RESUMEN
As two of important highly reactive species / nitrogen species, hypochloric acid (HClO) and peroxynitrite (ONOO-) are involved in various pathological and physiological processes, which are important factors that affect and reflect the functional state of lysosome. Nevertheless, many of their roles are still indefinite because of lack of suitable analytical methods for HClO and ONOO- detection in lysosome. Herein, we designed a lysosome-targeted probe to monitor HClO and ONOO-, which was a hydrid of the benzothiazole derivative, methyl thioether (HClO recognition site) and morpholino hydrazone (ONOO- recognition and lysosome target site). The probe exhibited high sensitivity, good selectivity and fast response toward HClO and ONOO- without spectral crosstalk, and can be employed for quantitative monitoring HClO and ONOO- with LOD of 63 and 83 nM, respectively. In addition, the dual-site probe was lysosome targetable and could be used for detection of HClO and ONOO- in living cells. Furthermore, the excellent behavior made it was suitable for imaging of HClO and ONOO- in zebrafish. Thus, the present probe provides a potent tool for distinguishing monitoring HClO and ONOO- and exploring the role of HClO and ONOO- in biological systems.
Asunto(s)
Colorantes Fluorescentes , Pez Cebra , Humanos , Animales , Lisosomas , Ácido Peroxinitroso , Células HeLa , Ácido HipoclorosoRESUMEN
Hypochloric acid (HOCl) plays vital roles in cell signaling and homeostasis which involved in many related diseases. Therefore, the accurate detected level of OCl- for discussing the complex contributions of OCl- to human health is of great significance. In this study, a novel mitochondrion-targeting fluorescent probe DMI bearing a carbon-carbon double bond as an OCl--responsive site has been developed. Probe DMI exhibited specific fluorescence response toward OCl- with fast response (within 4â¯s) and high sensitivity (detection limit is 0.05⯵M), where the obvious color changes could be observed by the naked eye. More importantly, DMI could be applied in the bioimaging of OCl- in living A549â¯cells successfully benefited from its good sensing properties and low toxicity. Fluorescence co-localization test was further carried out and confirmed mitochondria-targetable ability of DMI which can be used for investigating physiological function of HOCl at organelle levels.
Asunto(s)
Colorantes Fluorescentes/química , Ácido Hipocloroso/análisis , Mitocondrias/química , Imagen Óptica , Células A549 , Supervivencia Celular/efectos de los fármacos , Colorantes Fluorescentes/síntesis química , Colorantes Fluorescentes/farmacología , Humanos , Estructura Molecular , EspectrofotometríaRESUMEN
Two Schiff base fluorescein probes (FDA, FDH) based on fluorescein-aldehyde and nitroaniline derivatives were synthesized. The effects of amino and hydrazine substituents in fluorescein backbones were examined via fluorescence and absorbance spectra. In the presence of Ce4+, the fluorescence of FDA was quenched due to the ligand to metal charge transfer (LMCT). Hypochloric acid can react with the CN bond, and blocking the photo induced electron transfer (PET) of FDH leads to enhancement of the fluorescence. FDA showed detection limits for Ce4+ and OCl- as low as 63â¯nM in concentration range of 0-4⯵M. FDH showed detection limits for OCl- as low as 0.8⯵M in concentration rang 0-100⯵M. Polyvinylidene fluoride (PVDF) membrane containing the probes was prepared for the real-time qualitative detection of Ce4+ and OCl- in real water samples. The probes were successfully applied to biological imaging in vascular smooth muscle cells (VSMCs) and are expected to find applications in biosensing.
Asunto(s)
Aminas/química , Cerio/análisis , Colorantes Fluorescentes/síntesis química , Hidrazinas/química , Ácido Hipocloroso/análisis , Imagenología Tridimensional , Bases de Schiff/química , Agua/química , Animales , Colorantes Fluorescentes/química , Humanos , Concentración de Iones de Hidrógeno , Iones , Lagos/química , Modelos Moleculares , Músculo Liso Vascular/citología , Miocitos del Músculo Liso/citología , Polivinilos/química , Espectrometría de FluorescenciaRESUMEN
Cystatins are the inhibitors of thiol proteinases and are ubiquitously present in mammalian system. In brain, they put off unwanted proteolysis and are also involved in several neurodegenerative diseases. In the present study, it was demonstrated that photo-activated HOCl-induced modifications in brain cystatin leading to its inactivation and degradation due to hydroxyl radicals. It has been shown that oxidation of cystatin by ROS in vivo leads to oxidative modification which may direct the damage of this significant protein, as it is so well pronounced in vitro. The interplay between free radicals, antioxidants and co-factors is important in maintaining health, aging and age-related diseases. Body's endogenous antioxidant systems stabilize free radical-induced oxidative stress by the ingestion of exogenous antioxidants. If the generation of free radicals goes beyond the protective effect of antioxidants, this can cause oxidative damage which accumulates during the life cycle and has been implicated in aging and age-related diseases such as cardiovascular disease, cancer, neurodegenerative disorders and other chronic conditions. Activation of neutrophils in certain diseases (e.g., inflammatory conditions and atherosclerosis) results in the production of highly reactive species, such as OH⢠and the release of the enzyme myeloperoxidase. Stimulated monocytes and neutrophils generate hypochlorite (HOCl) via the release of the enzyme myeloperoxidase and hydrogen peroxide. Hypochlorous acid (HOCl) is a potent oxidant formed by myeloperoxidase that causes aggregation of many proteins and damage of proteins by reaction with amino-acid side-chains or backbone cleavage. Communicated by Ramaswamy H. Sarma.
Asunto(s)
Antioxidantes/química , Encéfalo/metabolismo , Cistatinas/química , Cistatinas/metabolismo , Radicales Libres/química , Ácido Hipocloroso/química , Oxidantes/química , Animales , Encéfalo/ultraestructura , Búfalos , Microscopía Electrónica de Transmisión , Oxidación-Reducción , ProteolisisRESUMEN
Mouse models resembling systemic sclerosis can be chemically induced by application of bleomycin or hypochloric acid (HOCl). To date, little is known about inflammatory cells and their potential role in scleroderma (Scl)-related fibrosis. Therefore, we compared both Scl models to define the early immune cell subsets in relation to fibrosis-related parameters. Both agents induced a significant increase in dermal thickness and collagen deposition after 4 weeks, as hallmarks of Scl. However, clinical skin thickness, densely packed, sirius red-stained collagen bundles and collagen cross-links were more pronounced in HOCl-induced Scl. In parallel, there was a significant upregulation of procollagen α1(I), α-SMA and TGF-ß transcripts in HOCl animals, whereas IL-1ß and MMP-13 mRNA levels were significantly increased in bleomycin-treated mice. Flow cytometric analysis of the Scl skin demonstrated an early cellular infiltrate containing mainly CD19+ B cells, CD4+ T cells, CD11c+ DC and CD11b+ myeloid cells, the latter ones being significantly more prominent after HOCl injection. Subanalysis revealed that Scl mice exhibited a significant increase of inflammatory myeloid CD11b+ Ly6Clow-high CD64low-high cells (HOCl>bleomycin). In particular, in the HOCl model, activated dermal macrophages (CCR2low MHCIIhigh ) and monocyte-derived DC (CCR2high MHCIIhigh ) predominated over less activated CD11b+ myeloid cells. In conclusion, the two models differ in certain aspects of the murine and human scleroderma but in the HOCl model, myeloid CD11b+ MHCIIhigh cells correlate with some fibrosis-related parameters. Therefore, analysis of both models is suggested to cover a comprehensive profile of Scl symptoms but with focus on the HOCl model when the role of early myeloid immune cells will be evaluated.