receptor 1 and ferritin H mRNA but decreased expression of ferroportin mRNA. MDA-MB-468 cells that were exposed to DIBI experienced double-strand DNA breaks during the S phase of the cell cycle. DNA damage was not mediated by reactive oxygen species (ROS) since DIBI-treated MDA-MB-468 cells exhibited a reduction in intracellular ROS. DIBI-treated MDA-MB-468 cells also showed increased sensitivity to growth inhibition by the chemotherapeutic drugs cisplatin, doxorubicin, and 4-hydroperoxy cyclophosphamide (active metabolite of cyclophosphamide). Combination treatment of MDA-MB-468 cells with DIBI and cisplatin caused greater DNA damage than either treatment alone, which was also associated with an increase in apoptotic cell death. Taken together, these findings suggest that DIBI-mediated iron withdrawal may enhance the effect of chemotherapeutic agents used in breast cancer treatment.

Periodontal ligament-derived mesenchymal stem cells modulate neutrophil responses via paracrine mechanisms.

Mesenchymal stem cells differentiate into distinct mesenchymal cell lineages and regulate the immune response. The aim of this study was to determine whether periodontal ligament-derived mesenchymal stem cells (PDLSCs) have the ability to modulate neutrophil responses via paracrine mechanisms. CD105-enriched PDLSCs were seeded for 24 h and challenged with Porphyromonas gingivalis total protein extract (PgPE) (0 or 2 ug/mL) for 3 h. Cells were then washed and further cultured for 18 h and the supernatants were collected and stored. Next, neutrophil-differentiated human promyelocytic leukemia HL-60 cells (HL60D) were treated with PDLSCs supernatants and HL-60D activation and functional responses were determined. PgPE treatment induced higher secretion of inflammatory markers and chemokines by PDLSCs, including RANTES, eotaxin, interferon (IFN)-γ- inducible protein 10 (IP-10), monocyte chemoattractant protein-1 (MCP-1), IFN-γ, interleukin (IL)-6, IL-8 and IL-1ra (P<0.05). HL-60D recruitment rate was increased by 4.7 ± 1.09-fold when exposed to PgPE-treated PDLSCs supernatants. PgPE-treated PDLSCs supernatants promoted a 1.78 ± 1.04-fold increase in the production of intracellular reactive oxygen species (ROS) by PMA-stimulated HL-60D, whereas PgPE-untreated PDLSCs supernatants led to a 16% reduction in intracellular ROS. In sharp contrast, neither PgPE-untreated nor PgPE-treated PDLSCs supernatants altered tumor necrosis factor (TNF)-α and IL-1β secretion by HL-60D cells. Together, these findings suggest an important role of PDLSCs in the recognition of P. gingivalis, paracrine recruitment and activation of antimicrobial mechanisms in innate immune cells, without interfering in cytokine responses.

Protective effect of crocin on ultraviolet B‑induced dermal fibroblast photoaging.

Ultraviolet B (UVB) radiation induces the production of reactive oxygen species (ROS), resulting in the aging of dermal fibroblasts. Crocin, a bioactive constituent of Crocus sativus, possesses anti-oxidation effects. The purpose of the present study was to evaluate the protective effect of crocin on UVB-induced dermal fibroblast photoaging. Human dermal fibroblasts were isolated and cultured with different concentrations of crocin prior to and following exposure to UVB irradiation. The senescent phenotypes of cells were evaluated, including cell proliferation, cell cycle, senescence-associated β-galactosidase (SA-β-gal) expression, intracellular ROS, expression of antioxidant protein glutathione peroxidase 1 (GPX-1) and extracellular matrix protein collagen type 1 (Col-1). Crocin rescued the cell proliferation inhibited by UVB irradiation, prevented cell cycle arrest and markedly decreased the number of SA-β-gal-positive cells. In addition, crocin reduced UVB-induced ROS by increasing GPX-1 expression and other direct neutralization effects. Furthermore, crocin promoted the expression of the extracellular matrix protein Col-1. Crocin could effectively prevent UVB-induced cell damage via the reduction of intracellular ROS; thus, it could potentially be used in the prevention of skin photoaging.

Dual Roles of Graphene Oxide To Attenuate Inflammation and Elicit Timely Polarization of Macrophage Phenotypes for Cardiac Repair.

Development of localized inflammatory environments by M1 macrophages in the cardiac infarction region exacerbates heart failure after myocardial infarction (MI). Therefore, the regulation of inflammation by M1 macrophages and their timely polarization toward regenerative M2 macrophages suggest an immunotherapy. Particularly, controlling cellular generation of reactive oxygen species (ROS), which cause M1 differentiation, and developing M2 macrophage phenotypes in macrophages propose a therapeutic approach. Previously, stem or dendritic cells were used in MI for their anti-inflammatory and cardioprotective potentials and showed inflammation modulation and M2 macrophage progression for cardiac repair. However, cell-based therapeutics are limited due to invasive cell isolation, time-consuming cell expansion, labor-intensive and costly ex vivo cell manipulation, and low grafting efficiency. Here, we report that graphene oxide (GO) can serve as an antioxidant and attenuate inflammation and inflammatory polarization of macrophages via reduction in intracellular ROS. In addition, GO functions as a carrier for interleukin-4 plasmid DNA (IL-4 pDNA) that propagates M2 macrophages. We synthesized a macrophage-targeting/polarizing GO complex (MGC) and demonstrated that MGC decreased ROS in immune-stimulated macrophages. Furthermore, DNA-functionalized MGC (MGC/IL-4 pDNA) polarized M1 to M2 macrophages and enhanced the secretion of cardiac repair-favorable cytokines. Accordingly, injection of MGC/IL-4 pDNA into mouse MI models attenuated inflammation, elicited early polarization toward M2 macrophages, mitigated fibrosis, and improved heart function. Taken together, the present study highlights a biological application of GO in timely modulation of the immune environment in MI for cardiac repair. Current therapy using off-the-shelf material GO may overcome the shortcomings of cell therapies for MI.

Metformin triggers apoptosis in PEL cells and alters bortezomib-induced Unfolded Protein Response increasing its cytotoxicity and inhibiting KSHV lytic cycle activation

Metformin, the most used drug for the treatment of diabetes type 2 patients, has been shown to have anti-cancer properties. In this study, we found that metformin induced apoptosis in Primary Effusion Lymphoma (PEL) cells, an aggressive B cell lymphoma associated with KSHV against which the conventional therapies usually fail. The cytotoxic effect of metformin correlated with intracellular reactive oxygen species reduction, activation of AMPK, the inhibition of pro-survival pathways such as mTOR and STAT3 and the down-regulation of v-FLIP, a latent viral antigen that also plays a pivotal role in PEL cell survival. Interestingly, we found that metformin could be used to potentiate the bortezomib-mediated cytotoxicity against PEL cells and to inhibit the activation of KSHV lytic cycle, a side effect of this treatment that resulted in a block of autophagy in these cells. Mechanistically, metformin altered UPR activated by bortezomib, leading to a reduced expression of BiP, up-regulation of CHOP and down-regulation of Bcl-2. In summary, this study suggests that metformin could represent a promising strategy for the treatment of PEL alone or in combination with bortezomib. In the latter case, besides exerting a stronger cytotoxic effect, it might be used to restrain bortezomib-induced viral replication that is involved in the maintenance and progression of KSHV-associated malignancies.

Reduction of Intracellular-Reactive Oxygen Species and Diminished Mitogen-Activated Protein Kinases (MAPKs) Activation are Associated with Oral Squamous Cell Carcinoma Cell Aggressiveness

Oral squamous cell carcinoma (OSCC) is a serious health problem in many countries. Several drugs have been used to treat head and neck and oral cavity cancers. However, the success rate has not been impressive because of the heterogeneity of cancerous cells, resulting in differential responsiveness to chemotherapy. Two distinct phenotypes of OSCC cells, the CLS-354/WT and CLS-354/DXcells, have been used as in vitro cell models for this study. CLS-354/DXcells were more aggressive than CLS-354/WTcells, supported by the observation that CLS-354/DXcells can undergo epithelial-mesenchymal transition (EMT), grow anchorage-independently, and increase invasiveness. We investigated the preliminary redox status of these 2 cell lines, including levels of reactive oxygen species (ROS) and cellular antioxidants, using flow cytometry analysis and ABTS+ free radical scavenging assay, respectively. A 7-fold decrease in ROS level was detected in CLS-354/DXcells, comparing with CLS-354/WTcells, while antioxidant capacity was not different from that of CLS-354/WT cells. Hydrogen peroxide, a ROS modulating agent, could induce ROS levels, and caused cell death in CLS-354/WT greater than that of CLS-354/DX cells. Of note, hydrogen peroxide-induced cytotoxicity could be rescued by N-acetyl cysteine, confirming ROS-mediated cytotoxicity in both cell lines. ROS-sensitive mitogen-activated protein kinases (MAPKs) were observed using immunoblot assay. The expressions of p-JNK1/2 and p-p38 MAPK in CLS-354/DX cells were absent, while these expressions were abundantly detected in CLS-354/WTcells. This suggests that lower ROS levels, with the concomitant reduction of JNK and p38 MAPK activation in CLS-354/DX cells, are associated with cancer cell aggressiveness. These findings provide significant evidence of the resistance to ROS-modulating agents in aggressive OSCC cells.

Evaluation of the antioxidant capacity, furan compounds and cytoprotective/cytotoxic effects upon Caco-2 cells of commercial Colombian coffee.

Antioxidant capacity (AC), total phenolics (TPs), furan compounds (HMF and furfural F) and cytoprotective/cytotoxic effects upon Caco-2 cells (MTT, cell cycle and reactive oxygen species (ROS)) were evaluated in Colombian coffee (2 ground and 4 soluble samples). The AC (ABTS and FRAP), TPs and HMF ranged between 124-722, 95-802μmoles Trolox/g, 21-100mg gallic acid/g and 69-2900mg/kg, respectively. Pretreatment of cells for 24h with lyophilized coffee infusions at the highest dose without cytotoxic effects (500μg/mL) significantly prevented the decrease in cell viability compared to control stress with H2O2 (5mM/2h), recovering viability to values between 34% and 45% and restoring the control values without stress induction in the G1 phase of cell cycle. After exposure to stress, four extracts decreased ROS values significantly to 22.5-24.9%. The coffee samples exerted a cytoprotective effect against oxidative stress, with improvement in cell viability and a reduction of intracellular ROS.

Glutamine Reduces the Apoptosis of H9C2 Cells Treated with High-Glucose and Reperfusion through an Oxidation-Related Mechanism.

Mitochondrial overproduction of reactive oxygen species (ROS) in diabetic hearts during ischemia/reperfusion injury and the anti-oxidative role of glutamine have been demonstrated. However, in diabetes mellitus the role of glutamine in cardiomyocytes during ischemia/reperfusion injury has not been explored. To examine the effects of glutamine and potential mechanisms, in the present study, rat cardiomyoblast H9C2 cells were exposed to high glucose (33 mM) and hypoxia-reoxygenation. Cell viability, apoptosis, intracellular glutamine, and mitochondrial and intracellular glutathione were determined. Moreover, ROS formation, complex I activity, membrane potential and adenosine triphosphate (ATP) content were also investigated. The levels of S-glutathionylated complex I and mitochondrial apoptosis-related proteins, including cytochrome c and caspase-3, were analyzed by western blot. Data indicated that high glucose and hypoxia-reoxygenation were associated with a dramatic decline of intercellular glutamine and increase in apoptosis. Glutamine supplementation correlated with a reduction in apoptosis and increase of glutathione and glutathione reduced/oxidized ratio in both cytoplasm and mitochondria, but a reduction of intracellular ROS. Glutamine supplementation was also associated with less S-glutathionylation and increased the activity of complex I, leading to less mitochondrial ROS formation. Furthermore, glutamine supplementation prevented from mitochondrial dysfunction presented as mitochondrial membrane potential and ATP levels and attenuated cytochrome c release into the cytosol and caspase-3 activation. We conclude that apoptosis induced by high glucose and hypoxia-reoxygenation was reduced by glutamine supplementation, via decreased oxidative stress and inactivation of the intrinsic apoptotic pathway.

Sodium l-ascorbate enhances elastic fibers deposition by fibroblasts from normal and pathologic human skin

BackgroundVitamin C (l-ascorbic acid), a known enhancer of collagen deposition, has also been identified as an inhibitor of elastogenesis. Objective Present studies explored whether and how the l-ascorbic acid derivative (+) sodium l-ascorbate (SA) would affect production of collagen and elastic fibers in cultures of fibroblasts derived from normal human skin and dermal fat, as well as in explants of normal human skin, stretch-marked skin and keloids.Methods Effects of SA on the extracellular matrix production were assessed quantitatively by PCR analyses, western blots, biochemical assay of insoluble elastin and by immuno-histochemistry. We also evaluated effects of SA on production of the reactive oxygen species (ROS) and phosphorylation of IGF-I and insulin receptors. Results SA, applied in 50–200μM concentrations, stimulates production of both collagen and elastic fibers in all tested cultures. Moreover, combination of SA with a proline hydroxylase inhibitor induces a beneficial remodelling in explants of dermal scars, resulting in the inhibition of collagen deposition and induction of new elastogenesis. Importantly, we revealed that SA stimulates elastogenesis only after intracellular influx of non-oxidized ascorbate anions (facilitated by the sodium-dependent ascorbate transporter), that causes reduction of intracellular ROS, activation of c-Src tyrosine kinase and the enhancement of IGF-1-induced phosphorylation of the IGF-1 receptor that ultimately triggers elastogenic signalling pathway. Conclusion Our results endorse the use of this potent stimulator of collagen and elastin production in the treatment of wrinkled and stretch-marked skin. They also encourage inclusion of SA into therapeutic combinations with collagenogenesis inhibitors to prevent formation of dermal scars and keloids.

Is the antioxidative effectiveness of a bilberry extract influenced by encapsulation?

Bilberries (Vaccinium myrtillus L.) have been suggested to have preventive properties against diseases associated with oxidative stress such as colon cancer or inflammatory bowel diseases. Therefore the gastrointestinal tract is regarded as a potential target for prevention. In this study the antioxidative properties of a commercially available anthocyanin-rich bilberry extract (BE) were investigated in comparison with four different BE-loaded microcapsule systems. As markers to describe the antioxidant status in this cellular system, intracellular reactive oxygen species (ROS) levels, oxidative DNA damage and total glutathione (tGSH) levels were monitored. Incubations with the BE-loaded capsule systems showed an increase in cellular glutathione levels and reduction of ROS levels at high BE concentrations (100-500 µg mL(-1) ) and a positive effect on the formation of DNA strand breaks (5-10 µg mL(-1) BE). The biological properties of BE-loaded pectin amide core-shell capsules, whey protein matrix capsules and coated apple pectin matrix capsules were comparable to those of the non-encapsulated BE. Overall, the BE and the encapsulated BE types tested have antioxidative activity under the studied assay conditions in terms of the prevention of oxidative DNA damage, the reduction of intracellular ROS and the enhancement of cellular tGSH.

Effect of cysteine and glutamine added to extender on post-thaw sperm functional parameters of buffalo bull

Amino acids seem to be crucial components for semen freezing extender due to antioxidant properties. Therefore, this study aimed to assess motility parameters, membrane integrity, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP) and DNA damage to detect the optimum concentrations of cysteine and glutamine for buffalo semen cryopreservation. Twenty ejaculates of four buffalo bulls were diluted in tris-egg yolk extender and divided into seven equal groups consisting of cysteine (5, 7.5 and 10 mmol), glutamine (10, 15 and 20 mmol) and no additive. Supplementation of 5 and 7.5 mmol cysteine and 15 mmol glutamine in cryopreservation extender significantly increased post-thaw motility and plasma membrane integrity of spermatozoa with significant reduction in intracellular ROS when compared with control groups (P < 0.05). Cysteine at 7.5 mmol concentration elevated progressive motility and MMP, compared with control (P < 0.05). No significant differences were observed for motion patterns and DNA damage of frozen-thawed buffalo spermatozoa in extender containing amino acids. The findings of this study showed that supplementation of 7.5 mmol cysteine and 15 mmol glutamine in semen cryopreservation extender has more potential to decrease intracellular ROS, and subsequently elevate motility and membrane integrity of buffalo frozen-thawed spermatozoa.