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.

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.

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.

低频脉冲磁场调节NO/ONOO-对缺氧/复氧条件下心肌细胞的保护作用

目的:观察低频脉冲磁场(LF-PMFs)对缺氧/复氧(H/R)条件下乳鼠心肌细胞增殖与凋亡的影响,并探讨一氧化氮/过氧亚硝基阴离子(NO/ONOO-)平衡在LF-PMFs保护心肌细胞的作用。方法:采用酶消化法分离SD乳鼠心肌细胞,随机分为对照组、H/R组和磁场组。用TUNEL法检测心肌细胞凋亡程度,MTT比色法检测心肌细胞增殖活性,以观察LF-PMFs对损伤心肌细胞的效果,用WST-1法检测超氧阴离子,Griess法测定NO和ONOO-。结果:LF-PMFs磁场可减少H/R诱导的心肌细胞凋亡,并促进心肌细胞的存活,以15 Hz,4.5 mT LF-PMFs磁场的作用效果最为显著。同时LF-PMFs可减少H/R条件下乳鼠心肌细胞超氧阴离子的生成,促进NO生成,提高NO/ONOO-比值。结论:LF-PMFs可通过抗氧化及调节NO/ONOO-平衡减轻H/R导致的心肌细胞损伤。

血红蛋白-维生素B6-ONOO-体系的荧光特性及ONOO-分析

在中性pH值的缓冲溶液中，血红蛋白作催化剂，维生素B。与过氧亚硝酸活性氧（ONOO-）发生反应，维生素B。原来位于391nm处的强烈的荧光发射峰消失，而在384nm和424nm处出现了两个新的荧光峰。据此建立了血红蛋白～维生素B。体系高灵敏检测ONOO-活性氧的新方法。方法的线性范围为5．24×10^-8～2．62×10^-6mol／L，检出限为2．62×10^-9mol／L，对1．31×10^-7mol／L的ONOO^=溶液测定的相对标准偏差为3．759／6（n=7）。

“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.

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。

一种新型ESIPT荧光探针用于诊断和评估铁死亡介导的HK-2细胞损伤

目的:急性肾损伤(AKI)由于缺乏早期诊断生物标志物和实时评估的可视化工具,给AKI早期诊断带来了重大挑战。因此,开发用于早期诊断和评估HK-2细胞损伤的新型探针至关重要。方法:基于ESTPT机制,合成了一种以2-(2'-羟基苯基)苯并噻唑(HBT)衍生物为荧光团的新型荧光探针(MEO-NO),用于检测铁死亡介导的HK-2细胞损伤中过氧亚硝基阴离子(ONOO^(-))。结果:当加入ONOO^(-)时,探针MEO-NO表现出明显的荧光变化,显著的斯托克斯位移(130 nm)和快速反应(约45 s),并具有极高的灵敏度(LOD=7.28 nmol/L)以及高选择性。此外,探针MEO-NO能够在细胞中检测ONOO^(-)。重要的是,MEO-NO可以检测索拉非尼诱导的铁死亡和顺铂(CP)诱导的HK-2细胞损伤过程中的ONOO^(-)。结论:在探针MEO-NO的帮助下,我们成功地在细胞模型中可视化并追踪了ONOO^(-)的变化,探针MEO-NO有望用于诊断和评估铁死亡介导的AKI中的ONOO^(-)水平变化。

丹皮酚拮抗过氧亚硝基阴离子致大鼠成骨细胞凋亡的作用

目的探讨丹皮酚拮抗过氧亚硝基阴离子(ONOO-)对体外培养大鼠成骨细胞凋亡的作用。方法用改良的组织块法分离培养新生大鼠颅骨成骨细胞,采用淬灭流动反应方法体外制备ONOO-,以1 mm o l/L终浓度加入成骨细胞培养体系,作用30 m in后弃去,继续常规培养12 h,用Hoechst33258染色法及TUNEL法检测成骨细胞的凋亡,并以0.1 mm o l/L丹皮酚消除ONOO-(1 mm o l/L)对大鼠成骨细胞凋亡的影响。结果1 mm o l/L的ONOO-可导致大鼠成骨细胞凋亡;0.1 mm o l/L丹皮酚能拮抗ONOO-(1 mm o l/L)所引起的大鼠成骨细胞凋亡。结论丹皮酚0.1 mm o l/L在体外具有拮抗ONOO-(1 mm o l/L)致大鼠成骨细胞凋亡的作用。

过氧亚硝基-鲁米诺化学发光体系的改进

建立了一个测定过氧亚硝基阴离子 (ONOO-)化学发光的改进体系 ,测试了某些抗氧化剂清除ONOO-的作用 ,其体系的组成和启动发光的程序如下 :向pH 10 5碳酸缓冲液配的 0 0 1mol/L浓度NaN3 溶液通O330s ,取其 80 0 μl原位注入含有 10 0 μl水配样品和 10 0 μl的 0 0 0 1mol/L鲁米诺溶液中 ,启动化学发光(chemiluminescence ,CL) ,立即测定每 6秒的脉冲数 (CP6S) ,连续测定 10～ 30次 .根据实际需要 ,选其某一次的CL强度作为评判指标 ,对比抗氧化剂的活性 .该发光体系灵敏、简便、且较稳定 ,最低可检测限为 8 74μmol/L的ONOO-量 ,线性范围为 8 74～ 74 0 4μmol/L .批内变异系数 3 35 % (n =10 ) ,批间变异系数 5 5 2 % (n =10 ) .测得维生素C (Vit.C )、茶多酚 (EGCG)、原花菁素、硫脲皆有抑制CL ,即清除ONOO-的作用 .