Dichlorofluorescein Fluorescence Research Articles

Redox modulation of oxidatively-induced DNA damage by ascorbate enhances both in vitro and ex-vivo DNA damage formation and cell death in melanoma cells

Elevated genomic instability in cancer cells suggests a possible model-scenario for their selective killing via the therapeutic delivery of well-defined levels of further DNA damage. To examine this scenario, this study investigated the potential for redox modulation of oxidatively-induced DNA damage by ascorbate in malignant melanoma (MM) cancer cells, to selectively enhance both DNA damage and MM cell killing. DNA damage was assessed by Comet and ɣH2AX assays, intracellular oxidising species by dichlorofluorescein fluorescence, a key antioxidant enzymatic defence by assessment of catalase activity and cell survival was determined by clonogenic assay. Comet revealed that MM cells had higher endogenous DNA damage levels than normal keratinocytes (HaCaT cells); this correlated MM cells having higher intracellular oxidising species and lower catalase activity, and ranked with MM cell melanin pigmentation. Comet also showed MM cells more sensitive towards the DNA damaging effects of exogenous H2O2, and that ascorbate further enhanced this H2O2-induced damage in MM cells; again, with MM cell sensitivity to induced damage ranking with degree of cell pigmentation. Furthermore, cell survival data indicated that ascorbate enhanced H2O2-induced clonogenic cell death selectively in MM cells whilst protecting HaCaT cells. Finally, we show that ascorbate serves to enhance the oxidising effects of the MM therapeutic drug Elesclomol in both established MM cells in vitro and primary cell cultures ex vivo. Together, these results suggest that ascorbate selectively enhances DNA damage and cell-killing in MM cells. This raises the option of incorporating ascorbate into clinical oxidative therapies to treat MM.

Quercetin antagonized advanced glycated end products induced apoptosis and functional inhibition of fibroblasts from the prolapsed uterosacral ligament.

The altered behaviors and functions of pelvic floor fibroblasts are pathophysiological changes of pelvic organ prolapse (POP). Our previous study showed that advanced glycated end products (AGEs) accumulated in the pelvic tissues of POP and induced fibroblast apoptosis. The study was designed to investigate whether quercetin antagonize AGEs-induced apoptosis and functional inhibition of fibroblasts. The uptake of 5-ethynyl-2'-deoxyuridine (EdU) was evaluated for cell proliferation. Flow cytometric analysis was applied for cell apoptosis. Intracellular reactive oxygen species (ROS) content was determined by the fluorescence of dichlorofluorescein (DCF). The contractility of fibroblasts was measured by collagen gel contraction assay. The expressions of extracellular matrix (ECM) related genes and the expression of miR-4429 and caspase-3 were quantified by qPCR. The expressions of phosphatase and tensin homolog (PTEN), phosphoinositide 3-kinase (PI3K), serine-threonine kinase (Akt), and phosphorylated Akt (p-Akt) were analyzed by Western Blot. The down-regulation of miR-4429 was achieved by cell transfection. Quercetin antagonized AGEs-induced apoptosis, proliferation inhibition, and ROS increase in fibroblasts. Quercetin did not alleviate AGEs-induced contractile impairment of fibroblasts. Quercetin reduced the gene expressions of lysyl oxidase like protein 1 (LOXL1)and matrix metallopeptidase 1 (MMP1), and increased the gene expressions of lysyl oxidase (LOX) and fibrillin 2 (FBN2) in fibroblasts. Quercetin reversed AGEs-induced upregulation of PTEN and downregulation of PI3K, P-Akt, and miR-4429 in fibroblasts. The inhibitory effect of quercetin on AGEs-induced fibroblast apoptosis was inhibited by downregulating the expression of miR-4429. In conclusion, quercetin antagonized AGEs-induced apoptosis and functional inhibition of fibroblasts from the prolapsed uterosacral ligament. And inhibiting AGEs-induced down-regulation of miR-4429/PTEN/PI3K/Akt pathway was the mechanism underlying the antagonistic effect of quercetin on AGEs-induced apoptosis.

Topical application of a TRPA1 antagonist reduced nociception and inflammation in a model of traumatic muscle injury in rats.

Musculoskeletal pain is a widely experienced public healthcare issue, especially after traumatic muscle injury. Besides, it is a common cause of disability, but this pain remains poorly managed. However, the pathophysiology of traumatic muscle injury-associated pain and inflammation has not been fully elucidated. In this regard, the transient receptor potential ankyrin 1 (TRPA1) has been studied in inflammatory and painful conditions. Thus, this study aimed to evaluate the antinociceptive and anti-inflammatory effect of the topical application of a TRPA1 antagonist in a model of traumatic muscle injury in rats. The mechanical trauma model was developed by a single blunt trauma impact on the right gastrocnemius muscle of Wistar male rats (250-350g). The animals were divided into four groups (Sham/Vehicle; Sham/HC-030031 0.05%; Injury/Vehicle, and Injury/HC-030031 0.05%) and topically treated with a Lanette® N cream base containing a TRPA1 antagonist (HC-030031, 0.05%; 200mg/muscle) or vehicle (Lanette® N cream base; 200mg/muscle), which was applied at 2, 6, 12, 24, and 46h after muscle injury. Furthermore, we evaluated the contribution of the TRPA1 channel on nociceptive, inflammatory, and oxidative parameters. The topical application of TRPA1 antagonist reduced biomarkers of muscle injury (lactate/glucose ratio), spontaneous nociception (rat grimace scale), inflammatory (inflammatory cell infiltration, cytokine levels, myeloperoxidase, and N-acetyl-β-D-glucosaminidase activities) and oxidative (nitrite levels and dichlorofluorescein fluorescence) parameters, and mRNA Trpa1 levels in the muscle tissue. Thus, these results demonstrate that TRPA1 may be a promising anti-inflammatory and antinociceptive target in treating muscle pain after traumatic muscle injury.

Exosomes derived from M2-type microglia ameliorate oxygen-glucose deprivation/reoxygenation-induced HT22 cell injury by regulating miR-124-3p/NCOA4-mediated ferroptosis

BackgroundAlthough it has been reported that miRNA carried by M2 microglial exosomes protects neurons from ischemia–reperfusion brain injury, the mechanism of action remains poorly understood. This study aimed to explore the miRNA signaling pathway by which M2-type microglia-derived exosomes (M2-exosomes) ameliorate oxygen–glucose deprivation/reoxygenation (OGD/R)-induced cytotoxicity in HT22 cells. MethodsBV2 microglia were induced by M2 polarization. Then, M2-exosomes were identified via transmission electron microscopy and special biomarker detection and co-cultured with HT22 cells. Cell proliferation was evaluated using the Cell Counting Kit-8 (CCK-8) assay. Intracellular concentrations of reactive oxygen species (ROS), Fe2+, glutathione (GSH), and malondialdehyde (MDA) were determined using dichlorofluorescein fluorescence and biochemical determination. miR-124-3p levels were determined using qRT-PCR, and protein expressions were examined via western blotting. ResultsOGD/R suppressed the proliferation and induced the accumulation of Fe2+, ROS, and MDA and reduction of GSH in mouse HT22 cells, suggesting ferroptosis of HT22 cells. OGD/R-induced changes in the above mentioned indexes was ameliorated by M2-exosomes but restored by the exosome inhibitor GW4869. M2-exosomes with (mimic-exo) or without miR-124-3p (inhibitor-exo) promoted and suppressed proliferation and ferroptosis-associated indexes of HT22 cells, respectively. Moreover, mimic-exo and inhibitor-exo inhibited and enhanced NCOA4 expression in HT22 cells, respectively. NCOA4 overexpression reversed the protective effects of miR-124-3p mimic-exo in OGD/R-conditioned cells. NCOA4 was targeted and regulated by miR-124-3p. ConclusionsM2-exosome protects HT22 cells against OGD/R-induced ferroptosis injury by transferring miR-124-3p and NCOA4 into HT22 cells, with the latter being a target gene for miR-124-3p.

Astaxanthin inhibits integrin α5 expression by suppressing activation of JAK1/STAT3 in Helicobacterpylori‑stimulated gastric epithelial cells.

Integrins act as cell‑matrix adhesion molecules involved in cell attachment to the extracellular matrix and generate signals that respond to cancer metastasis. Integrin α5β1 is a heterodimer with α5 and β1 subunits and mediates cell adhesion and migration of cancer cells. Integrins are transcriptionally regulated by the Janus kinase (JAK)/STAT signaling pathways. Our previous study revealed that Helicobacterpylori increased the levels of reactive oxygen species (ROS), which activate JAK1/STAT3 in gastric cancer AGS cells invitro. Astaxanthin (ASX) has been reported to be an effective antioxidant and anticancer nutrient. The present study investigated whether ASX suppresses H.pylori‑induced integrin α5 expression, cell adhesion and migration and whether ASX reduces ROS levels and suppresses phosphorylation of JAK1/STAT3 in gastric cancer AGS cells stimulated with H.pylori. The effect of ASX was determined using a dichlorofluorescein fluorescence assay, western blot analysis, adhesion assay and wound‑healing assay in AGS cells stimulated with H.pylori. The results demonstrated that H.pylori increased the expression levels of integrin α5, without affecting integrin β1, and increased cell adhesion and migration of AGS cells. ASX reduced ROS levels and suppressed JAK1/STAT3 activation, integrin α5 expression, cell adhesion and migration of H.pylori‑stimulated AGS cells. In addition, both a JAK/STAT inhibitor, AG490, and an integrin α5β1 antagonist, K34C, suppressed cell adhesion and migration in H.pylori‑stimulated AGS cells. AG490 inhibited integrin α5 expression in AGS cells stimulated with H.pylori. In conclusion, ASX inhibited H.pylori‑induced integrin α5‑mediated cell adhesion and migration by decreasing the levels of ROS and suppressing JAK1/STAT3 activation in gastric epithelial cells.

Antimicrobial particles based on Cu2ZnSnS4 monograins

In this research, Cu2ZnSnS4 (CZTS) particles were successfully fabricated via the molten salt approach from the copper, zinc and tin sulphides as raw precursors. SEM analysis revealed that CZTS particles are tetragonal-shaped with sharp edges, smooth flat plane morphology, and crystal size varying from 10.8 to 28.7 µm. The phase and crystalline structure of synthesized powders were investigated using XRD analysis, which confirms the presence of a tetragonal crystal structure kesterite phase. The chemical composition of CZTS particles was evaluated by EDX spectroscopy, which identified the nearly stoichiometric composition with an averaged formula of Cu1.88Zn1.04SnS3.97. The TG/DTA-MS and ICP-OES analysis showed the possible decomposition pathways and predicted their degradation rate in aqueous solutions. The CZTS particles possessed highly effective concentration and time-dependent antimicrobial properties against medically relevant bacteria and yeast strains. The CZTS particles (1 g L−1) exhibited over 95.7 ± 1.9% killing efficiency towards M. luteus. In contrast, higher dosages (3.5 and 5 g L−1) led to its complete inactivation and reduced the P. aeruginosa cell viability to 43.2 ± 3.2% and 4.1 ± 1.1%, respectively. Moreover, the CZTS particles (0.5 g L−1) are responsible for causing 54.8 ± 1.8% of C. krusei and 89.7 ± 2.1% of C. parapsilosis yeasts death within the 24 h of exposure, which expanded to almost 100% when yeasts were treated with two times higher CZTS concentration (1.0 g L−1). The mechanism of action has been proposed and evidenced by monitoring the 2′,7′-dichlorofluorescein (DCF) fluorescence, which revealed that the overproduction of reactive oxygen species (ROS) is responsible for microorganism death.

Mitochondrial connexin43 and mitochondrial KATP channels modulate triggered arrhythmias in mouse ventricular muscle.

Connexin43 (Cx43) exits as hemichannels in the inner mitochondrial membrane. We examined how mitochondrial Cx43 and mitochondrial KATP channels affect the occurrence of triggered arrhythmias. To generate cardiac-specific Cx43-deficient (cCx43-/-) mice, Cx43flox/flox mice were crossed with α-MHC (Myh6)-cre+/- mice. The resulting offspring, Cx43flox/flox/Myh6-cre+/- mice (cCx43-/- mice) and their littermates (cCx43+/+ mice), were used. Trabeculae were dissected from the right ventricles of mouse hearts. Cardiomyocytes were enzymatically isolated from the ventricles of mouse hearts. Force was measured with a strain gauge in trabeculae (22°C). To assess arrhythmia susceptibility, the minimal extracellular Ca2+ concentration ([Ca2+]o,min), at which arrhythmias were induced by electrical stimulation, was determined in trabeculae. ROS production was estimated with 2',7'-dichlorofluorescein (DCF), mitochondrial membrane potential with tetramethylrhodamine methyl ester (TMRM), and Ca2+ spark frequency with fluo-4 and confocal microscopy in cardiomyocytes. ROS production within the mitochondria was estimated with MitoSoxRed and mitochondrial Ca2+ with rhod-2 in trabeculae. Diazoxide was used to activate mitochondrial KATP. Most of cCx43-/- mice died suddenly within 8weeks. Cx43 was present in the inner mitochondrial membrane in cCx43+/+ mice but not in cCx43-/- mice. In cCx43-/- mice, the [Ca2+]o,min was lower, and Ca2+ spark frequency, the slope of DCF fluorescence intensity, MitoSoxRed fluorescence, and rhod-2 fluorescence were higher. TMRM fluorescence was more decreased in cCx43-/- mice. Most of these changes were suppressed by diazoxide. In addition, in cCx43-/- mice, antioxidant peptide SS-31 and N-acetyl-L-cysteine increased the [Ca2+]o,min. These results suggest that Cx43 deficiency activates Ca2+ leak from the SR, probably due to depolarization of mitochondrial membrane potential, an increase in mitochondrial Ca2+, and an increase in ROS production, thereby causing triggered arrhythmias, and that Cx43 hemichannel deficiency may be compensated by activation of mitochondrial KATP channels in mouse hearts.

Concurrent bioimaging of microalgal photophysiology and oxidative stress.

The production of reactive oxygen species (ROS) is an unavoidable consequence of oxygenic photosynthesis and represents a major cause of oxidative stress in phototrophs, having detrimental effects on the photosynthetic apparatus, limiting cell growth, and productivity. Several methods have been developed for the quantification of cellular ROS, however, most are invasive, requiring the destruction of the sample. Here, we present a new methodology that allows the concurrent quantification of ROS and photosynthetic activity, using the fluorochrome dichlorofluorescein (DCF) and in vivo chlorophyll a fluorescence, respectively. Both types of fluorescence were measured using an imaging Pulse Amplitude Modulation (PAM) fluorometer, modified by adding a UVA-excitation light source (385nm) and a green bandpass emission filter (530nm) to enable the sequential capture of red chlorophyll fluorescence and green DCF fluorescence in the same sample. The method was established on Phaeodactylum tricornutum Bohlin, an important marine model diatom species, by determining protocol conditions that permitted the detection of ROS without impacting photosynthetic activity. The utility of the method was validated by quantifying the effects of two herbicides (DCMU and methyl viologen) on the photosynthetic activity and ROS production in P. tricornutum and of light acclimation state in Navicula cf. recens Lange-Bertalot, a common benthic diatom. The developed method is rapid and non-destructive, allowing for the high-throughput screening of multiple samples over time.

Combined Application of Aminoglycosides and Ascorbic Acid in the Elimination of Proteus mirabilis Rods Responsible for Causing Catheter-Associated Urinary Tract Infections (CAUTIs)-A Molecular Approach.

Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTIs). In this study, we verified the effectiveness of amikacin or gentamicin and ascorbic acid (AA) co-therapy in eliminating uropathogenic cells, as well as searched for the molecular basis of AA activity by applying chromatographic and fluorescent techniques. Under simulated physiological conditions, a combined activity of the antibiotic and AA supported the growth (threefold) of the P. mirabilis C12 strain, but reduced catheter colonization (≤30%) in comparison to the drug monotherapy. Slight modifications in the phospholipid and fatty acid profiles, as well as limited (≤62%) 2',7'-dichlorofluorescein fluorescence, corresponding to the hydroxyl radical level, allowed for the exclusion of the hypothesis that the anti-biofilm effect of AA was related to membrane perturbations of the C12 strain. However, the reduced (≤20%) fluorescence intensity of propidium iodide, as a result of a decrease in membrane permeability, may be evidence of P. mirabilis cell defense against AA activity. Quantitative analyses of ascorbic acid over time with a simultaneous measurement of the pH values proved that AA can be an effective urine acidifier, provided that it is devoid of the presence of urease-positive cells. Therefore, it could be useful in a prevention of recurrent CAUTIs, rather than in their treatment.

Experimental Conditions That Influence the Utility of 2'7'-Dichlorodihydrofluorescein Diacetate (DCFH2-DA) as a Fluorogenic Biosensor for Mitochondrial Redox Status.

Oxidative stress has been causally linked to various diseases. Electron transport chain (ETC) inhibitors such as rotenone and antimycin A are frequently used in model systems to study oxidative stress. Oxidative stress that is provoked by ETC inhibitors can be visualized using the fluorogenic probe 2′,7′-dichlorodihydrofluorescein-diacetate (DCFH2-DA). Non-fluorescent DCFH2-DA crosses the plasma membrane, is deacetylated to 2′,7′-dichlorodihydrofluorescein (DCFH2) by esterases, and is oxidized to its fluorescent form 2′,7′-dichlorofluorescein (DCF) by intracellular ROS. DCF fluorescence can, therefore, be used as a semi-quantitative measure of general oxidative stress. However, the use of DCFH2-DA is complicated by various protocol-related factors that mediate DCFH2-to-DCF conversion independently of the degree of oxidative stress. This study therefore analyzed the influence of ancillary factors on DCF formation in the context of ETC inhibitors. It was found that ETC inhibitors trigger DCF formation in cell-free experiments when they are co-dissolved with DCFH2-DA. Moreover, the extent of DCF formation depended on the type of culture medium that was used, the pH of the assay system, the presence of fetal calf serum, and the final DCFH2-DA solvent concentration. Conclusively, experiments with DCFH2-DA should not discount the influence of protocol-related factors such as medium and mitochondrial inhibitors (and possibly other compounds) on the DCFH2-DA-DCF reaction and proper controls should always be built into the assay protocol.

Platelet CD40L Expression Response to Mixing of pRBCs and Washed Platelets but no Causality Association between Platelet ROS Generation and CD40L Expression: An In Vitro Study.

Platelets play a role in transfusion reaction via reactive oxygen species (ROS) generation and CD40 ligand (CD40L) expression. In this study, we aimed to test the hypothesis that the mixing of packed red blood cells (pRBCs) and washed platelets has a causal effect on platelet ROS generation and CD40L expression. Thus, a better understanding of this causality relationship may help interrupt the chain of events and avoid an uncontrollable transfusion reaction. We simulated transfusion in vitro by mixing pRBCs and washed platelets. Donor cross-matched stored pRBCs) from our blood bank and recipient whole blood from patients undergoing coronary artery bypass graft surgery prepared into washed platelets were used. Briefly, donor pRBCs were added to washed recipient platelets to form 1%, 5%, or 10% (v/v) mixtures. The mixed blood sample was used to determine platelet ROS generation (dichlorofluorescein fluorescence levels) and CD40L expression. The effect of antioxidants (20 mM glutamine and 20 mM dipeptiven) on ROS generation and CD40L expression was also evaluated. Platelet ROS generation was not significantly associated with the mixing of pRBCs and washed platelets (p = 0.755), glutamine treatment (p = 0.800), or dipeptiven treatment (p = 0.711). The expression of CD40L by platelets increased significantly (p < 0.001), and no significant difference was noted after treatment with glutamine (p = 0.560) or dipeptiven (p = 0.618). We observed that the mixing pRBCs and washed platelets had no effect via ROS, whereas CD40L could directly induce transfusion reactions. Furthermore, platelets did not causally express ROS or CD40L after being mixed with pRBCs. Although antioxidants are more accessible than anti-CD40L antibodies, platelet ROS may not serve as a therapeutic target for antioxidants. Nevertheless, CD40L expression may be a valuable therapeutic target for managing transfusion reactions.

Optimal Use of 2',7'-Dichlorofluorescein Diacetate in Cultured Hepatocytes.

Oxidative stress is a state that arises when the production of reactive transients overwhelms the cell's capacity to neutralize the oxidants and radicals. This state often coincides with the pathogenesis and perpetuation of numerous chronic diseases. On the other hand, medical interventions such as radiation therapy and photodynamic therapy generate radicals to selectively damage and kill diseased tissue. As a result, the qualification and quantification of oxidative stress are of great interest to those studying disease mechanisms as well as therapeutic interventions. 2',7'-Dichlorodihydrofluorescein-diacetate (DCFH2-DA) is one of the most widely used fluorogenic probes for the detection of reactive transients. The nonfluorescent DCFH2-DA crosses the plasma membrane and is deacetylated by cytosolic esterases to 2',7'-dichlorodihydrofluorescein (DCFH2). The nonfluorescent DCFH2 is subsequently oxidized by reactive transients to form the fluorescent 2',7'-dichlorofluorescein (DCF). The use of DCFH2-DA in hepatocyte-derived cell lines is more challenging because of membrane transport proteins that interfere with probe uptake and retention, among several other reasons. Cancer cells share some of the physiological and biochemical features with hepatocytes, so probe-related technical issues are applicable to cultured malignant cells as well. This study therefore analyzed the in vitro properties of DCFH2-DA in cultured human hepatocytes (HepG2 cells and differentiated and undifferentiated HepaRG cells) to identify methodological and technical features that could impair proper data analysis and interpretation. The main issues that were found and should therefore be accounted for in experimental design include the following: (1) both DCFH2-DA and DCF are taken up rapidly, (2) DCF is poorly retained in the cytosol and exits the cell, (3) the rate of DCFH2 oxidation is cell type-specific, (4) DCF fluorescence intensity is pH-dependent at pH < 7, and (5) the stability of DCFH2-DA in cell culture medium relies on medium composition. Based on the findings, the conditions for the use of DCFH2-DA in hepatocyte cell lines were optimized. Finally, the optimized protocol was reduced to practice and DCFH2-DA was applied to visualize and quantify oxidative stress in real time in HepG2 cells subjected to anoxia/reoxygenation as a source of reactive transients.

The potential of prodigiosin for control of Prorocentrum donghaiense blooms: Algicidal properties and acute toxicity to other marine organisms at various trophic levels

Prorocentrum donghaiense, a marine dinoflagellate, causes harmful algal blooms (HABs) characterised by the highest outbreak frequency and most extensive coverage among similar species in the East China Sea. Highly efficient and ecofriendly biocontrol strategies should be developed for HAB control. Prodigiosin is an efficient biological algicide that demonstrated strong algicidal activity towards P. donghaiense. However, the mechanism of its toxicity to P. donghaiense is unknown. These factors were investigated to evaluate potential use of prodigiosin for control of P. donghaiense blooms. Photosynthetic electron transport rate, maximum quantum yield and respiration rate of P. donghaiense decreased significantly upon exposure to prodigiosin, indicating that prodigiosin rapidly exerted adverse effects on the chloroplasts and mitochondria. Furthermore, a significant increase in dichlorofluorescein fluorescence intensity indicated an overproduction of reactive oxygen species (ROS). The antioxidant system of P. donghaiense scavenged ROS; however, an increase in malondialdehyde concentrations indicated that excessive ROS were still able to initiate lipid peroxidation. Thus, ROS production resulted in the formation of lipids with a reduced degree of unsaturation. Lipid peroxidation decreased lipid fluidity and rigidified the membrane system, causing serious functional destruction of the membrane. Flow cytometry analysis indicated that prodigiosin arrested the cell cycle of P. donghaiense. However, surviving algal cells were able to repair the damaged functions and resume the cell cycle after prodigiosin was removed by photodegradation. Otherwise, P. donghaiense cells lost their membrane integrity and died. To begin an evaluation of ecological safety of prodigiosin, we tested four marine organisms at various trophic levels. The results of these tests indicated that Chlorella vulgaris, Photobacterium phosphoreum, Artemia salina and Lateolabrax japonicus were less sensitive to prodigiosin than P. donghaiense. Toxicity to all five organisms declined after prodigiosin was exposed to sunlight for 6 h. Considering the toxic doses of prodigiosin to various organisms and its photodegradation characteristics, we suggest that prodigiosin has potential in controlling P. donghaiense blooms but should be applied at night, in small doses, with multiple applications.

Association of anti-oxidative capacity of HDL with subclinical atherosclerosis in subjects with and without non-alcoholic fatty liver disease

BackgroundNon-alcoholic fatty liver disease (NAFLD) patients are at a substantial risk for developing cardiovascular disease (CVD). High-density lipoprotein (HDL) is well known to have protective effects against the development of atherosclerotic CVD. One of the major antiatherogenic effects of HDL is its anti-oxidative function.ObjectivesThis study investigated the association of anti-oxidative capacity of HDL with subclinical atherosclerosis in NAFLD and non-NAFLD subjects.MethodsA total of 143 subjects including 51 NAFLD and 92 control subjects were included in this case–control study. HDL oxidative index (HOI) was determined spectrophotometrically using a cell-free method in the presence of a fluorescent substrate dichlorofluorescein diacetate (DCFDA). Paraoxonase 1 (PON1) activity, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) plasma levels were assessed in both groups.ResultsThe NAFLD patients with impaired HDL anti-oxidative function (HOI ≥ 1) had higher MDA levels, aspartate amino transferase (AST), liver stiffness (LS), and carotid intima-media thickness (cIMT) values compared to the controls. HDL oxidative index (HOI) was positively correlated with MDA levels and cIMT and negatively correlated with SOD activity.ConclusionsHigher circulating levels of MDA were associated with the impaired anti-oxidative function of HDL in NAFLD. The impaired anti-oxidative capacity of HDL might be related to NAFLD severity and subclinical atherosclerosis in NAFLD patients.

Lipid peroxidation reduction and hippocampal and cortical neurons protection against ischemic damage in animal model using Stellaria media

This study was aimed to determine the neuroprotective influence of Stellaria media in terms of restoring normal state of the rat’s hippocampus and cortex after oxidative insult caused by in vitro ischemia and reperfusion. Cell viability and membrane integrity were assessed using MTT and lactate dehydrogenase (LDH) assay, respectively. Ischemic insult was introduced in the rat brain’s hippocampal and cortical slivers by exposing oxygen and glucose deficiency (OGD) for 2 h, followed by 1 h of re-perfusion. Cellular oxidative stress levels were quantified by incorporating 2ʹ,7ʹ-dichlorofluorescein diacetate fluorescent probes. Additionally, the lipid peroxidation was assessed using TBARS assay. Findings revealed significant neuroprotection against OGD-induced mitochondrial impairment at 40 µg/mL of S. media in rat’s hippocampal and cortical slices. The LDH levels were decreased significantly (P < 0.001) during pre-incubation and reoxygenation periods using varied concentrations of S. media extract. Cellular oxidative stress levels results showed significant (P < 0.001) reduction in dichlorofluorescein fluorescence in slices homogenate of hippocampus and cortex using S. media extract. The lipid peroxidation assay results showed decreased (P < 0.01) levels of malondialdehyde in liver tissues of treated rats treated (200 mg/kg body weight) when compared to the ischemic animal. In summary, findings clearly indicated the neuroprotective effects of extract against in vitro ischemia in brain hippocampal and cortex slivers. S. media could undoubtedly be utilized as a healing agent in preventing neuronal cells’ loss during is chemic-reperfusion process.

Targeting multiple mediators of sepsis using multifunctional tannic acid-Zn2+-gentamicin nanoparticles

Targeting multiple mediators of sepsis using multifunctional tannic acid-Zn2+-gentamicin nanoparticles

Virgin coconut oil complements with its polyphenol components mitigate sodium fluoride toxicity in vitro and in vivo

Virgin coconut oil (VCO), prepared from fresh coconut kernel without any chemical refining, is an emerging functional food. The pharmacological benefits of VCO are believed to be due to the natural combination of phenolics. Although cell culture studies have demonstrated the antioxidant activity of VCO under different oxidative stress conditions, a valid in vivo demonstration of the antioxidant activity of VCO is yet to come. Sodium fluoride (NaF), an environmental pollutant, is widely used to induce oxidative stress in cell culture models and rodents to test the antioxidant potential of several compounds. Herein, VCO and its polyphenolic (VCOP) and non-phenolic oil fraction (VCOF) were individually tested in fluoride-exposed normal intestinal cells (IEC-6) and mice to address their contribution to the documented antioxidant potential. It was found that pretreatment of VCOP (40 µg/mL) was effective in mitigating the fluoride-induced cell death when compared to VCO (200 µg/mL) and VCOF (160 µg/mL). Further, exposure to fluoride (10 mM), increased the intracellular ROS measured based on the dichlorofluorescein (DCF) fluorescence, and this, in turn, was significantly reduced when the cells were supplemented with VCOP. Oral administration of VCO (2 mL/kg bwt) reversed the drop in the hepatic catalase and SOD activities to near normal with a minimal level of lipid peroxidation in fluoride intoxicated mice. However, VCOP and VCOF were less effective in lowering the fluoride-induced increase in hepatic oxidative stress markers. It is reasoned that the oil components of VCO complement the natural antioxidant molecules resulting in an overall increase in their bioavailability.

Effects of Superoxide Dismutase Inhibitors and Glucose on Cell Death and Generation of Reactive Oxygen Species in Pea Leaves.

The effects of superoxide dismutase (SOD) inhibitors, diethyldithiocarbamate (DDC), triethylenetetramine (trien), and their combination with glucose on cells of the epidermis from pea leaves of different age (rapidly growing young leaves and slowly growing old leaves) was investigated. DDC and trien caused death of the guard cells as determined by destruction of their nuclei. Glucose did not affect destruction of the nuclei induced by SOD inhibitors in the cells from old leaves, but intensified it in the cells from young leaves. 2-Deoxyglucose, an inhibitor of glycolysis, and propyl gallate, SOD-mimic and antioxidant, suppressed destruction of the nuclei that was caused by SOD inhibitors and glucose in cells of the epidermis from the young, but not from the old leaves. Glucose and trien stimulated, and propyl gallate reduced generation of reactive oxygen species (ROS) in the pea epidermis as determined by the fluorescence of 2',7'-dichlorofluorescein (DCF). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a protonophoric uncoupler of oxidative and photosynthetic phosphorylation, suppressed the DCF fluorescence in the guard cells. Treatment of the cells with CCCP followed by its removal with washing increased destruction of the nuclei caused by SOD inhibitors and glucose. In young leaves, CCCP was less effective than in old ones. The findings demonstrate the effects of SOD inhibitors and glucose on the cell death and generation of ROS and could indicate glycolysis-dependent ROS production.

Inhibitory Effect of Korean Red Ginseng Extract on Interleukin-8 Expression in Helicobacter pylori-Infected Gastric Epithelial Cells

ObjectivesInfection of Helicobacter pylori (H. pylori), a gram-negative bacterium, leads to various gastric diseases, such as gastritis, peptic ulcer and gastric cancer. H. pylori increases cytokine release and activates inflammatory mediators in gastric mucosa. Particularly, H. pylori upregulates the inflammatory chemokine interleukin-8 (IL-8), which are activated by oxidative stress. IL-8 can cause severe inflammation of the stomach and gastric cancer. Korean red ginseng is the steamed root of 6-year-old Korean ginseng (Panax ginseng Meyer). Ginsenosides, triterpene glycosides, are the active components of Korean red ginseng. Ginsenosides have antioxidant, anti-inflammatory, and antitumor activities. The present study is aimed at determining whether Korean red ginseng extract inhibits H. pylori-induced IL-8 expression in gastric epithelial cells. MethodsThe human gastric epithelial cell line AGS was used. Gastric epithelial AGS cells were treated with Korean red ginseng extract, and infected with H. pylori (NCTC 11,637). Reactive oxygen species (ROS) levels were determined using dichlorofluorescein fluorescence. NADPH oxidase activity was measured using lucigenin chemiluminescence. IL-8 mRNA expression was measured by using real-time PCR. NADPH oxidase subunits were determined in cytosolic extract and membrane extract by using Western blotting. ResultsH. pylori increased NADPH oxidase activity, ROS levels, and upregulated IL-8 expression in gastric epithelial cells. Korean red ginseng extract inhibited IL-8 expression by suppressing NADPH oxidase activity and reducing ROS levels in gastric epithelial cells. H. pylori induced translocation of NADPH oxidase cytosolic subunits to membrane, which is a marker of NADPH oxidase activation, in AGS cells. Korean red ginseng extract inhibited translocation of cytosolic subunits of NADPH oxidase to membrane in AGS cells. ConclusionsKorean red ginseng may be beneficial for preventing H. pylori-associated gastric inflammation by inhibiting oxidative stress and IL-8 expression. Funding SourcesThis study was supported by a Brain Korea 21 FOUR Project, Yonsei University, Seoul, Republic of Korea.

Intracellular ROS profile in hematopoietic progenitors of MDS patients: association with blast count and iron overload

ABSTRACT Objectives Reactive oxygen species (ROS) are under scrutiny as a participant in the pathophysiology of myelodysplastic syndrome (MDS) and the progression of MDS to acute myeloid leukemia (AML). Measurement of intracellular ROS (iROS) is particularly important since iROS is a direct indicator of cellular health and integrity. Methods We developed a technique to measure standardize iROS (siROS) level in lymphocytes and bone marrow (BM) CD34+ hematopoietic progenitors using the fluorescent probe dichlorofluorescein (DCF). We then quantified the siROS in 38 consecutive BM specimens from 27 MDS patients over the course of 10 months. Disease outcome of these patients were also assessed. Results High serum ferritin, high blast count and poor IPSS were associated with inferior survival and AML progression in this cohort. High blast MDS patients had lower siROS in their BM CD34+ cells than those of low blast patients, consistent with increased reliance on glycolysis and enhanced ROS defense in high blast MDS. We also observed narrower siROS distribution in the BM CD34+ cells of high blast patients, suggesting that loss of heterogeneity in ROS content accompanies the clonal evolution of MDS. Furthermore, we observed a strong correlation between CD34+ cells siROS and serum ferritin level in high blast patients. In one case, iron chelation therapy (ICT) resulted in parallel decreases in serum ferritin and CD34+ cells siROS. Conclusion Our findings established the siROS profile in early hematopoietic cells of MDS patients and its relationship with blast count and iron overload.