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The inositol 1,4,5-trisphosphate receptor (InsP(3)R) is up-regulated in patients with atrial fibrillation (AF) and InsP(3)-induced Ca(2+) release (IICR) is linked to pro-arrhythmic spontaneous Ca(2+) release events. Nevertheless, knowledge of the physiological relevance and regulation of InsP(3)Rs in atrial muscle is still limited. We hypothesize that InsP(3)R and NADPH oxidase 2 (NOX2) form a functional signaling domain where NOX2 derived reactive oxygen species (ROS) regulate InsP(3)R agonist affinity and thereby Ca(2+) release. To quantitate the contribution of IICR to atrial excitation-contraction coupling (ECC) atrial myocytes (AMs) were isolated from wild type and NOX2 deficient (Nox2(−/−)) mice and changes in the cytoplasmic Ca(2+) concentration ([Ca(2+)](i); fluo-4/AM, indo-1) or ROS (2’,7’-dichlorofluorescein, DCF) were monitored by fluorescence microscopy. Superfusion of AMs with angiotensin II (AngII: 1 μmol/L) significantly increased diastolic [Ca(2+)](i) (F/F(0), Ctrl: 1.00±0.01, AngII: 1.20±0.03; n=7; p

Background Non-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. Objectives This study investigated the association of anti-oxidative capacity of HDL with subclinical atherosclerosis in NAFLD and non-NAFLD subjects. Methods A 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. Results The 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. Conclusions Higher 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.

BACKGROUND Alzheimer's disease (AD) is a progressive dementia, and β-amyloid (Aβ) accumulation is widely regarded as the primary pathogenesis of AD. A new synthetic compound, 8-hydroxyquinoline-resveratrol derivative (E)-5-(4-hydroxystyryl)quinolin-8-ol (10c) was evaluated as a possible anti-AD agent. METHOD (I) The total amount of ROS in SH-SY5Y cells was detected by dichlorofluorescein diacetate (DCFH-DA), and the antioxidant activity and neuroprotective effect of 10c in SH-SY5Y cells were evaluated; (II) Griess reagent was used to test the activity of Compound 10c against NO production in LPS-induced BV-2 microglial cells; (III) An automatic digital stereotaxic instrument was used to inject Aβ25-35 into the brain to establish an AD animal model to evaluate the protective effect of compound 10c on Aβ25-35-induced learning and memory dysfunction in rats. RESULTS 10c exhibited far more potent antioxidant activity for both exogenous and endogenous reactive oxygen species (ROS) than trolox, resveratrol, and CQ (ROS production: 10c with 26.23% at 1.5 µM; resveratrol with 82.17% at 2.5 µM; CQ with 78.52% at 10 µM). 10c also shows good neuroprotective effects as an endogenous antioxidant in neuroblastoma cells. Moreover, Compound 10c also demonstrated effective inhibition of nitric oxide (NO) production (IC50 =3.10 µM) and IL-1β production in BV-2 microglial cells which were treated with lipopolysaccharide (LPS). In the water maze test, the numbers of rats who crossed the former platform were increased significantly in both the 10c group (5.7±1.6) and positive control group (CQ, 5.1±1.7). Meanwhile, both 10c (43.8±5.5 s) and CQ (44.1±6.6 s) treatment could significantly prolong the time rats spent in the target quadrant compared to the vehicle-treated model group. These results demonstrated that 10c could alleviate the learning and memory dysfunction of rats induced by Aβ25-35 to a certain extent. CONCLUSIONS Altogether, compound 10c is a promising compound for the treatment of AD.

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.

Triple-negative breast cancer (TNBC) remains as the deadliest cancer type due to the lack of treatment options. Hence, several attempts have been made to develop new anticancer for TNBC therapy. This study intended to challenge curcumin analog (CCA)-1.1, which is derived from pentagamavunone-1 structure, against the 4T1 cell line and TNBC cell model, covering the cytotoxic activity in correlation with cell cycle progression, apoptosis induction, reactive oxygen species (ROS) generation, and senescence evidence. The cell viability, cell cycle profile, apoptosis induction, intracellular ROS level, and senescence induction were determined in vitro using trypan blue exclusion, propidium iodide (PI) staining, Annexin-PI staining, dichlorofluorescein diacetate staining, and senescence-associated-β-gal method. CCA-1.1 showed cytotoxic activity on 4T1 cells, giving half maximal inhibitory concentration value of 3M, but was less toxic on non-cancerous 3T3-L1 cells. CCA-1.1 induced rapid cell death and inhibited cell cycle progression at the mitotic phase. Instead, of causing apoptosis, CCA-1.1 induced mitotic catastrophe. Furthermore, CCA-1.1 itself increased the intracellular ROS level and induced senescence, possibly through catastrophic cell death. Altogether, our preliminary study strengthens the potency of CCA-1.1 for its anticancer activities against TNBC cells and prospective to be pharmaceutically developed as a novel candidate for cancer therapy.

Various metabolites can modulate functioning of neuronal presynaptic terminals. We studied the effects of lactate and an agonist of its HCAR1 receptor, 3,5-dihydroxybenzoic acid, on the synaptic vesicle cycle and generation of reactive oxygen species in synaptosomes from the rat brain. In contrast to pyruvate, lactate did not affect the synaptic vesicle cycle at a 4 mM concentration. Using the acridine orange fluorescent probe, we revealed that 3,5-dihydroxybenzoic acid activated endocytosis at a 500 μM concentration. Lactate, but not 3,5-dihydroxybenzoic acid, increased fluorescence of 2',7'-dichlorofluorescein diacetate indicating the generation of reactive oxygen species. Using the MitoSOX fluorescent probe we also showed the formation of superoxide anion in mitochondria after the lactate treatment. Thus, lactate may affect the functioning of neuronal presynaptic terminals due to the formation of reactive oxygen species and activation of endocytosis, which is mediated by HCAR1 lactate receptor.

Depletion of reactive oxygen species and reduction of oxidative stress have been identified as key parameters in the prevention of cellular aging. In previous in vitro studies, the tea catechin epigallocatechin gallate (EGCG) was found to have both pro- and antioxidant properties, disregarding the low stability under cell culture conditions. Besides hydrogen peroxide, theasinensin dimers amongst other oxidation products are formed. Exact quantities, cellular uptake and antioxidant capacities of these dimeric oxidation products remain unknown. Via high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (MS/MS), formation kinetics and cellular uptake of EGCG and its major oxidation products are quantified. The antioxidant capacity is determined on a cellular level using a modified dichlorofluorescein (DCF) approach. As a first result, oxidation product quantities of up to 21 µM each are measured after incubation of 50 µM EGCG. While EGCG is taken up equimolarly, its major oxidation products are accumulated in hepatocarcinoma HepG2 cells at millimolar concentrations, especially theasinensin A (TSA). Lastly, the oxidation products show higher antioxidant properties than the monomer EGCG. In correlation with cellular uptake, TSA displays the highest capacity of all tested analytes. The findings reveal the strong influence of EGCG oxidation products on its bioactivity in vitro.

Purpose: To determine the apoptotic effect of trichlorophenyl-benzoxime (TCPB) on colorectal cancer (CRC) cells, and to elucidate the mechanism of action. Methods: Colon carcinoma cell lines (DLD-1 and HT-29) were used in this study. The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10 % fetal bovine serum (FBS) and 1 % penicillin/streptomycin at 37 ˚C in an atmosphere of 5 % CO2 and 95 % air. When the cells attained 60 - 70 % confluency, they were treated with serum-free medium and graded concentrations of TCPB (1.0 – 6.0 μM) for 24 h. Cell viability and apoptosis were assessed using 3-(4, 5-dimethylthiazol-2-yl) 2, 5-diphenyltetrazolium bromide (MTT) and flow cytometric assays, respectively. Western blotting and 2', 7' dichlorofluorescein diacetate (DCFH DA) assays were used for the determination of expression levels of apoptotic proteins, and levels of reactive oxygen species (ROS), respectively. Results: Treatment of DLD-1 and HT-29 cells with TCPB led to significant and dose-dependent reductions in their viability, as well as significant and dose-dependent increases in the number of apoptotic cells (p < 0.05). Treatment of HT-29 cells with TCPB led to significant increases in the population of cells in the G0/G1 phase, but significant reduction of cell proportion in S and G2/M phases (p < 0.05). It also significantly and dose-dependently upregulated the expressions of caspase-3 and bax, down-regulation of the expression of bcl-2 (p < 0.05). TCPB treatment upregulated the expressions of p53, cytochrome c (cyt c), procaspase-3, and procaspase-9, but down-regulated the expression of pAkt dose-dependently (p < 0.05). The expression of Akt in HT-29 cells was not significantly affected by TCPB (p > 0.05). However, TCPB significantly enhanced the cleavage of PARP1, and significantly and dose-dependently increased the levels of ROS in HT-29 cells (p < 0.05). Conclusion: These results suggest that TCPB exerts apoptotic effect on CRC cells via activation of mitochondria-dependent pathway, and thus can be suitably developed for the management of colon cancer.

Iron(III) complexes of curcumin (Hcur) having a tridentate NNN-donor dipicolylamine-based ligand L1 and its biotinylated analogue L2, namely, Fe(L1)(cur)Cl (1) and Fe(L2)(cur)Cl (2), were prepared, characterized and their photo-induced cytotoxicity studied. The complexes exhibited curcumin-based absorption band near 430 nm and emission maxima at 520 nm. Complex 2 with a biotin moiety showed enhanced cellular uptake and higher cytotoxicity compared to its non-biotin analogue 1. The visible light induced cytotoxicity was studied in HeLa, MCF-7 and HepG2 cell lines. Complex 2 displayed photodynamic effect (400â��700 nm, 10 J cmâ��2) giving an IC50 value of â�¼4 I¼M in HeLa and MCF-7 cells, while being significantly non-toxic in the dark (IC50>100 I¼M). A 5-fold increase in cytotoxicity was observed in HepG2 cells (IC50â�¼0.7 I¼M) for the biotin-appended complex 2. In vitro generation of reactive oxygen species (ROS) was observed from dichlorofluorescein diacetate (DCFDA) assay. The mode of cell death was apoptotic. Formation of hydroxyl radicals as the ROS was evidenced from the plasmid pUC19 DNA photo-cleavage studies. The complexes demonstrated the ability to act as targeted PDT (photodynamic therapy) drugs. © 2021 Wiley-VCH GmbH

Maloplatin-B, a cisplatin-based complex, namely [Pt(A-BOD)(NH3)2](NO3) (Pt-A-BOD) with a pendant boron-dipyrromethene (BODIPY) moiety, where HA-BOD is a methyl malonyl chloride derived monostyryl BODIPY ligand, was designed and developed as near-IR light (600-720 nm) organelle-targeting photodynamic therapy agent. The complex [Pt(acac)(NH3)2](NO3) (Pt-Ac) was used as a control. Pt-A-BOD displayed an absorption band at 616 nm (e = 2.9 × 104 M-1 cm-1) in 10% dimethyl sulfoxide/Dulbecco's Modified Eagle's Medium (DMSO/DMEM, pH 7.2). This complex displayed a broad emission band within 650-850 nm with a λem value of 720 nm in 10% DMSO-DMEM (pH 7.2) upon excitation (λex) at 615 nm with a large Stokes shift. The fluorescence quantum yield (ΦF) value for Pt-A-BOD is 0.032 and for the ligand HA-BOD is 0.24. The BODIPY complex and ligand showed the formation of singlet oxygen as the ROS (reactive oxygen species) on irradiation with near-IR red light of 660 nm, as evidenced from a 1,3-diphenylisobenzofuran (DPBF) assay. The complex displayed remarkable apoptotic NIR light-induced PDT activity with half-maximum inhibitory concentration values (IC50) of 1.6-2.4 μM in A549 lung and HeLa cervical cancer cells, while it was less active in the dark. The cellular ROS generation by the complex in red light was ascertained by a DCFDA (2',7'-dichlorofluorescein diacetate) assay. Cellular imaging showed its localization primarily in the mitochondria of A549 cancer cells. The JC1 and Annexin-V FITC/PI assays carried out for A549 cancer cells treated with the BODIPY complex showed the alteration of mitochondrial membrane potential and apoptotic cell death on near-IR red light (600-720 nm) irradiation, respectively.

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). Me...

Backgrounds Preeclampsia (PE) is characterized as placental vascular disturbance and excessive secretion of soluble fms-like tyrosine kinase 1 (sFlt-1) into the maternal circulation. Trimethylamine N-oxide (TMAO, a gut microbe-derived metabolite) is strongly associated with various cardiovascular and cerebrovascular diseases. Recently, we observe that higher maternal circulating TMAO and sFlt-1 in patients with PE. The aims of the present study are to explore the effects of TMAO on placental sFlt-1 production and the underlying mechanism in human placenta. Methods Human placental explants, human placental primary trophoblasts and the extravillous trophoblasts (EVT) cell line (HRT-8/SVneo) were exposured to various concentrations of TMAO (100, 150, 300, and 600 μM). The mRNA expression and protein secretion of sFlt-1 in placental explants, primary trophoblasts and HRT-8/SVneo cells were determined with qPCR and ELISA, respectively. The levels of intracellular reactive oxygen species (ROS) production in primary trophoblasts and HRT-8/SVneo cells were measured by peroxide-sensitive fluorescent probe dichlorofluorescein diacetate. Results Exposure of placental explants, primary trophoblasts and HRT-8/SVneo cells to TMAO significantly enhanced sFlt-1 at both mRNA and protein levels in a dose dependent manner. Moreover, inhibition of NADPH oxidase with apocynin significantly attenuated TMAO-induced ROS production in primary trophoblasts and HRT-8/SVneo, and suppressed sFlt-1 secretion in placental explants, primary trophoblasts and HRT-8/SVneo. Conclusions Our findings indicated the NADPH oxidase dependent ROS pathway played a critical role in mediating TMAO-induced sFlt-1 generation in human placenta. TMAO may become a potential novel target for pharmacological or dietary interventions to reduce the risk of developing PE.

Linearolactone (1) and kaempferol (2) have amebicidal activity in in vitro studies. The type of cell death induced by 1 and 2 and their effects on the virulence of E. histolytica were analyzed by transmission and confocal electron microscopy, reactive oxygen species (ROS) production, and apoptosis, detected by flow cytometry with dichlorofluorescein 2',7'-diacetate and annexin-V binding, respectively, and confirmed by TUNEL. The interaction of 1 and 2 with actin was analyzed by docking, and the in vivo amoebicidal activity was established with the Mesocricetus auratus model; amebic liver abscess (ALA) development was evaluated by magnetic resonance (MR) and validated post mortem. In vitro, compounds 1 and 2 caused chromatin condensation, intracellular ROS, and loss of actin structures. Coupling analysis showed that they bind to the allosteric and catalytic sites of actin with binding energies of -11.30 and -8.45 kcal/mol, respectively. Treatments with 1 and 2 induced a decrease in ALA formation without toxic effects on the liver and kidney. Thus, compound 1, but not 2, was able to induce apoptosis-like effects in E. histolytica trophozoites by intracellular production of ROS that affected the actin cytoskeleton structuration. In vivo, compound 1 was more active than compound 2 to reduce the development of ALA.

In this study, we have studied the cytotoxicity and genotoxic potency of 3 pro-oxidants; H2O2, menadione and KBrO3 in different dosing scenarios, namely acute (1-day dosing) and chronic (5-days). For this purpose, relative population doubling (RPD%) and mononucleated micronucleus (MN) test were used. TK6 cells and NH32 were employed in in vitro experiments. In the study, the total acute dose was divided into 5 days for each prooxidant chemicals by dose fractionation (1/5th per day) method. Acute dosing was compared to chronic dosing. The oxidative stress caused by the exposure of cells with pro-oxidant chemicals to the cells was determined by an optimized 2′,7′-dichlorofluorescein diacetate (DCFHDA) test method. The antioxidant levels of the cell lines were altered with buthionine sulfoxide (BSO) and N-acetyl cysteine (NAC), and the effect of antioxidant capacity on the MN formation in the cells was observed with this method. In the case of H2O2 and menadione, fractional dosing has been observed to result in lower toxicity and lower genotoxicity. But in the case of KBrO3, unlike the other 2 pro-oxidants, higher MN induction was observed with fractionated doses. DCFHDA test clearly demonstrated ROS induction with H2O2 and menadione but not with KBrO3. Unexpectedly, DCFHDA test demonstrated that KBrO3 did not cause an increase ROS levels in both acute and chronic dosing, suggesting an alternative ROS induction mechanism. It was also observed that, treatment with BSO and NAC, caused increasing and decreasing of MN fold change respectively, allowing further ROS specific mechanisms to be explored. Hence, dose fractionation expectedly caused less MN, cytotoxicity and ROS formation with H2O2 and menadione exposure, but not with KBrO3. This implies a unique mechanism of action for KBrO3 induced genotoxicity. Chronic dosing in vitro may be a valuable approach allowing better understanding of how chemicals damage DNA and pose human hazards.

The potent bacterial infections serve as the primary cause of mortality due to the absence of novel antibacterial therapy. The present investigation synthesized citric acid dendrimers through the divergent method that serves as a suitable platform in fulfilling the prerequisites of drug delivery. The cytotoxicity studies revealed 5.4 ± 0.3% of cell viability at 0.5 μg/ml and 35±0.3% at 300 μg/ml after 36 h. The IC50 values on Human Alveolar Epithelial (HAE) cells for unmodified dendrimers were 1.36±0.06 mg ml−1while for PEG CZ G5 dendrimers were 35.19±0.15 mg/mlwith 9 ± 0.18% hemolysis. The significant differences were due to the variations in drug content and PEG shield density at the terminal surface. The Reactive Oxygen Species (ROS) uptake and dichlorofluorescein intensity for PEG CZ G2 dendrimers was 24.15 ± 1.24% and 42.3 ± 0.12%whichincreased significantly with the degree of pegylation and revealed 72.5 ± 1.03% and 79.2 ± 0.24%for PEG CZ G5 dendrimers. The increased ROS concentrations altered the mitochondrial functioning and served as a proof for antibacterial effect. Drug release pattern when heuristically fitted into various kinetic models specified zero-order release with non-fickian diffusion. Antibacterial investigations and pharmacokinetic studies revealed that the synthesized generations were quite effective against various bacterial pathogens with optimized concentrations.

Candida albicans (C. albicans) infection shows a growing burden on human health, and it has become challenging to search for treatment. Therefore, this work focused on the antifungal activity, and cytotoxic effect of biosynthesised nanostructures on human ovarian tetracarcinoma cells PA1 and their corresponding mechanism of cell death. Herein, the authors fabricated advanced biosynthesis of uncoated α-Fe2O3 and coated α-Fe2O3 nanostructures by using the carbohydrate of Spirulina platensis. The physicochemical features of nanostructures were characterised by UV-visible, high resolution transmission electron microscopy, Fourier transform infrared spectroscopy, and X-ray diffraction. The antifungal activity of these nanostructures against C. albicans was studied by the broth dilution method, and examined by 2', 7'-dichlorofluorescein diacetate staining. However, their cytotoxic effects against PA1 cell lines were evaluated by MTT and comet assays. Results indicated characteristic rod-shaped nanostructures, and increasing the average size of α-Fe2O3@ZnO nanocomposite (105.2 nm × 29.1 nm) to five times as compared to α-Fe2O3 nanoparticles (20.73nm × 5.25 nm). The surface coating of α-Fe2O3 by ZnO has increased its antifungal efficiency against C. albicans. Moreover, the MTT results revealed that α-Fe2O3@ZnO nanocomposite reduces PA1 cell proliferation due to DNA fragmentation (IC50 18.5 μg/ml). Continual advances of green nanotechnology and promising findings of this study are in favour of using the construction of rod-shaped nanostructures for therapeutic applications.

BACKGROUND AND OBJECTIVE Leaves of basil plant (Ocimum basilicum) are often used for medicinal purposes because of their bioactive constituents, yet the antioxidant properties of this plant are not fully studied in the field of diabetes. The present study investigated the antioxidant property and anti-diabetic effect of two basil cultivars of Ocimum basilicum, 'Italian Genovese' and 'Thyrsiflora', in a Streptozotocin (STZ) rat model of diabetes . MATERIALS AND METHODS Sixty adult Sprague Dawley rats (n = 10/group) were divided into 6 groups: Three non-diabetic and three diabetic groups that either did not receive any supplementation or were supplemented with the leaves extract of one or the other cultivar. After 13 weeks of feeding, all rats were sacrificed, pancreatic tissues were homogenized and used for evaluating oxidative DNA damage and dichlorofluorescein fluorescence (DCF) assay. Blood was collected for the measurements of glucose and insulin. RESULTS The STZ caused oxidative stress in the diabetic group as evidenced by an increase in oxidative DNA damage and also caused DCF production in pancreatic tissues as compared to non-diabetic groups, (p

Background: Biosurfactants are the surface-active molecules produced by the living micro-organisms. They have wide application in the food, cosmetic, pharmaceutical and petrochemical industry due to its physiological and functional properties. Objectives: In the present study, biosurfactant produced from Pseudomonas sp. was used for its physiochemical characterizations and anti-tumergenic property against colon cancer cell line (SW480). Materials and Methods: The biosurfactant was produced in Bushnell Haas Broth media and extracted by solvent extraction method. The physiochemical characterizations such as water solubility index, water activity, foaming index, oil holding capacity of extracted biosurfactant were performed. The degree of toxicity of biosurfactant was measured using a conversion assay of 3-(4, 5-dimethylthiazole-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) dye against SW480 and mouse L292 fibroblastic cell line (non-tumergenic). Furthermore, acridine orange-EtBr (ethidium bromide) staining, Dichlorofluorescein Diacetate (DCF-DA) staining, wound healing assay were performed to determine the anti-colon cancer activity of biosurfactant. Results: The oil holding capacity, water activity (aw), foaming capacity and water solubility index for biosurfactant were found to be 4.36 ± 0.05 g/g, 0.32 ± 0.03, 52.28 ± 0.10 % and 80.86 ± 0.20 % respectively. In the MTT assay, after 48 h of incubation, 84.7% of cell viability on L292 was observed at 250 μg/ml (maximum concentration) for biosurfactant. Whereas, only 50% cell viability was observed after 48 h of incubation on SW480 at 168.52 μg/ml biosurfactant concentration. Conclusion: The biosurfactant could be a bench-mark as a non-cytotoxic biosurfactant which may be used as a possible biological substance in the food industry as a stabilizing agent and in pharmaceutical industry as an anti-colon cancer agent.

Cardiotoxicity is the major challenge in chemotherapy with doxorubicin (DOX) or adriamycin. Doxorubicin manifests oxidative stress via an uncontrolled progression of reactive oxygen species in cardiomyocytes; thereby, dysregulation and dysfunction of myocardium thus lead to apoptosis. Several attempts have been made to overcome this side effect in patients with antioxidant-rich supplements to control the free radicals. Plant-based or plant-derived compounds pay more attention to cure such complications in patients for supporting the treatment, revitalizing or regulating the normal metabolism. Hence, our study focused on pretreatment of embryonically derived rat cardiomyocytes (H9c2) with phytocompound lotusine to prevent DOX-mediated oxidative stress. From the experiment, the DOX-exposed cells have shown morphological abnormalities such as reduced cell size, shrinkage, blebbing, and chromatin condensation, whereas no such deformities were observed in lotusine-pretreated cells even after the exposure to DOX. Increased endogenous antioxidants with reduced lipid peroxidation were observed in lotusine-pretreated cells, whereas the antioxidants were reduced along with increased lipid peroxidation in doxorubicin-exposed cells. A decreased reactive oxygen species generation was evidenced with the 2',7'-dichlorofluorescein diacetate (DCF-DA) staining method. In qPCR analysis, the lotusine-pretreated cells have mitigated doxorubicin-mediated apoptosis by downregulating the pro-apoptotic gene Bax and apoptotic executor caspase-3. It was further confirmed with the luminometric assay, which resulted in lesser luminescence in lotusine-pretreated cells, whereas higher luminescence was recorded in doxorubicin-alone-treated cells. In conclusion, the present study revealed that the lotusine pretreatment has exhibited potential cardioprotective activity against DOX-induced oxidative stress by increasing the intracellular antioxidant defense.

Aim: Intervertebral disc (IVD) degeneration (IDD) is one of the main causes for spinal degenerative diseases, such as disk herniation, spinal canal stenosis, and spinal deformities. Growing evidence has highlighted the contribution of oxidative stress in pathogenesis of IDD, and antioxidant treatment is thus considered to be a promising therapeutic strategy for IDD. The aim of this study was to investigate whether N-tert-butyl-α-phenylnitrone (PBN), a free radical scavenger, could attenuate the pathological changes of IDD by alleviating oxidative stress.Materials and Methods: Nucleus pulposus (NP) cells were isolated from rabbit lumbar disks. MTT assay, real-time PCR and western blotting were employed to evaluate the effects of PBN on oxidative damages induced by 2,2'-azobis (2-amidinopropane) dihydrochloride (AAPH) in NP cells.Results: AAPH induced oxidative stress and the subsequent degenerative changes in NP cells via the ERK/MAPK pathway. On the contrary, the oxidative stress induced by AAPH was significantly ameliorated by PBN. Moreover, PBN also attenuated AAPH-induced expression of matrix degradation proteases and apoptosis. PBN suppresses AAPH-induced activation of ERK/MAPK pathway, which may be the underlying mechanism for the protective effects of PBN.Conclusions: Our study for the first time identified a novel role and mechanism for PBN in protecting the IVD against oxidative stress, matrix catabolism and apoptosis, which may have implications for its further application in combating IVD degenerative diseases.Abbreviations: AAPH: 2,2'-azobis(2-methylpropanimidamidine) dihydrochloride; ADAMTS: a disintegrin and metalloproteinase with thrombospondin motifs; AF: annulus fibrosus; CEP: cartilage endplate; DCF: 2'7'-dichlorofluorescein; IDD: intervertebral disc degeneration; IVD: intervertebral disc; LPS: lipopolysaccharide; MMP: matrix metalloproteinase; MTT: methyl-thiazolyl-tetrazolium; NP: nucleus pulposus; PBN: N-tert-butyl-alfa-phenylnitrone; PGs: proteoglycans; ROS: reactive oxygen species; SDS: sodium dodecyl sulfate.

Objectives The purpose of this study is to investigate the antifibrosis and anti-oxidation of rhein in vivo and in vitro, and to evaluate potential mechanisms involved in the treatment of chronic kidney disease (CKD). Methods In experimental animal studies, CKD was established by 5/6 nephrectomy (5/6Nx). Serum creatinine (Scr) and blood urea nitrogen (BUN) were determined. Histopathologic tests were performed by HE and Masson trichrome stained. The level of ROS was investigated by fluorescence microplate with the probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA). The protein expressions of p47phox and gp91phox were measured in 5/6Nx rats. In HK-2 cells, the expression of SIRT3 and Foxo3α was measured in SIRT3 knockdown conditions. The indicators of oxidation and fibrosisi were measured in SIRT3 knockdown conditions. Key findings The results showed that, in addition to reducing renal interstitial pathologic injury and collagen fibrils, rhein administration improved renal function. The protective mechanisms were attributed to active SIRT3/FOXO3α signalling pathway and then play the anti-oxidative capacity of rhein, as well as to subsequent antifibrotic effect. Conclusion Taken together, rhein protected kidney through SIRT3/FOXO3a involvement. The anti-oxidative capacity of rhein contributed to the protective effects including the subsequent antifibrotic responses.

Silver nanoparticles (Hematite (α-Fe2O3) have been used as an antimicrobial and as disinfectant. Nevertheless, there is limited data about its antitumor potential. This study focused on investigating the cytotoxic effects of Hematite (α-Fe2O3) from Butea monosperma flower extract on MCF-7 breast cancer cells and its mechanism of action. Green method was created for the synthesis of Hematite (α-Fe2O3) using an aqueous extract of B. monosperma flower. Synthesis of hematite (α-Fe2O3) was described by different analytical techniques including ultraviolet-visible spectrophotometer, field-emission scanning electron microscopy, X-ray diffraction, and Fourier transforms infrared spectroscopy. Cell viability was determined by the 3-[4, 5-dimethylthiazol-2-yl]-a 2,5-diphenyltetrazolium bromide assay. Reactive oxygen species (ROS) formation was measured using probe 2',7'-dichlorofluorescein diacetate and intracellular calcium (Cai2+) was evaluated with probe flu3-AM. Cells were treated with different concentrations of hematite (α-Fe2O3) (1, 3, 6, 10, 15, 25, 50 and 100 μg/mL). The results showed that hematite (α-Fe2O3) hindered cell growth in a dose-dependent manner. Hematite (α-Fe2O3) appeared to have dose-dependent cytotoxicity against MCF-7 cells through activation of the ROS generation and an increase in the intracellular Cai2+ (IC50 52 ± 3.14). In conclusion, the results of this preliminary study demonstrated that hematite (α-Fe2O3) from B. monosperma flower extract may be a potential therapeutic potential medicament for human breast cancer treatment. Key words: Cytotoxicity, MCF-7 cell line, Butea monosperma, Hematite (α-Fe2O3), nanoparticles.

The production of reactive species over physiological levels associated to pathogenic bacteria could represent a high risk for many diseases. The Rosmarinus officinalis L. is used around the world due its pharmacological proprieties. So, in this study our aim is to test for the first time if R. officinalis L. extract (eeRo) and its fractions (DCM, EA, ButOH) could have better or similar antioxidant action to standars and among themselves in vitro or ex vivo, in brain, stomach and liver of rats. Moreover, we intend to clarify their possible effects on pathogenic bacteria. The eeRo was obtained from the dried leaves subjected to an alcoholic extraction and fractioned. The quantification of the constituents of eeRo and fractions were done by HPLC. The antioxidant proprieties of R. officinalis was analyzed by DPPH•- radical scavenging, total antioxidant, dichlorofluorescein, lipid peroxidation and sodium nitroprusside -induced lipid peroxidation assays. The Minimum inhibitory concentrations of R. officinalis L. were tested with standard strains of danger bacteria. The eeRo, DCM, EA had significant total antioxidant and DPPH•- radical scavenging activities. The DCM and eeRo got significant effects against basal levels of reactive species in liver, stomach and brain. The eeRo and DCM protected the liver and brain against lipid peroxidation. The eeRo, DCM, EA and ButOH had inhibitory effect in the Gram-positive and Gram-negative bacteria. In general way, the DCM and eeRo had the best antioxidant and antibacterial effects among all tested fractions. Keywords: Microorganisms, Medicine, Food, Plant, Free radicals

Plasmalogens are alkenyl-acyl glycerophospholipids and decreased in post-mortem Alzheimer’s disease (AD) brains. The aim of this study is to investigate the time-dependent changes of plasmalogens in the hippocampus of an AD model mouse (J20). Plasmalogen levels at 3, 6, 9, 12 and 15 months were analyzed by liquid-chromatography-targeted-multiplexed-selected-reaction-monitoring-tandem-mass-spectrometry (LC-SRM/MS). Reactive oxygen species (ROS) levels were evaluated using dichlorofluorescein diacetate (DCF-DA). Plasmalogen synthesizing enzyme glycerone-phosphate O-acyltransferase (GNPAT) and late endosome marker Rab7 levels were quantified by Western blotting. GNPAT localization, changes of neuronal and glial cell numbers were evaluated by immunostaining. Compared to wild-type mice (WT), total plasmalogen-ethanolamine, but not plasmalogen-choline levels, were increased at 9 months and subsequently decreased at 15 months in J20 mice. A principal component analysis of plasmalogen-ethanolamine species could separate WT and J20 mice both at 9 and 15 months. Both GNPAT and Rab7 protein were increased in J20 mice at 9 months, whereas GNPAT was decreased at 15 months. ROS levels were increased in J20 mice except for 9 months. Our results suggest that increased plasmalogen-ethanolamine could counteract ROS levels and contribute to the phagocytosis process in J20 mice at 9 months. Such results might indicate a transient protective response of plasmalogen-ethanolamine in AD conditions.

To date, organoseleniums are pre-eminent for peroxide decomposition and radical quenching antioxidant activities. On the contrary, here, a series of Janus-faced aminophenolic diselenides have been prepared from substituted 2-iodoaniline and selenium powder using copper-catalyzed methodology. Subsequently, condensation with substituted salicylaldehyde afforded the Schiff base, which on reduction, yielded the desired substituted aminophenolic diselenides in 72%–88% yields. The generation of reactive oxygen species (ROS) from oxygen gas by the synthesized aminophenolic diselenides was studied by analyzing the oxidation of dichlorofluorescein diacetate (DCFDA) dye and para-nitro-thiophenol by fluorescence and UV-Visible spectroscopic methods. Furthermore, density functional theory calculations and crystal structure analysis revealed the role of functional amine and hydroxyl sites present in the Janus-faced organoselenium catalyst for the activation of molecular oxygen, where NH and phenolic groups bring the oxygen molecule close to the catalyst by N–H⋯O and O–H⋯O intermolecular interactions. Additionally, these functionalities stabilize the selenium-centered radical in the formed transition states. Antioxidant activities of the synthesized diselenides have been explored as the catalyst for the decomposition of hydrogen peroxide using benzenethiol sacrificial co-reductant by a well-established thiol assay. Radical quenching antioxidant activity was studied by the quenching of DPPH radicals at 516 nm by UV-Visible spectroscopy. The structure activity correlation suggests that the electron-rich phenol and electron-rich and sterically hindered selenium center enhance the oxidizing property of the aminophenolic diselenides. Janus-faced diselenides were also evaluated for their cytotoxic effect on HeLa cancer cells via MTT assay, which suggests that the compounds are effective at 15–18 μM concentration against cancer cells. Moreover, the combination with therapeutic anticancer drugs Erlotinib and Doxorubicin showed promising cytotoxicity at the nanomolar concentration (8–28 nM), which is sufficient to suppress the growth of the cancer cells.

The main objective of this study was to investigate whether dichlorofluorescein (DCF) is adequate for the formulation of stable dichlorofluorescein-induced silver nanoparticles under the boiling method to analyze their effects on the seed germination of Mung seeds (Vigna radiata). Preliminary dichlorofluoresceine nanoparticles (DCF-SNPs) synthesis evidence by noticing the solution color transformed from a light green color to a dark brown color. The 2.5 ml of dichlorofluoresceine (DCF) solution was found sufficient for the formulation of dichlorofluoresceine induced silver nanoparticles at boiling conditions. Purified dichlorofluoresceine nanoparticles (DCF-SNPs) measure an average diameter of 293 nm where the majority of nanoparticles were around 159 nm in size with the surface load of-9.35 mV zeta potential value. The impact of dichlorofluorescein silver nanoparticles (DCF-SNPs) on the germination percentage of V. radiata has shown that, the 25% concentration of DCF-SNPs is excellent for the growth of Mung seeds (V. radiata). Overall, the dichlorofluorescein silver nanoparticles may be constructive for improving the percentage of seed germination at 25% of its concentration and may also be useful for fluorescent measurement using the confocal microscopy technique. Hence, dichlorofluorescein silver nanoparticles (DCF-SNPs) are proposed as an efficient detection system for nanoparticles in agrochemicals for plants.

In recent years, epidemiological studies have identified a relationship between diet and cerebro–cardiovascular disease (CVD). In this regard, there is a promising dietary group for cardiovascular protection are polyphenols, especially anthocyanins. Vascular reactivity studies were performed using Healthberry 865® and constituent single anthocyanins to characterize vasomotor responses, immunofluorescence analysis with dichlorofluorescein diacetate and dihydroethidium were used to evaluate nitric oxide and oxidative stress, lucigenin assay was used to measure NADPH oxidase activity, and gel electrophoresis and immunoblotting were used to dissect the molecular mechanisms involved. We demonstrated that Healthberry 865® exerts an important vasorelaxant effect of resistance artery functions in mice. Its action is mediated by nitric oxide release through the intracellular signaling PI3K/Akt. Moreover, behind its capability of modulating vascular tone, it also exerts an important antioxidant effect though the modulation of the NADPH oxidase enzyme. Interestingly, its cardiovascular properties are mediated by the selective action of different anthocyanins. Finally, the exposure of human dysfunctional vessels to Healthberry 865® significantly reduces oxidative stress and improves NO bioavailability. Although further investigations are needed, our data demonstrate the direct role of Healthberry 865® on the modulation of vasculature, both on the vasorelaxation and on oxidative stress, thus, supporting the concept that a pure mixture of anthocyanins could be helpful in preventing the onset of vascular dysfunction associated with the development of CVD.

Several automated instruments exist to measure the acellular oxidative potential (OP) of ambient particulate matter (PM). However, cellular OP of the ambient PM is still measured manually, which severely limits the comparison between two types of assays. Cellular assays could provide a more comprehensive assessment of the PM-induced oxidative stress, as they incorporate more biological processes involved in the PM-catalyzed reactive oxygen species (ROS) generation. Considering this need, we developed a semi-automated instrument, the first of its kind, for measuring the cellular OP based on a macrophage ROS assay using rat alveolar macrophages. The instrument named SCOPE – semi-automated instrument for cellular oxidative potential evaluation – uses dichlorofluorescein diacetate (DCFH-DA) as a probe to detect the OP of PM samples extracted in water. SCOPE is capable of analyzing a batch of six samples (including one negative and one positive control) in 5 h and is equipped to operate continuously for 24 h with minimal manual intervention after every batch of analysis, i.e., after every 5 h. SCOPE has a high analytical precision as assessed from both positive controls and ambient PM samples (coefficient of variation (CoV) %). The results obtained from the instrument were in good agreement with manual measurements using tert-butyl hydroperoxide (t-BOOH) as the positive control (slope =0.83 for automated vs. manual, R2=0.99) and ambient samples (slope =0.83, R2=0.71). We further demonstrated the ability of SCOPE to analyze a large number of both ambient and laboratory samples and developed a dataset on the intrinsic cellular OP of several compounds, such as metals, quinones, polycyclic aromatic hydrocarbons (PAHs) and inorganic salts, commonly known to be present in ambient PM. This dataset is potentially useful in future studies to apportion the contribution of key chemical species in the overall cellular OP of ambient PM.

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.

Background: Lacidipine, a third-generation calcium channel blocker, exerts beneficial effects on the endothelium of hypertensive patients in addition to blood pressure lowering. However, the detailed mechanism underlying Lacidipine-related endothelial protection is still elusive.Methods: Sixteen spontaneous hypertensive rats (SHRs) were randomly divided into two groups: Lacidipine-treated SHR group and saline-treated control group. Tail systolic blood pressure was monitored for four consecutive weeks. Endothelial cells (ECs) were pretreated with Lacidipine prior to being stimulated with H2O2, bleomycin, or Lipopolysaccharides (LPS) in vitro. Then, cell activity, migration, and senescence were measured by Cell Counting Kit-8 assay, transwell assay, and β-galactosidase staining, respectively. The fluorescent probe 2′, 7′-dichlorofluorescein diacetate (DCFH-DA) was used to assess the intracellular reactive oxygen species (ROS). Related protein expression was detected by Western blotting and immunofluorescence.Results: Our data showed that Lacidipine treatment lowered the blood pressure of SHRs accompanied by the elevation of CXCR7 expression and suppression of P38 and CCAAT/enhancer-binding protein beta (C/EBP-β) compared with the control group. In vitro experiments further demonstrated that Lacidipine increased the cell viability and function of ECs under oxidative stress, cell senescence, and inflammatory activation via the CXCR7/P38/signaling pathway.Conclusions: Our results suggested that Lacidipine plays a protective role in EC senescence, oxidative stress, and inflammatory injury through the regulation of CXCR7/P38/C/EBP-β signaling pathway.

Singlet oxygen (1O2) plays a vital role in pathophysiological processes and is the dominant executor of photodynamic therapy (PDT). Several small molecular probes have been designed to detect singlet oxygen for the evaluation of PDT efficacy. However, little attention was paid to the precise visualization of the 1O2 signal at the subcellular organelle level in living biological systems. Herein, a super-pH-resolved (SPR) nanosensor was developed to specifically illuminate 1O2 in endocytic organelles through encoding the cell-impermeant singlet oxygen sensor green (SOSG) into pH-sensitive micelles. The acid-activatable SPR-SOSG achieved more than 10-fold amplification of the 1O2 signal, leading to extremely higher sensitivity of singlet oxygen detection in specific endocytic organelles of living cells and animals, as compared with the nonactivatable nanoprobe and the commercially available 2',7'-dichlorofluorescein diacetate (DCFH-DA) probe. Hence, the SPR-SOSG nanoplatform provides a promising tool to evaluate the efficacy and mechanism of nanocarrier-based photodynamic therapy.

The biological response of bronchial epithelial cells to particles is associated with a sequestration of cell metal by the particle surface and a subsequent disruption in host iron homeostasis. The macrophage is the cell type resident in the respiratory tract that is most likely to make initial contact with inhaled particles. We tested the postulates that (1) silica, a prototypical particle, disrupts iron homeostasis in alveolar macrophages (AMs); and (2) the altered iron homeostasis results in both an oxidative stress and pro-inflammatory effects. Human AMs (1.0 × 106/mL) demonstrated an increased import of iron following particle exposure with nonheme iron concentrations of 0.57 ± 0.03, 1.72 ± 0.09, 0.88 ± 0.09, and 3.21 ± 0.11 ppm in cells exposed for 4 h to media, 500 μM ferric ammonium citrate (FAC), 100 μg/mL silica, and both silica and FAC, respectively. Intracellular ferritin concentrations and iron release were similarly increased after AM exposure to FAC and silica. Silica increased oxidant generation by AMs measured using both dichlorofluorescein diacetate fluorescence and reduction of nitroblue tetrazolium salt. Concentrations of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-α in macrophage supernatant increased following 100 μg/mL silica exposure for 24 h. Treatment of AMs with 500 μM FAC decreased both oxidant generation and cytokine release associated with silica exposure, supporting a dependence of these effects on sequestration of cell metal by the particle surface. We conclude that (1) silica exposure disrupts iron homeostasis resulting in increased import, accumulation, and release of the metal; and (2) the altered iron homeostasis following silica exposure impacts oxidant generation and pro-inflammatory effects.

—The production of reactive oxygen species in human erythrocytes during incubation in the presence of a glycerol–mannitol mixture and polyethylene glycol with a molecular weight of 1500 Da, as well as during hypothermic storage of cells cryopreserved under their protection, was studied. Analysis of the production of reactive oxygen species was carried out by flow cytometry using fluorescence indices of dichlorofluorescein. It was found that incubation in the glycerol–mannitol mixture, as well as cryopreservation and subsequent hypothermic storage, did not cause activation of the production of reactive oxygen species in erythrocytes. Polyethylene glycol caused the intensification of the production of reactive oxygen species both during incubation and storage of cryopreserved erythrocytes under hypothermia. Peculiarities of changes in the intensity of the production of reactive oxygen species in erythrocytes under the influence of cryoprotective agents and cryopreservation may have a significant impact on the stability of cells under stress and their ability to maintain functional integrity after returning to physiological conditions.

Background NAD(P)H:quinone oxidoreductase-1 (NQO1) is a widely-distributed flavin adenine dinucleotide-dependent flavoprotein that promotes obligatory 2-electron reductions of quinones, quinoneimines, nitroaromatics, and azo dyes. This reduces quinone levels and thereby minimizes generation of excess reactive oxygen species (ROS) formed by redox cycling, and concurrent depletion of intracellular thiol pools. Ajoene is derived from crushed garlic. It is formed by a reaction involving two allicin molecules, and is composed of allyl sulfide and vinyl disulfide. Ajoene is present in two isomers, E- and Z-form. Methods Expression of antioxidant enzymes and nuclear factor E2-related factor-2 (Nrf2) was measured by Western blot analysis. NQO1 promoter activity was assessed by the luciferase reporter gene assay. ROS accumulation was monitored by using the fluorescence-generating probe 2',7'-dichlorofluorescein diacetate. The intracellular glutathione levels were measured by using a commercially available kit. Results Z-ajoene significantly up-regulated the expression of representative antioxidant enzyme NQO1 in non-tumorigenic breast epithelial MCF-10A cells at non-toxic concentrations. Z-ajoene enhanced up-regulation and nuclear translocation of Nrf2, which plays a pivotal role in the induction of many genes encoding antioxidant enzymes and other cytoprotective proteins. Z-ajoene treatment also increased the activity of nqo1-promoter harboring antioxidant response element consensus sequences in MCF-10A cells. Silencing of Nrf2 by small interfering RNA abrogated ajoene-induced expression of NQO1. Z-ajoene activated extracellular signal-regulated kinase (ERK). Inhibition of ERK activation by U0126 abrogated ability of Z-ajoene to activate Nrf2 and to induce NQO1 expression. Intracellular ROS accumulation was observed after treatment with Z-ajoene, whereas the E-isoform was not effective. The inhibition of ROS by treatment with N-acetylcysteine, a radical scavenger, abrogated Z-ajoene-induced expression of NQO1 as well as activation of ERK and Nrf2, suggesting that Z-ajoene augments the Nrf2-dependent antioxidant defense via ROS generation and ERK activation. Conclusions Z-ajoene induces NQO1 expression in MCF-10A cells through ROS-mediated activation of Nrf2.

BACKGROUND/AIMS Recently, we have demonstrated that episodic hypoxia occurs in kidneys of mice challenged repetitively with the immunosuppressant cyclosporine A (CsA), in analogy to humans on CsA treatment. However, the molecular consequences of episodic hypoxia remain poorly defined, as is its impact on cell survival. Here, we systematically study cell response to episodic, as compared to single course hypoxia. METHODS In vivo, kidneys of mice challenged daily with CsA for one week were analyzed by microarray analysis, gene ontology analysis, and qPCR. In vitro, renal cells were subjected to hypoxia (1 % O₂) which was either episodic (4 h for 6 consecutive days), short-term (4 h), or sustained (24 h). Western blot analysis quantified hypoxia-inducible factor-1α (HIF-1α). 2',7'-dichlorofluorescein diacetate detected intracellular ROS. After re-oxygenation, staurosporine served to induce apoptosis, quantified by active caspase-3. RESULTS In vivo, HIF target gene expression was suppressed by daily CsA treatment. Yet, we found up-regulation of genes involved in defence against cellular stress, notably against ROS. Renal cells in vitro behaved largely different under episodic and sustained hypoxia, while their response to short-term hypoxia oscillated between the previous two. Episodic hypoxia exhibited the highest total HIF-1α protein level, lowest nucleus-to-cytoplasm ratio, and lowest HIF target gene expression. When compared with normoxia, re-oxygenation after sustained hypoxia increased ROS by 3.04 ± 1.04 fold (p

Corneal wounds usually heal quickly; but diabetic patients have more fragile corneas and experience delayed and painful healing. In the present study, we compared the healing capacity of corneal epithelial cells (CECs) between normal and diabetic conditions and the potential mechanisms. Primary murine CEC derived from wild-type and diabetic (db/db) mice, as well as primary human CEC were prepared. Human CEC were exposed to high glucose (30 mM) to mimic diabetic conditions. Cell migration and proliferation were assessed using Scratch test and MTT assays, respectively. Reactive oxygen species (ROS) production in the cells was measured using dichlorofluorescein reagent. Western blot was used to evaluate the expression levels of Akt. Transepithelial electrical resistance (TEER) and zonula occludens-1 (ZO-1) expression were used to determine tight junction integrity. We found that the diabetic CEC displayed significantly slower cell proliferation and migration compared with the normal CEC from both mice and humans. Furthermore, ROS production was markedly increased in CEC grown under diabetic conditions. Treatment with an antioxidant N-acetyl cysteine (NAC, 100 μM) significantly decreased ROS production and increased wound healing in diabetic CEC. Barrier function was significantly reduced in both diabetic mouse and human CEC, while NAC treatment mitigated these effects. We further showed that Akt signaling was impaired in diabetic CEC, which was partially improved by NAC treatment. These results show that diabetic conditions lead to delayed wound-healing capacity of CEC and impaired tight junction formation in both mice and human. Increased ROS production and inhibited Akt signaling may contribute to this outcome, implicating these as potential targets for treating corneal wounds in diabetic patients.

Lipid metabolic disorders play critical roles in atherogenesis. Traditionally, it has been suggested that reduced high density lipoprotein (HDL) levels might be an important morbidity indicator for cardiovascular diseases. Therefore, it has been argued that therapeutically raising HDL levels may reduce atherogenesis in patients with dyslipidemia. However, recent clinical trials to elevate serum HDL levels by pharmacologic approaches failed to demonstrate clinical efficacy. Thus, to investigate the functionality of HDL and to explore the possible clinical relevance as well as to define an effective indicator that can represent HDL function may provide another key and reference to disclose the clinical treatment of dyslipidemia. We analyzed the association between the data of dichlorofluorescein assay (assay the functionality of HDL), the effect of HDL on oxidized low density lipoprotein (oxLDL)-stimulated endothelial progenitor cells (EPCs) in vitro, levels of circulating EPCs, and ex vitro EPC colony forming units of each case, we defined the indicator (relative HDL index (RHDL index) = dichlorofluorescein assay result of each subject/dichlorofluorescein assay reading of our young healthy controls) that may represent functionality of HDL. HDL from healthy adults protected oxLDL-treated EPCs by modulating p38 mitogen-activated protein kinase and Rho activation and by promoting nitric oxide production. HDL from subject with RHDL index ≧2 also failed to restore the functionality of oxLDL-treated EPCs via cell-signaling pathways in vitro. The RHDL index significantly correlated with patients' circulating EPC number or EPC colony forming units ex vivo. In conclusions, we explored the RHDL index as a score to predict a patient's EPC functions in vivo and ex vitro.

Background Pyridoxine (VB6 ), which acts as a coenzyme in the biosynthesis of niacin, is formulated in pharmaceuticals to treat skin roughness. However, the mechanism of action of VB6 is not known precisely. Objective This study was conducted to clarify the influence of highly oxidative conditions on the expression of skin moisture-related mRNAs and to evaluate the preventive effects of VB6 focusing on antioxidant behaviour. Methods Intracellular levels of reactive oxygen species (ROS) in normal human epidermal keratinocytes (NHEKs) were determined using the 2',7'-dichlorofluorescein diacetate assay. Real-time PCR was employed to investigate the influence of higher oxidative conditions on the expression of mRNAs encoding serine palmitoyl transferase (SPT) and filaggrin, and to characterize the mechanism of the antioxidant effect of VB6 . Intracellular glutathione was quantified using an assay based on the glutathione recycling system with 5,5'-dithiobis (2-nitrobenzoic acid) reagent and glutathione reductase. Carbonylated proteins (CPs) were semi-quantified by detecting aldehyde residues. Results Treatment of NHEKs with BSO increased the level of intracellular CPs by interfering with intracellular glutathione synthesis. Further, treatment with BSO down-regulated the expression level of SPT mRNA, but VB6 restored SPT mRNA expression in BSO-treated NHEKs. VB6 decreased the level of intracellular CPs with or without BSO treatment in a dose-dependent manner. In addition, VB6 increased levels of intracellular NADH/NADPH and glutathione through the activation of nuclear factor E2-related factor 2 (Nrf2) signalling. Conclusion These results suggest that highly oxidative conditions cause an impaired skin barrier function due to the down-regulation of SPT that results in skin roughness. VB6 improved the down-regulation of SPT mRNA expression initiated by highly oxidative conditions by enhancing the intracellular antioxidant system.

PURPOSE/OBJECTIVE(S) Gold nanoparticles (GNPs) are effective radiosensitizers due to their ability to enhance ionizing radiation absorptance and deposit elevated dose in their vicinity. Radiation induced bystander effect (RIBE) expands radiation effects to non-targeted cells close to radiation-targeted cells, further improving radiation outcome. In this study, we investigated the impact of PSMA-targeted GNPs on RIBE. We hypothesize that the PSMA-targeted GNPs would intensify radiation effects by augmenting RIBE consequence as well as enhancing sensitivity of bystander cells to RIBE. MATERIALS/METHODS Anti-prostate-specific membrane antigen (PSMA) antibodies were conjugated onto PE Gylated GNPs through EDC/NHS chemistry. UV-Vis spectrophotometry and real time cell analysis were employed to evaluate the cellular uptake and biocompatibility, respectively, when LNCaP prostate cancer cells were treated with PSMA-targeted GNPs at varying concentrations. Then GNPs with biocompatible dosage were used for cancer cell treatments. The media transfer technique was performed to induce the RIBE effects on bystander cells. First, LNCaP cells pretreated with phosphate buffered saline (PBS) or GNPs for 24hrs were irradiated with 160kvP X-rays at varying doses (0∼8 Gy). The culture media from irradiated cells were collected and filtered (sterile 0.2µm polyethersulfone) to acquire PBS conditioned media (PBS-CM) and GNP conditioned media (GNP-CM). Then, the collected PBS-/GNP-CMs were transferred to non-irradiated LNCaP cells with/without GNPs for 24hrs to investigate RIBE effect. The final GNP-induced RIBE was evaluated via MTT cellular viability assay, clonogenic assay, γ-H2AX assay, as well as reactive oxygen species (ROS) assessment through 2',7'-dichlorofluorescein diacetate staining. RESULTS PSMA-targeted GNPs showed minimal cytotoxicity in LNCaP cells up to 1mg/mL concentrations. Cellar uptake analysis demonstrated that GNPs were bound to cells within 30 mins, with maximum uptake at 250µg/mL. Compared to 2 Gy PBS-CM, 8 Gy PBS-CM demonstrated much higher RIBE response on the non-irradiated cells, showing the dose dependent RIBE in LNCaP cells. Compared to PBS-CM, GNP-CM exhibited much higher cell death in MTT assay, as well as demonstrated RIBE enhancement in γ-H2AX (133%) and clonogenic assays (120%) analysis. Moreover, GNP-CM showed elevated ROS expression (125%) than PBS-CM. In the presence of PBS-CM, the non-irradiated cells with GNPs showed increased cell death compared to that without GNPs, indicating that GNP would enhance the sensitivity of bystander cells to RIBE. This sensitivity enhancement ratio was also validated in γ-H2AX (120%) and clonogenic assays (120%). CONCLUSION PSMA directed GNPs were associated with physical dose enhancement along with the enhanced RIBE, playing a critical role in increasing the overall radiosensitivity of prostate cancer cells. Our results also showed that PSMA-targeted GNPs enhanced the sensitivity of bystander cells to RIBE.