Simvastatin inhibits osteoclast differentiation by scavenging reactive oxygen species

Osteoclasts, together with osteoblasts, control the amount of bone tissue and regulate bone remodeling. Osteoclast differentiation is an important factor related to the pathogenesis of bone-loss related diseases. Reactive oxygen species (ROS) acts as a signal mediator in osteoclast differentiation. Simvastatin, which inhibits 3-hydroxy-3-methylglutaryl coenzyme A, is a hypolipidemic drug which is known to affect bone metabolism and suppresses osteoclastogenesis induced by receptor activator of nuclear factor-κB ligand (RANKL). In this study, we analyzed whether simvastatin can inhibit RANKL-induced osteoclastogenesis through suppression of the subsequently formed ROS and investigated whether simvastatin can inhibit H2O2-induced signaling pathways in osteoclast differentiation. We found that simvastatin decreased expression of tartrate-resistant acid phosphatase (TRAP), a genetic marker of osteoclast differentiation, and inhibited intracellular ROS generation in RAW 264.7 cell lines. ROS generation activated NF-κB, protein kinases B (AKT), mitogen-activated protein kinases signaling pathways such as c-JUN N-terminal kinases, p38 MAP kinases as well as extracellular signal- regulated kinase. Simvastatin was found to suppress these H2O2-induced signaling pathways in osteoclastogenesis. Together, these results indicate that simvastatin acts as an osteoclastogenesis inhibitor through suppression of ROS-mediated signaling pathways. This indicates that simvastatin has potential usefulness for osteoporosis and pathological bone resorption.     

Novel oligonucleotide carrier as scavenger for reactive oxygen species

A novel oligonucleotide carrier which can scavenge ROS is described. The synthesized graft polymer is composed of a PEG segment and a TEMPO‐containing hydrophobic segment for scavenging ROS. This graft polymer can form a PIC through electrostatic interaction with oligonucleotides such as siRNA. The amount of ROS was monitored by fluorescence measurements using H₂DCFDA as a probe, and it was confirmed that the ROS level was effectively suppressed. The cellular uptake of PIC containing the fluorescence‐labeled oligonucleotide was evaluated by fluorescence microscopy. Delivered siRNA suppressed the expression of the mRNA. The prepared graft copolymer is thus a promising candidate as a novel oligonucleotide carrier which also reduces ROS damage generated by cationic polymer carriers.

     

Rhodamine B degradation and reactive oxygen species generation by a ZnSe-graphene/TiO2 sonocatalyst

Nanostructured ZnSe-graphene/TiO2 was synthesized by a hydrothermal-assisted approach. ZnSe-graphene/TiO2 exhibited favorable adsorption of rhodamine B, a wide wavelength absorption range, and efficien...     

Analysis of intracellular reactive oxygen species by micellar electrokinetic capillary chromatography with laser-induced-fluorescence detector

Reactive Oxygen Species (ROS) are an important part of the normal cell growth cycle and play essential roles in many biological functions. Many techniques have been developed to detect and measure the amount of ROS present in cells. These techniques include spectrophotometry, high performance liquid chromatography (HPLC), capillary electrophoresis with laser-induced-fluorescence detector (CE-LIF) and capillary electrophoresis-on-a-chip with LIF. As ROS has a short half-life outside of the cell, various fluorescent probes, such as dihydrorhodamine 123 (DHR 123), that are membrane permeable have been used in the detection of intracellular ROS. In this paper, micellar electrokinetic capillary chromatography (MEKC) was coupled with LIF detector. Fluorescent probe, 3′-(p-aminophenyl)-fluorescein (APF), was used to detect specific ROS in Chinese Hamster Ovary (CHO) suspension cells as well as attached mouse bone marrow-derived dendritic cells (BMDCs). Separation buffer composition was optimized at a concentration of 25?mM tetraborate buffer with 50?mM sodium dodecyl sulfate at pH 9.3 and lysis of the cells was done successfully with a buffer of 70% ethanol and 0.1mM sodium dodecyl sulfate using a cell amount of 1?×?10⁷. ROS in cells was successfully analyzed by MEKC-LIF method developed, with acceptable RSD for time at 1.54% and area at 2.81%.     

Removal of lipopolysaccharide and reactive oxygen species using sialic acid immobilized polysulfone dialyzer

Sialic acid (N‐acetylneuraminic acid, NANA) was covalently immobilized onto the surface of a polysulfone (PSF) hollow fiber membrane. Prior to the immobilization, the surface of PSF was treated with ozone, followed by grafting with acrylic acid, and then the esterification of NANA. The surface concentration of NANA was determined by 2‐thiobarbituric acid (TBA) test. Hemocompatibility, the capability of suppressing oxidative stress, and clearance of lipopolysaccharide (LPS) from the resulting hollow fiber membrane were evaluated. The results show that by immobilizing NANA onto PSF hollow fiber, the adhesion of platelet was reduced, while both APTT and PT were little affected. Furthermore, oxidative stress was suppressed by NANA‐immobilized PSF hollow fibers. The level of LPS was also greatly reduced. Copyright © 2009 John Wiley & Sons, Ltd.     

Synthesis of new bicarbazole-linked triazoles as non-cytotoxic reactive oxygen species (ROS) inhibitors

Abstract

Carbazole analogs 3 and 4 and a new library of bicarbazole-linked triazoles 6–11 were prepared via new synthetic methodology. Metal-catalyzed oxidative coupling reaction was utilized for the synthesis of bicarbazole acetylene 4 and different metals (Zn⁺², Co⁺², Fe⁺³, Ni⁺², Cu⁺², Mn⁺²) as catalyst were screened. Only Fe-catalyzed reaction was found to be excellent and gave homocoupled product 4. Cu-catalyzed 1,3-dipolar cycloaddition was also utilized to install triazole moiety for the synthesis of hybrid molecules 6–11. Based on reported anti-inflammatory activity of carbazole and triazole scaffolds, all compounds were screened for their reactive oxygen species (ROS) inhibitory potential. Results from these studies revealed triazole 9 as most active compound (IC₅₀ value of 7.6?±?0.1?µg/mL on human whole blood and 2.7?±?0.09?µg/mL on isolated neutrophils) using ibuprofen as a standard. Therefore, class described herein can serve as attractive structural element for further studies on ROS inhibition.     

The effect of covalent immobilization of sialic acid on the removal of lipopolysaccharide and reactive oxygen species for polyethylene terephthalate

Polyethylene terephthalate (PET) was aminolyzed with 1,6‐diaminohexane (DAH) and then sialic acid (NANA) was immobilized via amidation onto the surface. The surface concentration of NANA was determined by 2‐thiobarbituric acid (TBA) test. The hemocompatibility of the resulting PET fabrics was evaluated based on complete blood count (CBC), coagulating times, and protein adsorption. The ability to remove lipopolysaccharide (LPS) was also determined. In addition, the effect of contacting NANA‐immobilizing PET on the suppression of reactive oxygen species (ROS) production was measured by the chemiluminescence (CL) method. The results show that by immobilizing NANA onto PET, the adhesion of platelet (PLt) was reduced, and oxidative stress was suppressed. The level of LPS was also greatly reduced. Copyright © 2010 John Wiley & Sons, Ltd.     

荧光法研究抗癌药物更生霉素D与小牛胸腺DNA的作用机理

以溴化乙锭(ethidiumbromide,EB)为荧光探针,研究了更生霉素D(actinomyinD,ACTD)与小牛胸腺DNA(calfthymusDNA,CTDNA,以下简称DNA)的作用机理。对荧光光谱、荧光偏振、Scatchard图、DNA热变性曲线、DNA盐效应曲线等研究表明,ACTD与DNA之间主要存在嵌入和沟槽两种作用,并证明ACTD与DNA磷酸基之间不存在静电作用。     

Regulation of pro-inflammatory responses by lipoxygenases via intracellular reactive oxygen species in vitro and in vivo

Reactive oxygen species (ROS) performs a pivotal function as a signaling mediator in receptor-mediated signaling. However, the sources of ROS in this signaling have yet to be determined, but may include lipoxygenases (LOXs) and NADPH oxidase. The stimulation of lymphoid cells with TNF-alpha, IL-1beta, and LPS resulted in significant ROS production and NF-kappaB activation. Intriguingly, these responses were markedly abolished via treatment with the LOXs inhibitor nordihydroguaiaretic acid (NDGA). We further examined in vivo anti-inflammatory effects of NDGA in allergic airway inflammation. Both intraperitoneal and intravenous NDGA administration attenuated ovalbumin (OVA)-induced influx into the lungs of total leukocytes, as well as IL-4, IL-5, IL-13, and TNF-alpha levels. NDGA also significantly reduced serum levels of OVA-specific IgE and suppressed OVA-induced airway hyperresponsiveness to inhaled methacholine. The results of our histological studies and flow cytometric analyses showed that NDGA inhibits OVA-induced lung inflammation and the infiltration of CD11b+ macrophages into the lung. Collectively, our findings indicate that LOXs performs an essential function in pro-inflammatory signaling via the regulation of ROS regulation, and also that the inhibition of LOXs activity may have therapeutic potential with regard to the treatment of allergic airway inflammation.     

Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases

Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to gamma-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases activities.     

Rutin inhibits osteoclast formation by decreasing reactive oxygen species and TNF-alpha by inhibiting activation of NF-kappaB

Rutin, a glycoside of flavonol, inhibits osteoclast formation induced by receptor activator of NF-kappaB ligand (RANKL) in bone marrow-derived macrophages. It reduces reactive oxygen species produced by RANKL and its inhibitory effect results from reduced levels of TNF-alpha. Rutin also lowers NF-kappaB activation in response to RANKL.     

活性氧刺激响应纳米载体

纳米载体一般是由天然高分子或人工合成高分子组成的、纳米级范畴的运输系统,具有减少药物毒性、提高药物的靶向性、增加药物有效性等优点。随着生物医学技术的进步,有研究表明,作为氧化代谢产物的活性氧(ROS)在疾病部位常常伴随着过表达的异常现象。基于此,近年来ROS刺激响应纳米载体获得了关注和发展,以不同响应机制的ROS响应基团为基础,发展了一系列的ROS响应纳米载体,实现了疾病部位ROS刺激下的药物特异性可控释放。该文聚焦于近年来常用于纳米载体的ROS响应基团,依据元素划分为两大类:硫族元素类响应基团(硫醚、缩硫酮、硒化物、二硒化物、碲化物)和其他元素类响应基团(芳香硼酸酯、过氧草酸酯、二茂铁);通过不同的设计理念将其引入纳米载体,根据ROS响应纳米载体的不同响应机制(疏水-亲水相变、断裂),探讨了载体各自的ROS响应情况、体外药物释放情况,以及在活体中的应用情况。     

Antifungal effects of actinomycin D on Verticillium dahliae via a membrane-splitting mechanism

Antifungal bioassays led to the isolation of actinomycins D and A₁ from Streptomyces luteus TRM45540 collected from Norpo in Xinjiang, and these compounds were identified by nuclear magnetic resonance spectroscopy. The antifungal activity of actinomycin D was higher than that of actinomycin A₁. Actinomycin D clearly inhibited the spore germination, hyphal growth and biomass accumulation of Verticillium dahliae in a dose-dependent manner. Flow cytometric analysis with propidium iodide, total ergosterol measurement, cell leakage and scanning electron microscopy experiments demonstrated that the plasma membrane of this fungus was damaged by actinomycin D, resulting in swollen cells and cellular content leakage. Transmission electron microscopy revealed that parts of the plasma membrane infolded after being treated with actinomycin D. The antifungal activity of actinomycin D damaged the fungal plasma membrane of V. dahliae via a membrane-splitting mechanism, which provided new insights into the functional mechanism of actinomycin D.     

Photoactivation by visible light of CdTe quantum dots for inline generation of reactive oxygen species in an automated multipumping flow system

Quantum dots (QD) are semiconductor nanocrystals able to generate free radical species upon exposure to an electromagnetic radiation, usually in the ultraviolet wavelength range. In this work, CdTe QD were used as highly reactive oxygen species (ROS) generators for the control of pharmaceutical formulations containing epinephrine. The developed approach was based on the chemiluminometric monitoring of the quenching effect of epinephrine on the oxidation of luminol by the produced ROS. Due to the relatively low energy band-gap of this chalcogenide a high power visible light emitting diode (LED) lamp was used as photoirradiation element and assembled in a laboratory-made photocatalytic unit. Owing to the very short lifetime of ROS and to ensure both reproducible generation and time-controlled reaction implementation and development, all reactional processes were implemented inline by using an automated multipumping micro-flow system. A linear working range for epinephrine concentration of up to 2.28 × 10⁻⁶ mol L⁻¹ (r = 0.9953; n = 5) was verified. The determination rate was about 79 determinations per hour and the detection limit was about 8.69 × 10⁻⁸ mol L⁻¹. The results obtained in the analysis of epinephrine pharmaceutical formulations by using the proposed methodology were in good agreement with those furnished by the reference procedure, with relative deviations lower than 4.80%.     

The microenvironment effect on the generation of reactive oxygen species by Pd-bacteriopheophorbide

Generation of reactive oxygen species (ROS) is the hallmark of important biological processes and photodynamic therapy (PDT), where ROS production results from in situ illumination of certain dyes. Here we test the hypothesis that the yield, fate, and efficacy of the species evolved highly depend on the dye's environment. We show that Pd-bacteriopheophorbide (Pd-Bpheid), a useful reagent for vascular targeted PDT (VTP) of solid tumors, which has recently entered into phase II clinical trials under the code name WST09 (trade name TOOKAD), forms appreciable amounts of hydroxyl radicals, superoxide radicals, and probably hydrogen peroxide in aqueous medium but not in organic solvents where singlet oxygen almost exclusively forms. Evidence is provided by pico- and nanosecond time-resolved spectroscopies, ESR spectroscopy with spin-traps, time-resolved singlet oxygen phosphorescence, and chemical product analysis. The quantum yield for singlet oxygen formation falls from approximately 1 in organic solvents to approximately 0.5 in membrane-like systems (micelles or liposomes), where superoxide and hydroxyl radicals form at a minimal quantum yield of 0.1%. Analysis of photochemical products suggests that the formation of oxygen radicals involves both electron and proton transfer from (3)Pd-Bpheid at the membrane/water interface to a colliding oxygen molecule, consequently forming superoxide, then hydrogen peroxide, and finally hydroxyl radicals, with no need for metal catalysis. The ability of bacteriochlorophyll (Bchl) derivatives to form such radicals upon excitation at the near infrared (NIR) domain opens new avenues in PDT and research of redox regulation in animals and plants.     

A luminol-based micro-flow-injection electrochemiluminescent system to determine reactive oxygen species

A flow injection analysis (FIA) system with electrochemiluminescent (ECL) detection has been established. Based on a specially designed flow-through ECL cell with a very simple structure, the system possesses rapid response and high sensitivity. With luminol as the ECL reagent, the response of hydrogen peroxide (H₂O₂) was investigated on the developed FIA-ECL system. After optimizing the experimental conditions, such as the electric parameters, the buffer condition and the flow rate, it was demonstrated that the developed FIA-ECL system works well for quantified assays. Compared with reported works, the present results indicate that the developed FIA-ECL system has the lowest limit of detection (S/N = 3) of 3.0 × 10⁻⁹ mol/L for H₂O₂, which is equal to the level of chemiluminescence (CL). The developed system was successfully used to monitor the yield of reactive oxygen species (ROSs) in water vapour during the work of an ultrasonic humidifier with H₂O₂ as index. And the amount of ROSs in some other real samples, including tap water, drinking water and river water was detected with recoveries from 92.0% to 106%.     

Simultaneous determination of glutathione and reactive oxygen species in individual cells by microchip electrophoresis

A microchip electrophoresis method was developed for simultaneous determination of reactive oxygen species (ROS) and reduced glutathione (GSH) in the individual erythrocyte cell. In this method, cell sampling, single-cell loading, docking, lysing, and capillary electrophoretic separation with LIF detection were integrated on a microfluidic chip with crossed channels. ROS was labeled with dihydrorhodamine 123 in the intact cell, while GSH was on-chip labeled with 2,3-naphthalene-dicarboxaldehyde, which was included in the separation medium. On-chip electrical lysis, characterized by extremely fast disruption of the cellular membrane (<40 ms), was exploited to minimize enzymatic effects on analyte concentrations during the determination. The microfluidic network was optimized to prevent cell leaking from the sample reservoir (S) into separation during the separation phase. The structure of the S was modified to avoid blockage of its outlet by deposited cells. Detection limits of 0.5 and 6.9 amol for ROS and GSH, respectively, were achieved. The average cell throughput was 25 cells/h. The effectiveness of the method was demonstrated in the simultaneous determination of GSH and ROS in individual cells and the variations of cellular GSH and ROS contents in response to external stimuli.     

Evaluation of chemiluminescence reagents for selective detection of reactive oxygen species

In order to evaluate the chemiluminescence (CL) reagents for selective detection of reactive oxygen species (ROS), we comprehensively measured the CL responses of 20 CL reagents (three luminol derivatives, two imidazopyrazinone derivatives, eight lophine derivatives, six acridinium ester derivatives and lucigenin) against six types of ROS (superoxide anion: O₂⁻, hydroxyl radical: OH, hydrogen peroxide: H₂O₂, hypochlorite anion: ClO⁻, singlet oxygen: ¹O₂, and nitric oxide: NO). As a result of the screening, it was found that nine CL reagents selectively detected O₂⁻ while one CL reagent selectively detected OH. However, no CL reagent had selectivity on the detection of H₂O₂, ClO⁻, ¹O₂ and NO. Our screening results could help to select the most suitable CL reagent for selective determination of different ROS.As an application study, 4-methoxyphenyl-10-methylacridinium-9-carboxylate (MMAC), one of the acridinium ester derivatives, showed high selectivity on the detection of O₂⁻, and thus was applied to the assay of superoxide dismutase (SOD) activity. The dynamic range and detection limit of the developed CL assay were 0.1-10 and 0.06 U mL⁻¹, respectively. Significant correlation (r = 0.997) was observed between the results by the CL assay using MMAC and the spectrophotometric assay using 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt.     

Analysis of oxidation process of cholecystokinin octapeptide with reactive oxygen species by high‐performance liquid chromatography and subsequent electrospray ionization mass spectrometry

The C‐terminal octapeptide of cholecystokinin (CCK8) includes some easily oxidizable amino acids. The oxidation of CCK8 by reactive oxygen species (ROS) such as hydrogen peroxide (H₂O₂) and hydroxyl radicals (OH^?) was investigated using reversed‐phase high performance liquid chromatography (RP‐HPLC) and subsequent electrospray ionization mass spectrometry. The mechanism of oxidation of CCK8 in the H₂O₂ system differed from that of CCK8 in the Fenton system, in which OH^? are produced. In the H₂O₂ system, ²⁸Met and ³¹Met were oxidized to methionine sulfoxide, and no further oxidation or degradation/hydrolysis occurred. On the other hand, in the Fenton system, ²⁸Met and ³¹Met residues were oxidized to methionine sulfone via the formation of methionine sulfoxide. In addition, the oxidized product was observed at the Trp residue but not at the Tyr residue, and small peptide fragments from CCK8 were observed in the Fenton system. From these results, it was concluded that ²⁸Met and ³¹Met residues of CCK8 are susceptible to oxidation by ROS. Copyright © 2009 John Wiley & Sons, Ltd.     

Nanozymes for regulation of reactive oxygen species and disease therapy

With high catalytic activity and stability, nanozymes have huge advantage in generating or eliminating the reactive oxygen species (ROS) due to their intrinsic enzyme-mimicking abilities, therefore attracting wide attention in ROS-related disease therapy. To better design nanozyme-based platforms for ROS-related biological application, we firstly illustrate the catalytic mechanism of different activities, and then introduce different strategies for using nanozymes to augment or reduce ROS level for the applications in cancer therapy, pathogen infection, neurodegeneration, etc. Finally, the challenges and future opportunities are proposed for the development and application of nanozymes.