RESUMO
BACKGROUND: Limb ischemia-reperfusion is a common phenomenon in clinical surgery, which disrupts the balanced physiological response process and ultimately leads to changes in intracellular viscosity. Intracellular viscosity is an important microenvironmental parameter that affects the normal function of organisms, and its level is closely related to many diseases. In addition, oxidative stress in the lower limbs can impair body function, and changes in pressure can lead to changes in the viscosity of limb tissues. Therefore, it is necessary to develop effective tools to detect changes in intracellular viscosity and visualize the progression of hind limb ischemia-reperfusion injury. RESULTS: In order to solve this problem, a near infrared viscometry sensitive fluorescence probe (PH-XQ) with long emission wavelength and stable luminescence performance was designed and synthesized by using oxanthracene derivatives and malononitrile. The fluorescence probe (PH-XQ) has excellent selectivity, high sensitivity, low toxicity, high biocompatibility and excellent detection performance. The fluorescence intensity of the PH-XQ probe at 667 nm is highly sensitive to the change of viscosity. With the increase of viscosity, the fluorescence intensity of probe PH-XQ was significantly enhanced, and the fluorescence enhancement ratio was about 14-fold. In addition, PH-XQ can detect not only changes in viscosity between normal cells and drug-induced inflammatory cells, but also changes in the viscosity of the hind limbs of normal mice and mice after ischemia reperfusion. SIGNIFICANCE: In particular, we are the first to successfully detect changes in handlimb viscosity after ischemia-reperfusion in mice using a probe. This study clearly elucidates changes in viscosity during ischemia-reperfusion of mouse limbs, providing favorable support for the relationship between viscosity and related diseases, and further providing a potential tool for the diagnosis of viscosity-related diseases.
Assuntos
Corantes Fluorescentes , Traumatismo por Reperfusão , Animais , Viscosidade , Corantes Fluorescentes/química , Corantes Fluorescentes/síntese química , Camundongos , Traumatismo por Reperfusão/diagnóstico por imagem , Membro Posterior , Masculino , Imagem Óptica , Raios Infravermelhos , HumanosRESUMO
Research on ferroptosis in myocardial ischemia/reperfusion injury (MIRI) using mitochondrial viscosity as a nexus holds great promise for MIRI therapy. However, high-precision visualisation of mitochondrial viscosity remains a formidable task owing to the debilitating electrostatic interactions caused by damaged mitochondrial membrane potential. Herein, we propose a dual-locking mitochondria-targeting strategy that incorporates electrostatic forces and probe-protein molecular docking. Even in damaged mitochondria, stable and precise visualisation of mitochondrial viscosity in triggered and medicated MIRI was achieved owing to the sustained driving forces (e.g., pi-cation, pi-alkyl interactions, etc.) between the developed probe, CBS, and the mitochondrial membrane protein. Moreover, complemented by a western blot, we confirmed that ferrostatin-1 exerts its therapeutic effect on MIRI by improving the system xc-/GSH/GPX4 antioxidant system, confirming the therapeutic value of ferroptosis in MIRI. This study presents a novel strategy for developing robust mitochondrial probes, thereby advancing MIRI treatment.
Assuntos
Ferroptose , Traumatismo por Reperfusão Miocárdica , Ferroptose/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/patologia , Simulação de Acoplamento Molecular , Animais , Mitocôndrias/metabolismo , Mitocôndrias/efeitos dos fármacos , Humanos , Cicloexilaminas/química , Cicloexilaminas/farmacologia , Fenilenodiaminas/química , Fenilenodiaminas/farmacologiaRESUMO
Cysteine (Cys) is a critical amino acid that involves in many physiological and pathological processes in the human body, and it plays an important role in maintaining redox homeostasis in living systems. The concentration of intracellular Cys is abnormal under oxidative stress thus leading to many diseases. Therefore, it is significant to develop an effective method for detection of Cys under oxidative stress. In this work, we propose a new polymer-based ratiometric fluorescent probe with good selectivity and sensitivity for detecting Cys. The bioimaging experiments results show that the novel probe has a rapid ratiometric response to Cys, which can be used to monitor Cys level changes during LPS or H2O2 induced oxidative stress in living cells and zebrafish.
Assuntos
Cisteína , Corantes Fluorescentes , Animais , Cisteína/metabolismo , Células HeLa , Humanos , Peróxido de Hidrogênio/toxicidade , Lipopolissacarídeos/toxicidade , Estresse Oxidativo , Polímeros/toxicidade , Espectrometria de Fluorescência , Peixe-ZebraRESUMO
PURPOSE: Cellular senescence is a physiological phenomenon by which cells irreversibly lose their proliferative potential. It is not clear whether senescent cells are related to malignant transformation in oral precancerous lesions. The role of peroxiredoxin1 (Prx1)-induced cell senescence in OLK malignant transformation has not been reported. The aim of this study is to investigate the role and mechanism of cell senescence in oral carcinogenesis. METHODS: In this study, 4-nitro-quinoline-1-oxide (4NQO) induced tongue carcinogenesis model in Prx1+/+ and Prx1+/- mice and dysplastic oral keratinocyte (DOK) were used. Prx1 knockdown DOK cells were harvested with shRNA injection, and cell senescence was detected via the senescence-associated ß-galactosidase (SA ß-gal) assay. The senescence and mitophagy-related proteins were observed by immunohistochemistry (IHC), Western blot and qRT-PCR. The binding of Prx1 with prohibitin 2 (PHB2) and light chain 3 (LC3) was predicted via ZDOCK and measured in mice by Duolink analysis. RESULTS: Histologically, 4NQO treatment induced epithelial hyperplasia, dysplasia (mild, moderate and severe), carcinomas in situ and oral squamous cell carcinoma (OSCC) in mouse tongue mucosa. The malignant transformation rate in Prx1+/- mice (37.5%) was significantly lower compared with Prx1+/+ mice (57.1%). In Prx1+/+ mice, a higher number of senescent cells and greater expression of p53 and p21 were observed in hyperplastic and dysplastic tongue tissues when compared with those in OSCC tissues. Prx1 knockdown induced a greater number of senescent cells in hyperplastic tissues, and DOK cells accompanied cell cycle arrest at the G1 phase and PHB2/LC3II downregulation. Prx1 was predicted to dock with PHB2 and LC3 via ZDOCK, and the interactions were confirmed by in situ Duolink analysis. CONCLUSION: Prx1 silencing inhibits the oral carcinogenesis by inducing cell senescence dependent on mitophagy.
RESUMO
Biothiols, as part of the reactive sulfur species (RSS), are a class of bioactive molecules that play important physiological roles in human body. However, due to the similarity in structure and reaction sites of biothiols, it is difficult to differentiated detection them at the same time. In this work, a fluorescent probe CM-NBD combined coumarin derivative and 7-nitrobenzofurazan has been developed, which can effectively detect biothiols through simple ether cleavage. Because of a specific location group, CM-NBD can well localize in lysosomes with a high co-localization coefficient. Interesting, due to the weakly acidic environment of lysosomes, Cys can be distinguished from Hcy/GSH and H2S via dual-color mode. The probe is able not only to image exogenous biothiols but also to discriminate Cys from Hcy/GSH and H2S in cells and zebrafish model.
Assuntos
Cisteína , Corantes Fluorescentes , Animais , Glutationa , Homocisteína , Humanos , Lisossomos , Espectrometria de Fluorescência , Peixe-ZebraRESUMO
As a weak acidic organelle, lysosomes maintain acidic pH ranging from 4.5 to 5.5 and abnormal lysosomal pH levels can result in functional deficiency of lysosomes. Tracking the pH changes in lysosomes will help us to understand lysosome-related biological processes and diseases. However, pH-stimuli-responsive polymer-based lysosomal pH fluorescent probe with good water solubility and biocompatibility have rarely been reported. In this work, on the basis of naphthalimide chromophore and poly(N,N-dimethylaminoethyl methacrylate), we designed lysosome-targeted fluorescent probe NapBr-PDM or NapMl-PDM with single- or dual-responsive sites for monitoring pH changes in lysosomes. Both NapBr-PDM and NapMl-PDM exhibited a narrow polydispersity index, excellent fluorescence properties, water solubility, and high photostability. The synthesized probe NapBr-PDM, with a single-responsive site, showed fast response to pH changes and well-stained lysosomes, which could monitor lysosomal pH changes in cells after incubation with chloroquine. NapMl-PDM had good lysosomes-targeting property and highly sensitive response to pH, which could track lysosomal pH changes. Moreover, NapMl-PDM achieved tracking of lysosomal pH changes in cells during lysosomal storage disorder induced by high-concentration sucrose solution for the first time. Therefore, this work provided useful tools for monitoring intracellular pH changes as well as studying the relationship between lysosomal pH and its related diseases.
Assuntos
Fluorescência , Corantes Fluorescentes/química , Metacrilatos/química , Naftalimidas/química , Nylons/química , Imagem Óptica , Corantes Fluorescentes/síntese química , Células HeLa , Humanos , Concentração de Íons de Hidrogênio , Lisossomos/química , Estrutura MolecularRESUMO
Tobacco smoking is the major risk factor for oral squamous cell carcinoma (OSCC). Previously, we found that nicotine up-regulates peroxiredoxin 1 (Prx1), an important antioxidant enzyme, and nuclear factor kappa B (NFκB) in OSCC cells. However, the molecular mechanism of Prx1 in oral carcinogenesis remains obscure. To improve our understanding of the functional role of Prx1 during the cascade of tobacco-associated oral carcinogenesis, we characterized Prx1, NFκB, and epithelial-to-mesenchymal transition (EMT) markers including E-cadherin, vimentin and Snail in 30 primary oral tumors (15 from smokers with OSCC and 15 from non-smokers with OSCC) and 10 normal oral mucosa specimens from healthy individuals. The expression levels of Prx1, nuclear NFκB, vimentin and Snail were higher in the tumors from smokers with OSCC than in those from non-smokers with OSCC or the healthy controls. The expression levels of E-cadherin showed an opposite trend. Prx1 silencing suppressed the nicotine-induced EMT, cell invasion and migration in SCC15 cells in vitro. Furthermore, Prx1 activated the NFκB pathway in SCC15 cells. Prx1 might therefore play an oncogenic role in tobacco-related OSCC and thus serve as a target for chemopreventive and therapeutic interventions.
Assuntos
Carcinogênese/patologia , Carcinoma de Células Escamosas/patologia , Transição Epitelial-Mesenquimal , Neoplasias Bucais/patologia , Peroxirredoxinas/metabolismo , Adulto , Idoso , Antígenos CD , Caderinas/metabolismo , Carcinogênese/induzido quimicamente , Linhagem Celular Tumoral , Regulação para Baixo , Feminino , Técnicas de Silenciamento de Genes , Humanos , Masculino , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Invasividade Neoplásica/patologia , Peroxirredoxinas/genética , Transdução de Sinais , Fatores de Transcrição da Família Snail/metabolismo , Fumar Tabaco/efeitos adversos , Regulação para Cima , Vimentina/metabolismoRESUMO
Peroxiredoxin 1 (Prx1) is important in the protection of cells from oxidative damage and the regulation of cell proliferation and apoptosis. Prx1 is overexpressed in oral precancerous lesions of oral leukoplakia (OLK) and oral cancer; however, the association between Prx1 expression and OLK pathogenesis remains unknown. The present study investigated the role of Prx1 and its molecular mechanisms in oxidative stress-induced apoptosis during the pathogenesis of OLK. Wild-type and Prx1 knockout mice were treated with 50 µg/ml 4-nitroquinoline-1-oxide (4NQO) or 4NQO + H2O2 for 16 weeks to establish mouse models with tongue precancerous lesions. Apoptotic cells were detected using terminal deoxynucleotidyl transferase dUTP nick-end labeling assay. The expression of Prx1, apoptosis signal-regulating kinase 1 (ASK1), phosphor-ASK1, p38 and phosphor-p38 was analyzed using immunohistochemical staining, and their mRNA expression levels were evaluated by reverse transcription quantitative polymerase chain reaction. The present results demonstrated that 4NQO or 4NQO + H2O2 induced the development of tongue precancerous lesions in Prx1 knockout and wild-type mice. Prx1 was overexpressed in tongue precancerous lesions compared with normal tongue mucosa. There was a significant decrease in the degree of moderate or severe epithelial dysplasia, and mild epithelial dysplasia was clearly elevated, in Prx1 knockout mice treated with 4NQO + H2O2 compared with wild-type mice treated with 4NQO + H2O2. Prx1 suppressed apoptosis and upregulated phosphor-ASK1 and phosphor-p38 expression in tongue precancerous lesions. The present results suggest that Prx1 suppresses oxidative stress-induced apoptosis via the ASK1/p38 signalling pathway in mouse tongue precancerous lesions. In conclusion, Prx1 and H2O2 have a coordination role in promoting the progression of tongue precancerous mucosa lesions. The present findings provide novel insight into Prx1 function and the mechanisms of Prx1 in OLK pathogenesis.
RESUMO
Overexpression of peroxiredoxin 1 (Prx1) has been observed in numerous cancers including oral squamous cell carcinoma (OSCC). The precise molecular mechanism of up-regulation of Prx1 in carcinogenesis, however, is still poorly understood. The objective of this study is to investigate the relationship between Prx1 and hypoxia, and potential mechanism(s) of Prx1 in OSCC cell line SCC15 and xenograft model. We treated wild-type and Prx1 knockdown SCC15 cells with transient hypoxia followed by reoxygenation. We detected the condition of hypoxia, production of reactive oxygen species (ROS), and expression and/or activity of Prx1, heme oxygenase 1 (HO-1) and nuclear factor-kappa B (NF-κB). We found that hypoxia induces ROS accumulation, up-regulates Prx1, increases NF-κB translocation and DNA binding activity, and down-regulates HO-1 in vitro. In Prx1 knockdown cells, the expression level of HO-1 was increased, while NFκB translocation and DNA binding activity were decreased after hypoxia or hypoxia/reoxygenation treatment. Moreover, we mimicked the dynamic oxygenation tumor microenvironment in xenograft model and assessed the above indices in tumors with the maximal diameter of 2 mm, 5 mm, 10 mm or 15 mm, respectively. Our data showed that tumor hypoxic condition and expression of Prx1 are significantly associated with tumor growth. The expression of HO-1 and NF-κB, and NF-κB DNA binding activity were significantly elevated in 15 mm tumors, and the level of 8-hydroxydeoxyguanosine was increased in 10 mm and 15 mm tumors, compared to those in size of 2 mm. The results from this study provide experimental evidence that overexpression of Prx1 is associated with hypoxia, and Prx1/NF-κB/HO-1 signaling pathway may be involved in oral carcinogenesis.