A highly sensitive fluorescent probe RN-NA reveals peroxynitrite as a novel biomarker for primary open angle glaucoma

AIM:To directly quantify peroxynitrite(ONOO-)using a highly sensitive fluorescence resonance energy transfer probe RN-NA,investigate the association between ONOOand primary open angle glaucoma(POAG),and clarify whether RN-NA could be used as a potential tool for POAG diagnosis.METHODS:Plasma and aqueous humor(AH)samples were collected from POAG patients(n=100,age:59.70±6.87y)and age-related cataract(ARC)patients(n=100,age:61.15±4.60y)admitted to our hospital.Next,RN-NA was used to detect ONOO-in plasma and AH samples,and the relationship between ONOO-level and POAG was analyzed using binary logistic regression.Besides,Pearson correlation analysis was applied to characterize the correlation of the levels of ONOO-with the patients’age,intraocular pressure(IOP),and mean deviation of visual field testing.The ONOO-scavenger MnTMPyP was employed to treat the 3-morpholinosyndnomine(SIN-1)-induced ocular hypertension in mice(n=7,6-8wk).Finally,the IOP and ONOO-in both eyes were measured 30min after the last drug treatment.RESULTS:ONOO-levels of AH and plasma were significantly higher in the POAG group than in the ARC group(P<0.01).Additionally,ONOO-levels were closely correlated with POAG in a binary logistic regression analysis[odds ratio(OR)=1.008,95%confidence interval(CI):1.002-1.013,P<0.01 for AH;OR=1.004,95%CI:1.002-1.006,P<0.001 for plasma].Pearson correlation analysis showed that ONOO-levels in AH or plasma were positively associated with visual field defects(R=0.51,P<0.01 for AH;R=0.45,P<0.001 for plasma),and ONOO-levels in plasma and AH were correlated in the POAG group(R=0.69,P<0.001).However,administering MnTMPyP to mouse eyes reversed the elevated IOP caused by SIN-1(P<0.05).CONCLUSION:ONOO-levels in AH and plasma,detected by RN-NA,are significantly related to POAG and positively correlated with visual field defects in POAG patients.Hence,ONOO-is a potential biomarker of POAG,especially advanced POAG.Besides,anti-nitration compounds may be novel ocular hypotensive agents based on the animal study.

Regulatory role of peroxynitrite in advanced glycation end products mediated diabetic cardiovascular complications

The Advanced Glycation End Products(AGE)binding with its receptor can increase reactive oxygen species(ROS)generation through specific signaling mediators.The effect of superoxide(O2-)and O2-mediated ROS and reactive nitrogen species depends on their concentration and location of formation.Nitric oxide(NO)has anti-inflammatory and anticoagulant properties and a vasodilation effect,but NO can be deactivated by reacting with O_(2)^(-).This reaction between NO and O2-produces the potent oxidant ONOO−.Therefore,ONOO-'s regulatory role in AGEs in diabetic cardiovascular complications must considered as a regulator of cardiovascular complications in diabetes.

A novelα-ketoamide reactivity-based two-photon fluorogenic probe for visualizing peroxynitrite in Parkinson's disease models

Peroxynitrite(ONOO^(-))contributes to oxidative stress and neurodegeneration in Parkinson's disease(PD).Developing a peroxynitrite probe would enable in situ visualization of the overwhelming ONOO^(-)flux and understanding of the ONOO^(-)stress-induced neuropathology of PD.Herein,a novelα-ketoamide-based fluorogenic probe(DFlu)was designed for ONOO^(-)
monitoring in multiple PD models.The results demonstrated that DFlu exhibits a fluorescence turn-on response to ONOO^(-)with high specificity and sensitivity.The efficacy of DFlu for intracellular ONOO^(-)
imaging was demonstrated systematically.The results showed that DFlu can successfully visualize endogenous and exogenous ONOO^(-)in cells derived from chemical and biochemical routes.More importantly,the two-photon excitation ability of DFlu has been well demonstrated by monitoring exogenous/endogenous ONOO^(-)production and scavenging in live zebraflsh PD models.This work provides a reliable and promising
α-ketoamide-based optical tool for identifying variations of ONOO^(-)in PD models.

Peroxynitrite Scavenging Activities of Resveratrol and Piceid

In vitro antioxidant activities of resveratrol and piceid against peroxynitrite（ONOO-） were examined by the inhibition of 3-nitrotyrosine formation.Trolox was used as a positive control.Resveratrol and piceid exhibited high ONOO--scavenging activities in a concentration dependent manner.The antioxidant activities（the concentration of test compound required to yield a 50% inhibition of tyrosine nitration,IC 50） of resveratrol and piceid against ONOO-were（48.34±0.97） and（74.69±1.49） μmol/L,respectively.Compared with that of trolox[（105.40±1.16） μmol/L],their scavenging activities were 2.2-and 1.5-fold higher for resveratrol and piceid.Formation of nitroresveratrol as shown by UV-Vis spectroscopy and liquid chromatography-tandom mass spectrometry（LC-MS/MS） analysis indicates that resveratrol could directly scavenge ONOO-via nitration reaction.Our results demonstrate that foods and medicinal herbs with resveratrol and piceid as stronger ONOO-scavengers are valuable ingredients and have healthy application in preventing humans from peroxynitrite-mediated oxidative damage by scavenging peroxynitrite efficiently.

Identification and quantification of flavonoids in Carica papaya leaf and peroxynitrite-scavenging activity

Objective: To characterize the types, contents, and peroxynitrite-scavenging activities of flavonoids in the leaf of Carica papaya(C. papaya).Methods: Chromatographic and spectroscopic techniques along with high performance liquid chromatography quantitative analysis and peroxynitrite-scavenging assay were performed to isolate and quantify flavonoid compounds in the flavonoid-rich fraction(Bu OH fraction) derived from Me OH extract of C. papaya leaves and evaluate their peroxynitrite-scavenging activities.Results: Seven flavonoids were isolated from the leaves of C. papaya, including quercetin 3-(2~G-rhamnosylrutinoside), kaempferol 3-(2~G-rhamnosylrutinoside), quercetin 3-rutinoside, myricetin 3-rhamnoside, kaempferol 3-rutinoside, quercetin, and kaempferol. All of the substances exhibited potent activities on peroxynitrite scavenging(IC50 4.15 mmol/L), which were stronger than the positive control, L-penicillamine(6.90 mmol/L). The content of kaempferol 3-(2~G-rhamnosylrutinoside) was significantly higher than other identified compounds(123.18 mg/g Bu OH fraction and 7.23 mg/g Me OH extract).Conclusions: The results of the present study demonstrate the potent antioxidant flavonoids of C. papaya leaf, with kaempferol 3-(2~G-rhamnosylrutinoside) as the major one.

Peroxynitrite induced decrease in Na^+, K^+-ATPase activity is restored by taurine

AIM: Peroxynitrite (ONOO-) is a powerful oxidant shown to damage membranes. In the present study, the effect of taurine on changes of liver plasma membrane Na+, K+-ATPase induced by ONOO- was investigated. METHODS: Liver plasma membrane was exposed toONOO-with or without taurine. Na+, K+-ATPase activity and lipid peroxidation as thiobarbituric acid reactive substances (TBARS) levels were measured.RESULTS: Different concentrations of ONOO- (100, 200,500, and 1 000 μmol/L) were found to decrease liver plasma membrane Na+, K+-ATPase activity significantly. The depletion of enzyme activity was not concentration dependent. Effects of different concentrations of taurine on liver plasma membrane Na+, K+-ATPase activity were also measured. Taurine did not cause any increase in enzyme activity. When plasma membranes were treated with 200 μmol/L ONOO- with different concentrations of taurine, a restoring effect of taurine on enzyme activity was observed. TBARS levels were also measured and taurine was found to decrease the elevated values. CONCLUSION: Taurine is observed to act as an antioxidant of ONOO-to decrease lipid peroxidation and thus affect liver plasma membrane Na+, K+-ATPase by restoring its activity.

Theoretical Studies on the Isomerization of Peroxynitrite to Nitrate Mediated by Peroxynitrous Acid

The conversion of peroxynitrite （ONOO-） to nitrate （NO3-） mediated by peroxynitrous acid （ONOOH） has been investigated at the CCSD/6-311G（d）//B3LYP/6-31 1＋G（d,p） level. Two kinds of pathways for the title reaction were found. The results show that the energy barrier of isomerization through pathway 1 is around 25 kcal/mol in the gas phase. This value is significantly lower than that of isomerization without any catalysts. Thus, it indicates that ONOOH definitely makes the conversion from ONOO- to NO3- feasible. Although pathway 2 does not decrease the energy barrier of this isomerization, peroxynitric acid （O2NOOH） was obtained; moreover, this is a new pathway for this formation. In view of the results that peroxynitrate anion can decompose into nitrite and dioxygen, we conclude that our results are consistent with the experimental observation that nitrate, nitrite, and dioxygen are the main final products of the decay of peroxynitrite around pH 7.

Metabolism of benzo[a]pyrene in peroxynitrite/Fe(III) porphyrin system

The peroxynitrite/porphyrin biomimetic system was established to investigate the effects of peroxynitrite on benzo[a]pyrene （B[a]P） metabolism. Three model systems consisting of different iron porphyrins were compared, and the results showed that the peroxynitdte/T（p-Cl）PPFeCl system was the highest catalytic efficiency in the metabolism of B [a]P. We analyzed the B [a]P metabolites produced from this system by RP-HPLC method and firstly identified the formation of nitrobenzo[a]pyrenes which are the special metabolites of B [a]P induced by peroxynitrite.

Serum E3 SUMO-protein ligase NSE2 level and peroxynitrite related to oxidative stress in nephrolithiasis patients

Objective: To prove probable relations between serum E3 SUMO-protein ligase NSE2(NSMCE2) concentration, peroxynitrite related to oxidative stress in nephrolithiasis patients.Methods: A total of 60 patients with nephrolithiasis and 50 healthy volunteers were involved in this study. Colorimetric method was used to detect blood urea, creatinine, uric acid, protein, albumin, total antioxidant status, total oxidant status, peroxynitrite, nitric oxide and oxidative stress index. Glutathione, NSMCE2 and superoxide dismutase were measured by ELISA.Results: A significant increase in level of peroxynitrite, total oxidant status, NSMCE2 and oxidative stress index in patients was observed, while total antioxidant status and glutathione were significantly decreased.Conclusions: The study concluded that serum NSMCE2 significantly correlated with peroxynitrite and oxidative stress in patients with nephrolithiasis.

Nitric Oxide/Peroxynitrite Redox Imbalance in Endothelial Cells Measured with Amperometric Nanosensors

The cytoprotective messenger nitric oxide (NO) and cytotoxic peroxynitrite (ONOO-) are the main components of oxidative stress and can be generated by endothelial cells. A tandem of electrochemical nanosensors (diameter 200-300 nm) were used to measure, in situ, the balance between NO and ONOO-produced by human umbilical vein endothelial cells (HUVEC’s). The amperometric nanosensors were placed 5 ± 2 μm from the surface of the endothelial cells and the concentration of NO and ONOO- was measured at 630 mV and -300 mV (vs Ag/AgCl) respectively. Normal, functional, endothelial cells produced maximal 450 ± 25 nmol.L-1 of NO and 180 ± 15 nmol.L-1 of ONOO- in about 3 s, after stimulation with calcium ionophore. The in situ measurements of NO and ONOO- were validated using nitric oxide synthase inhibitor L-NMMA, ONOO- scavenger Mn(III) porphyrin, and superoxide dismutase (PEG-SOD). The ratio of NO concentration to ONOO- concentration ([NO]/[ONOO-]) was introduced for quantification of both, the redox balance and the level of the nitroxidative stress in the endothelium. [NO]/[ONOO-] was 2.7 ± 0.1 in a functional endothelium. The model of the dysfunctional endothelium was made by the treatment of HUVEC’s with angiotensin II for 20 min. Dysfunctional HUVEC’s produced only 115 ± 15 nmol.L-1 of NO, but generated a significantly higher concentration of ONOO- of 490 ± 30 nmol.L-1. The [NO]/[ONOO-] ratio decreased to 0.23 ± 0.14 in the dysfunctional endothelium. Electrochemical nanosensors can be effectively used for in situ monitoring of changing levels of nitroxidative/ oxidative stress, and may be useful in early medical diagnosis of the cardiovascular system.

Effect of Peroxynitrite (ONOO^-) on the Function of Murine Perivascular Adipose Tissue

Perivascular adipose tissue (PVAT) surrounds the exterior of blood vessels and releases numerous substances such as adiponectin which positively modulate blood vessel tone. In some cardiovascular diseases such as diabetes and high blood pressure, the function of PVAT changes and we speculate that oxidant stress may play a role in this change. PVAT has the ability to generate both superoxide and nitric oxide and these can combine rapidly under physiological conditions to form peroxynitrite (ONOO-). In disease states, the production of ONOO- may be increased and so its effect on the function of PVAT is of great interest. Consequently, we studied the effects of acute addition of the oxidant species ONOO- on vascular tone and production of adiponectin by mouse thoracic aortic PVAT. Murine PVAT was immunostained for nitrotyrosine, indicating that ONOO- is formed in the PVAT. Exogenous ONOO- significantly increased the anticontractile effect of PVAT via increased adiponectin content but had no effect on eNOS expression or phosphorylation. These results suggest that generation of ONOO- within PVAT may be an important regulatory mechanism which influences the activity of PVAT. The effect of chronic exposure to raised levels of ONOO- on PVAT function remains to be determined.

ONOO^-活性氧的荧光光谱分析进展

系统介绍了ONOO-活性氧的化学性质,并对其参与的主要化学反应进行了分类和总结.同时,比较了近年来用于ONOO-检测的方法,并重点论述了荧光光谱分析方法尤其是酶催化动力学荧光分析法在检测ONOO-活性氧方面的优势和最新的研究进展.

Ebselen 抑制 ONOO^- 对神经元[Ca^(2+)]_i 的影响

Ｆｕｒａ－２显微荧光测钙技术研究发现，过氧亚硝基阴离子（ＯＮＯＯ－）作用于ＭＮ９Ｄ细胞，数ｓ内即可导致其胞内游离钙离子浓度（［Ｃａ２＋］ｉ）的急剧升高．胞外液换为无钙液或向胞外液中加入硝苯吡啶（Ｎｉｆｅｄｉｐ－ｉｎｅ）、二硫苏糖醇（ＤＴＴ）均可抑制ＯＮＯＯ－对［Ｃａ２＋］ｉ的影响，提示Ｌ－型钙通道的激活是ＯＮＯＯ－引起［Ｃａ２＋］ｉ升高的主要原因，ＯＮＯＯ－的这种作用可能与其氧化特性有关．Ｅｂｓｅｌｅｎ（２－苯基－１，２－苯并异硒唑－３（２Ｈ）酮）明显抑制ＯＮＯＯ－对［Ｃａ２＋］ｉ的影响，并且存在一定的剂量效应关系．

NO、ONOO^-在大鼠离体肾缺血再灌注肾功能损伤中的作用

[目的]探讨一氧化氮(nitricoxide,NO)、过氧亚硝基阴离子(peroxynitrite,ONOO-)在大鼠离体肾缺血再灌注损伤时对肾功能的影响。[方法]应用离体灌流肾(isolatedperfusedkidney,IPK)技术观察肾I-R对菊粉清除率(Cin)、尿钠排泄指数(FENa)、肾血管阻力(RVR)的影响;阻断内源性NO生成对肾I-R所致的上述指标变化的影响;外源性直接给予NO供体硝普钠(SNP)、ONOO-对大鼠IPK肾功能的影响。[结果]①缺血再灌注导致大鼠IPK肾血管阻力(RVR)明显增大(P<0.05),IPK的Cin降低(P<0.05),肾I-R后IPK的FENa有较大幅度的增加(P<0.05)。②SNP能够使健康大鼠IPK的RVR降低、Cin增加和FENa增大(P<0.05)。而ONOO-使RVR增高、Cin降低和FENa增加(P<0.05)。③iNOS抑制剂氨基胍(aminoguanidine,AG)使大鼠IPK的功能I-R后5hRVR降低、Cin增大和尿钠减少(P<0.05);而应用NOS抑制剂N-硝基-L-精氨酸(L-NNA),加重了肾I-R大鼠IPK肾机能的损伤,I-R1h和I-R5h均出现了RVR增大、Cin降低和尿钠排泄增多(P<0.05)。[结论]肾I-R所致NO大量生成后产生的ONOO-损伤了大鼠的肾功能。

青光眼患者小梁网中ONOO^-的表达及与眼压升高关系的研究

目的检测正常人和原发性急性闭角型青光眼(primary acute angle closure glaucoma,PAACG)及原发性慢性闭角型青光眼(primary chronic angle closure glaucoma,PCACG)患者小梁网组织标本中过氧亚硝基阴离子(peroxynitrite,ONOO-)的表达,探讨小梁网中ONOO-的变化、分布及与青光眼的关系。方法取正常眼6例及PAACG患者14例18只眼及PCACG患者12例15只眼小梁网组织标本,应用形态学观察,免疫组织化学方法检测小梁细胞ONOO-的标志物硝基酪氨酸(nitrotyrosine,NT)的表达,并采用计算机图像分析系统对检测结果进行分析。结果正常人的小梁网组织标本NT的表达呈弱阳性或不表达,PAACG组小梁网组织中NT呈强阳性表达,PCACG组小梁网组织中NT呈阳性表达,3组间的差异均具有统计学意义(F=25.32,P<0.01),眼压与小梁网NT着色强度呈相关关系(P<0.01)。结论正常人的小梁网细胞ONOO-微弱表达而PAACG与PCACG患者小梁网细胞ONOO-大量表达且其表达量随眼压的升高而增多,表明眼压升高可导致ONOO-过量生成,从而损伤小梁网细胞,进一步加重青光眼眼压升高。

“Three-in-one” strategy of trifluoromethyl regulated blood-brain barrier permeable fluorescent probe for peroxynitrite and antiepileptic evaluation of edaravone

Epilepsy,as a chronic neurological disease of the brain,is closely related to oxidative stress,and the peroxynitrite(ONOO-)significantly rise up in this event.Therefore,ONOO-is considered as a potential biomarker for early prediction of epilepsy.However,some potential diagnostic reagents for epilepsy are hindered by the blood-brain barrier(BBB).Meanwhile,“drug repurposing”is attracting a growing interest.Edaravone(EDA),as a first-line drug in the clinical treatment of cerebral ischemia,plays antioxidant roles in scavenging free radicals,promising potential antiepileptic activity.Thus,it is imperative to develop fluorescent probes for monitoring ONOO-fluctuations in the epileptic brain.Hence,we proposed a novel fluorescent probe with the thiocarbonate as the promising recognition unit for ONOO-and dicyanoisophorone derivative as the fluorophore.Moreover,by the“three-in-one”strategy,the introduction of trifluoromethyl into DCI-ONOO-3 can extend the emission wavelength of the fluorophore,shorten the response and increase lipophilicity.Consequently,DCI-ONOO-3 was used for monitoring ONOO-fluxes in brain of epileptic mice and evaluating the antiepileptic efficacy of EDA.It opens up a new way for the design of BBB permeable fluorescent probes,and provides a convincing new method for the diagnosis and treatment of epilepsy.

Mitochondria-specific near-infrared photoactivation of peroxynitrite upconversion luminescent nanogenerator for precision cancer gas therapy

Gas therapy is emerging as a highly promising therapeutic strategy for cancer treatment.However,there are limitations,including the lack of targeted subcellular organelle accuracy and spatiotemporal release precision,associated with gas therapy.In this study,we developed a series of photoactivatable nitric oxide(NO)donors NRh-R-NO(R=Me,Et,Bn,iPr,and Ph)based on an N-nitrosated upconversion luminescent rhodamine scaffold.Under the irradiation of 808 nm light,only NRh-Ph-NO could effectively release NO and NRh-Ph with a significant turn-on frequency upconversion luminescence(FUCL)signal at 740 nm,ascribed to lower N-N bond dissociation energy.We also investigated the involved multistage near-infrared-controlled cascade release of gas therapy,including the NO released from NRh-Ph-NO along with one NRh-Ph molecule generation,the superoxide anion O_(2)^(⋅−)produced by the photodynamic therapy(PDT)effect of NRh-Ph,and highly toxic peroxynitrite anion(ONOO‒)generated from the co-existence of NO and O_(2)^(⋅−).After mild nano-modification,the nanogenerator(NRh-Ph-NO NPs)empowered with superior biocompatibility could target mitochondria.Under an 808 nm laser irradiation,NRh-Ph-NO NPs could induce NO/ROS to generate RNS,causing a decrease in the mitochondrial membrane potential and initiating apoptosis by caspase-3 activation,which further induced tumor immunogenic cell death(ICD).In vivo therapeutic results of NRh-Ph-NO NPs showed augmented RNS-potentiated gas therapy,demonstrating excellent biocompatibility and effective tumor inhibition guided by real-time FUCL imaging.Collectively,this versatile strategy defines the targeted RNS-mediated cancer therapy.

四羧基锰(Ⅲ)酞菁与ONOO^-的相互作用

研究了四羧基锰(Ⅲ)酞菁(Mn(Ⅲ)TcPc)与ONOO-的相互作用及其对ONOO-硝基化酪氨酸(Tyr)和损伤DNA的影响.结果表明Mn(Ⅲ)TcPc能与ONOO-作用,并可促进ONOO-对Tyr的硝基化,该硝基化作用能被谷胱甘肽(GSH)或抗坏血酸(VitC)等生物还原剂抑制;该硝基化作用与pH值有关,以pH7为最合适.Mn(Ⅲ)TcPc能抑制ONOO-对DNA的损伤作用.GSH对Mn(Ⅲ)TcPc有还原作用.并对上述的促进或抑制Tyr硝基化反应的机理进行了理论解释.

一种用于过氧亚硝酸根检测的近红外比率荧光探针的研制

设计合成了一种基于半花菁的近红外比率荧光探针分子Cy-P,并将其用于活细胞中的外源性和内源性过氧亚硝酸根(ONOO-)荧光成像检测.研究结果表明:探针分子Cy-P自身具有较大的π共轭结构,其最大发射波长在近红外区域;加入ONOO-后,强氧化性的ONOO-会导致Cy-P的π共轭体系被破坏,并生成蓝色荧光物质,最大发射波长蓝移了268 nm;探针分子Cy-P对ONOO-线性检测范围为0~15μmol/L,检测下限为13 nmol/L;该探针分子对ONOO-的识别性能优于其他各种可能干扰的生物分析物;该探针分子具有低细胞毒性,适用于活细胞中的外源性和内源性ONOO-的成像检测.

靶向细胞器的过氧亚硝酸盐荧光探针研究进展

过氧亚硝酸盐(ONOO^(-))作为氧代谢的重要产物,在免疫应答、细胞信号转导等许多生理活动中起着重要作用。然而,ONOO^(-)的水平异常会损伤细胞生物分子,与许多疾病密切相关。因此,迫切需要监测活细胞中ONOO^(-)的含量变化,而ONOO^(-)的功能很大程度上依赖于亚细胞区,所以开发亚细胞的ONOO^(-)水平变化的监测手段更有研究价值。为此,多种具有靶向细胞器的ONOO^(-)荧光探针被设计、开发和应用。重点介绍了近6年来具有线粒体、溶酶体和内质网靶向能力的ONOO^(-)荧光探针的代表性例子。综述了这些荧光探针的细胞器靶向策略、结构、荧光行为和生物学应用。