Peroxynitrite Scavenger Research Articles

Peroxynitrite-Promoted Persulfide Prodrugs with Protective Potential against Paracetamol Poisoning.

The newly emerging persulfide prodrugs provide additional options for the profound study of persulfide, a fascinating molecule expected to intervene in biological functions and even diseases. Peroxynitrite is often the culprit in pathological processes characterized by oxidative stress, while the persulfide prodrug responsive to it is still pending. To enrich the family of redox-activated prodrugs, we designed prodrugs with a 2-oxo-2-phenylacetamide trigger, which achieved the release of persulfide via 1, 6-N, S-relay. The degradation of prodrugs and the formation of persulfides were confirmed to be peroxynitrite-responsible by the qualitative and quantitative studies based on LC-MS/MS methods and a spectrophotometry-based tag-switch strategy. Furthermore, these prodrugs showed potent peroxynitrite scavenging activity, cellular therapeutic potential against paracetamol poisoning in HepG2 and oxidative stress in H9c2, as well as desirable in vitro metabolic properties.

Angong Niuhuang Wan reduces hemorrhagic transformation and mortality in ischemic stroke rats with delayed thrombolysis: involvement of peroxynitrite-mediated MMP-9 activation

BackgroundHemorrhagic transformation (HT) is a common complication of delayed tissue plasminogen activator (t-PA) treatment for ischemic stroke. Peroxynitrite plays an important role in the breakdown of blood–brain barrier (BBB) and the development of HT. We tested the hypothesis that Angong Niuhuang Wan (AGNHW), a traditional Chinese medicinal formula, could be used in conjunction with t-PA to protect the BBB, minimize HT, and improve neurological function by suppressing peroxynitrite-mediated matrix metalloproteinase-9 (MMP-9) activation.MethodsWe first performed quality control study and chemical identification of AGNHW by using UPLC. In animal experiments, male Sprague–Dawley rats were subjected to 5 h of middle cerebral artery occlusion (MCAO) followed by 19 h of reperfusion plus t-PA infusion (10 mg/kg) at 5 h of cerebral ischemia. AGNHW (257 mg/kg) was given orally at 2 h after MCAO. Hemorrhagic transformation was measured using hemorrhagic scores and hemoglobin levels in ischemic brains. Evans blue leakage was utilized to assess the severity of the blood–brain barrier (BBB) damage. The modified neurologic severity score (mNSS) test was used to assess neurological functions. Peroxynitrite and superoxide was detected by using fluorescent probes. MMP-9 activity and expression were examined by gelatin zymography and immunostaining. The antioxidant effects were also studied by using brain microvascular endothelial b.End3 cells exposed to 5 h of oxygen and glucose deprivation (OGD) plus 5 h of reoxygenation with t-PA treatment (20 µg/ml).ResultsAGNHW significantly reduced the BBB damage, brain edema, reduced hemorrhagic transformation, enhanced neurological function, and reduced mortality rate in the ischemic stroke rats with t-PA treatment. AGNHW reduced peroxynitrite and superoxide in vivo and in vitro and six active chemical compounds were identified from AGNHW with peroxynitrite scavenging activity. Furthermore, AGNHW inhibited MMP-9 activity, and preserved tight junction protein claudin-5 and collagen IV in the ischemic brains.ConclusionAGNHW could be a potential adjuvant therapy with t-PA to protect the BBB integrity, reduce HT, and improve therapeutic outcome in ischemic stroke treatment via inhibiting peroxynitrite-mediated MMP-9 activation.Graphical

Thromboxane-dependent coronary vasoconstriction in obese mice: Role of peroxynitrite

Obesity leads to chronic oxidative stress promoting the development of cardiovascular diseases including coronary artery disease and endothelial dysfunction. Increased reactive oxygen species production associated with obesity might lead to endothelial dysfunction through cyclooxygenase (COX) pathway. We evaluated arachidonic acid (AA)-dependent coronary vascular responses and explored COX metabolism in obese C57BL/6 mice. In response to arachidonic acid (AA), isolated hearts from obese mice showed increased vasoconstriction compared with control mice. Released thromboxane (TX) A2 during AA-induced vasoconstriction phase was increased in heart perfusates from obese mice. Indomethacin and 1-benzylimidazole, both reduced vasoconstriction response in control and obese mice. Vasoconstriction response to TXA2 mimetic analog U46619 was 2.7 higher in obese mice. Obesity increased COX-2, TXS and TX receptor protein expression as well as oxidative stress evaluated by nitrotyrosine and peroxynitrite levels, compared with control mice. Obese mice treated with FeTMPyP, a peroxynitrite scavenger, reversed all these parameters to control levels. These data suggest that alterations in COX pathway may be associated with increased generation of free radicals, including peroxynitrite, that result from the oxidative stress observed in obesity.

토마토 추출물의 라디칼 소거 및 항염증 활성

This study compares the antioxidant and anti-inflammatory capacities of acetone extracts obtained from commercially available tomatoes (gangwon chal, wansuk, cherry, daechu cherry, plum, and campari). Campari and cherry tomatoes had the highest levels of 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical, 2,2’-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid (ABTS) radical, and hydroxyl radical scavenging activities, as well as cupric reducing antioxidant capacity (CUPRAC), followed sequentially by gangwon chal, daechu cherry, plum and wansuk varieties. Highest peroxynitrite scavenging activity, oxygen radical absorbance capacity (ORAC), and total phenolic contents were obtained in campari tomatoes, followed by cherry, gangwon chal, daechu cherry, plum and wansuk varieties. Lycopene contents were highest in cherry tomato, followed by daechu cherry and campari, and were lowest in the gangwon chal, plum and wansuk varieties. Maximum inhibition of the lipopolysaccharide-induced nitric oxide production in RAW 264.7 cells was observed in campari tomatoes, followed by cherry, daechu cherry, gangwon chal, plum and wansuk varieties. Antioxidant activities, including hydroxyl radical and peroxynitrite scavenging, ORAC, and CUPRAC, showed a high correlation with the total phenolic or lycopene contents. Anti-inflammatory capacity was highly correlated with antioxidant activities, total phenolic, and lycopene contents. Taken together, findings of the current study demonstrate that campari and cherry tomatoes have the most potent antioxidant and anti-inflammatory effects, which are closely related to the lycopene as well as polyphenolic contents.

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Alcohol exacerbated biochemical and biophysical alterations in liver mitochondrial membrane of diabetic male wistar rats – A possible amelioration by green tea

Inhibition of peroxidases and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in intact barley root tips.

NBT and HE may be efficiently used for the detection of superoxide, while DCDHF-DA and DHR123 for the detection of peroxynitrite in intact barley root tips, only ifPRXs and oxidoreductases are inhibited to avoid false-positive reactions. Strong peroxidase (PRX) and oxidoreductase activities were observed in the barley root tips that were markedly inhibited by NaN3. Rapid and strong nitro-blue tetrazolium chloride (NBT) reduction is associated mainly with the vital functions of root cells but not with superoxide formation. In turn, the inhibition of root surface redox activity by NaN3 strongly reduced the formation of formazan, but its slight accumulation, observed in the root elongation zone, was a result of NADPH oxidase-mediated apoplastic superoxide formation. A longer staining time period with NBT was required for the detection of antimycin A-mediated superoxide formation inside the cells. This antimycin A-induced superoxide was clearly detectable by hydroethidine (HE) after the inhibition of PRXs by NaN3, and it was restricted into the root transition zone. TEMPOL, a superoxide scavenger, strongly inhibited both NBT reduction and HE oxidation in the presence of NaN3. Similarly, the DCDHF-DA and DHR123 oxidation was markedly reduced after the inhibition of apoplastic PRXs by NaN3 and was detectable mainly in the root transition zone. This fluorescence signal was not influenced by the application of pyruvate but was strongly reduced by urea, a peroxynitrite scavenger. The presented results suggest that if the root PRXs and oxidoreductases are inhibited, both NBT and HE detect mainly superoxide, whereas both DCDHF-DA and DHR123 may be efficiently used for the detection of peroxynitrite in intact barley root tips. The inhibition of PRXs and oxidoreductases is crucial for avoiding false-positive reactions in the localization of reactive oxygen species in the intact barley root tip.

Abstract WP245: Sex-dependent Regulation Of Mitochondrial Respiratory Function In Mouse Brain Microvessels By Peroxynitrite Decomposition Catalyst

Background: Peroxynitrite (PN) is a strong oxidizing and nitrating molecule. PN is a potent inhibitor of mitochondrial respiration and mediates the ischemia-reperfusion injury following stroke. In isolated mouse brain mitochondria, we observed that PN donors inhibit mitochondrial respiratiory function whereas PN decomposition catalyst, Fe (III) tetrakis (1-methyl-4-pyridyl) porphyrin pentachlorideporphyrin pentachloride (FeTMPyP), enhanced mitochondrial state III and state IVo mitochondrial respiration. In addition, we demonstrated that mitochondrial respiration is the primary contributor of cellular energy in brain microvessels (BMVs). The present study tested the hypothesis that FeTMPyP negatively regulates the mitochondrial respiration in the mouse BMVs. Methods: BMVs were isolated from male and female mice (C57Bl/6, 2-4 months) using a combination of filters with pore sizes of 300μm and 40μm followed by gradient centrifugation. BMVs were treated with FeTMPyP at 37 o C for 60 minutes. Oxygen consumption rates (OCR) were measured using the Agilent Seahorse XFe24 analyzer and various respiratory parameters were determined following Mitostress test. Results: In the male BMVs, basal respiration, ATP production, and non-mitochondrial respiration were not altered by FeTMPyP treatment. Contrary to our hypothesis, in the male BMVs, PN scavenger decreased the mitochondrial maximal respiration by 24.6% (2.1 ± 0.4 vs 2.8 ± 0.3 picomoles of O 2 /min/μg protein;) whereas the spare respiratory capacity was reduced by 33.3% (1.2 ± 0.3 vs 1.8 ± 0.3 picomoles of O 2 /min/μg protein) (n=17 each, p<0.05). Proton leak was elevated by 70% (0.7 ± 0.1 vs 0.4 ± 0.1 picomoles of O 2 /min/μg protein ) by PN scavenger in male BMVs. In contrast, in the female BMVs, the PN scavenger failed to alter the mitochondrial respiratory parameters (n=15 each, p=NS). Interestingly, FeTMPyP increased non-mitochondrial respiration by 63.8% (0.95 ± 0.2 vs 0.58 ± 0.1 picomoles of O 2 /min/μg protein) in the female BMVs (n=17 each, p<0.05). Conclusion: BMVs display sex-dependent respenses to endogenous PN. Notably, in female mouse BMVs, PN appears to act as an antioxidant as PN inhibited the non-mitochondrial respiration which contributes to extramitochondrial superoxide generation.

SIRT2 tyrosine nitration by peroxynitrite in response to renal ischaemia/reperfusion injury

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the production of renal ischaemia/reperfusion (I/R). The current study is to elucidate a mechanism of SIRT2 tyrosine nitration to accelerate the cell apoptosis induced by peroxynitrite (ONOO‾), the most reactive and deleterious RNS type in renal ischaemia/reperfusion (I/R) injury. Our results demonstrate that there is a significant enhancement of the 3-nitrotyrosine levels in renal tissues of Acute Kidney Injury (AKI) patients and rats that underwent renal I/R, and a positive correlation between the 3-nitrotyrosine level and renal function impairment, indicative of an accumulation of peroxynitrite. Notably, peroxynitrite-evoked nitration of SIRT2 destroyed its enzymatic activity and the capability to deacetylate FOXO3a, and enhanced expression of Bim and caspase3, facilitating renal cell apoptosis in renal ischaemia/reperfusion and SIN-1(peroxynitrite donor) treatment in vitro, and these effects were reversed by FeTMPyP, a peroxynitrite decomposition scavenger. Importantly, we identified that the tyrosine 86 is responsible for SIRT2 nitration and inactivation using site-mutation assay and Mass Spectrography analysis. Altogether, these findings point to a novel protective mechanism that an inhibition of SIRT2 tyrosine nitration can be a promising strategy to prevent ischaemic renal diseases involving AKI.

Structural and (Pseudo-)Enzymatic Properties of Neuroglobin: Its Possible Role in Neuroprotection.

Neuroglobin (Ngb), the third member of the globin family, was discovered in human and murine brains in 2000. This monomeric globin is structurally similar to myoglobin (Mb) and hemoglobin (Hb) α and β subunits, but it hosts a bis-histidyl six-coordinated heme-Fe atom. Therefore, the heme-based reactivity of Ngb is modulated by the dissociation of the distal HisE7-heme-Fe bond, which reflects in turn the redox state of the cell. The high Ngb levels (~100–200 μM) present in the retinal ganglion cell layer and in the optic nerve facilitate the O2 buffer and delivery. In contrast, the very low levels of Ngb (~1 μM) in most tissues and organs support (pseudo-)enzymatic properties including NO/O2 metabolism, peroxynitrite and free radical scavenging, nitrite, hydroxylamine, hydrogen sulfide reduction, and the nitration of aromatic compounds. Here, structural and (pseudo-)enzymatic properties of Ngb, which are at the root of tissue and organ protection, are reviewed, envisaging a possible role in the protection from neuronal degeneration of the retina and the optic nerve.

Abstract 13269: Combination Therapy With Sodium Nitrite and Hydralazine Ameliorates the Severity of Heart Failure With Preserved Ejection Fraction

Background: Recent studies suggest that nitrosative stress resulting from an imbalance in nitric oxide (NO) is the primary driver of heart failure with preserved ejection fraction (HFpEF); however, many of the comorbidities present in HFpEF patients are driven by oxidative stress. Due to the failure of nitric oxide (NO) based therapeutics in HFpEF clinical trials and the role of oxidative stress in each of the different HFpEF comorbidities, we sought to investigate the effects of sodium nitrite (NaNO2) therapy alone or in combination with the antioxidant and peroxynitrite scavenger, hydralazine, in a novel, “two-hit” murine model of HFpEF. Methods: HFpEF was induced in nine-week-old C57/BL6N mice (n = 13-15 per group) by high fat diet and L-NAME (0.5 g/L/day) via drinking water for 10 weeks. At 5 weeks, mice were treated with vehicle, sodium nitrite(75 mg/L) via drinking water, hydralazine (2 mg/kg/day B.I.D., I.P, or a combination of sodium nitrite + hydralazine. Cardiac structure and function via echocardiography and exercise capacity were measured at baseline, week 5 (prior to treatment), and week 10 (final endpoint). At week 10, invasive hemodynamic and in-vitro vascular reactivity studies were performed. Results: Sodium nitrite and hydralazine combination therapy resulted in significant (p < 0.01) improvement inE/E’ that was superior to either therapy alone. Hydralazine as a monotherapy and in combination with NaNO2lowered the E/A ratio compared to NaNO2 monotherapy (p <0.05). Aortic vasorelaxation maximal responses to acetylcholine were significantly improved in all study groups with the greatest improvement observed withNaNO2 + hydralazine. Left ventricular end-diastolic pressure (LVEDP) was significantly (p < 0.01) reduced with either therapy alone, but our data indicate that combination therapy was superior to either drug alone. Conclusion: Our data suggest that antioxidant therapy is required in combination with NO based therapeutics in HFpEF.

Abstract 6980: Peroxynitrite-Mediated Mitochondrial Autophagy is Activated for the Development of Global Cerebral Ischemia Induced by Cardiac Arrest

Introduction: Mitochondrial dysfunction is implicated in ischemic brain after cardiac arrest, and the timely removal of damaged mitochondria is critical for cellular homeostasis. Mitochondrial autophagy is a mitochondrial quality control mechanism via selective degradation of mitochondria but its role in the ischemic brain remains controversial. Peroxynitrite, as a representative of reactive nitrogen species, is an important neurologic damage factor in the brain injury but its role in cardiac arrest is unknown. Methods: Male Sprague-Dawley rats underwent 8-minute asphyxial cardiac arrest and cardiopulmonary resuscitation. Survival, neurological deficit and mitochondrial function were assessed for up to 72 hours following cardiac arrest. Western blotting, immunofluorescence and enzyme-linked immunosorbent assay were used to evaluate activated mitochondrial autophagy and peroxynitrite production. To further explore the underlying mechanism, FeTMPyP was used to act as a peroxynitrite decomposition catalyst. Results: After resuscitation from cardiac arrest, PINK1/Parkin-induced mitochondrial autophagy was activated and peroxynitrite was increased in the ischemic brain. Peroxynitrite scavenger with FeTMPyP treatment significantly prevented the generation of peroxynitrite, inhibited mitochondrial autophagy activation, reduced neurological damages and improved mitochondrial and neurological outcome after cardiac arrest. Moreover, peroxynitrite induced serine 616 phosphorylation of Drp1 and Drp1 translocation to the mitochondria, which was responsible for mitochondrial autophagy activation. Conclusions: Mitochondrial autophagy activation mediated by peroxynitrite could be a key event in the pathogenesis of brain injury induced by cardiac arrest and provides a promising therapeutic strategy.

Lifetime exposure of ambient PM2.5 elevates intraocular pressure in young mice

Epidemiological studies suggest that ambient particulate matter exposure may be a new risk factor of glaucoma, but it lacks solid experimental evidence to establish a causal relationship. In this study, young mice (4 weeks old) were exposed concentrated ambient PM2.5 (CAP) for 9 months, which is throughout most of the life span of a mouse under heavy pollution. CAP was introduced using a versatile aerosol concentration enrichment system which mimics natural PM2.5 exposure. CAP exposure caused a gradual elevation of intraocular pressure (IOP) and an increase in aqueous humor outflow resistance. In the conventional outflow tissues that regulates IOP, inducible nitric oxide synthase (iNOS) was up-regulated and 3-nitrotyrosine (3-NT) formation increased. At the cellular level, PM2.5 exposure increased the transendothelial electrical resistance of cells that control IOP (AAP cells). This is accompanied by increased reactive oxygen species (ROS), iNOS and 3-NT levels. Peroxynitrite scavenger MnTMPyP successfully treated the IOP elevation and restored it to normal levels by reducing 3-NT formation in outflow tissues. This study provides the novel evidence that in young mice, lifetime whole-body PM2.5 exposure has a direct toxic effect on intraocular tissues, which imposes a significant risk of IOP elevation and may initiate the development of ocular hypertension and glaucoma. This occurs as a result of protein nitration of conventional aqueous humor outflow tissues.

Nitration of protein kinase G-Iα modulates cyclic nucleotide crosstalk via phosphodiesterase 3A: Implications for acute lung injury

Phosphodiesterase 3A (PDE3A) selectively cleaves the phosphodiester bond of cAMP and is inhibited by cGMP, making it an important regulator of cAMP–cGMP signaling crosstalk in the pulmonary vasculature. In addition, the nitric oxide–cGMP axis is known to play an important role in maintaining endothelial barrier function. However, the potential role of protein kinase G-Iα (PKG-Iα) in this protective process is unresolved and was the focus of our study. We describe here a novel mechanism regulating PDE3A activity, which involves a PKG-Iα–dependent inhibitory phosphorylation of PDE3A at serine 654. We also show that this phosphorylation is critical for maintaining intracellular cAMP levels in the pulmonary endothelium and endothelial barrier integrity. In an animal model of acute lung injury (ALI) induced by challenging mice with lipopolysaccharide (LPS), an increase in PDE3 activity and a decrease in cAMP levels in lung tissue was associated with reduced PKG activity upon PKG-Iα nitration at tyrosine 247. The peroxynitrite scavenger manganese (III) tetrakis(1-methyl-4-pyridyl)porphyrin prevented this increase in PDE3 activity in LPS-exposed lungs. In addition, site-directed mutagenesis of PDE3A to replace serine 654 with alanine yielded a mutant protein that was insensitive to PKG-dependent regulation. Taken together, our data demonstrate a novel functional link between nitrosative stress induced by LPS during ALI and the downregulation of barrier-protective intracellular cAMP levels. Our data also provide new evidence that PKG-Iα is critical for endothelial barrier maintenance and that preservation of its catalytic activity may be efficacious in ALI therapy.

Inhibition of Angiotensin-I Converting Enzyme by Ginsenosides: Structure-Activity Relationships and Inhibitory Mechanism.

Ginseng (Panax ginseng C. A. Meyer) extract has been reported to inhibit the angiotensin converting enzyme (ACE); however, the possible inhibitory action of most of its constituents (ginsenosides) against ACE remains unknown. Thus, in this study, we investigated ginsenoside derivatives' inhibitory effect on ACE. We assessed the activities of 22 ginsenosides, most of which inhibited ACE significantly. Notably, protopanaxatriol, protopanaxadiol, and ginsenoside Rh2 exhibited the most potent ACE inhibitory potential, with IC50 values of 1.57, 2.22, and 5.60 μM, respectively. Further, a kinetic study revealed different modes of inhibition against ACE. Molecular docking studies have confirmed that ginsenosides inhibit ACE via many hydrogen bonds and hydrophobic interactions with catalytic residues and zinc ion of C- and N-domain ACE that block the catalytic activity of ACE. In addition, we found that the active ginsenosides stimulated glucose uptake in insulin-resistant C2C12 skeletal muscle cells in a dose-dependent manner. Moreover, the most active ginsenosides' reactive oxygen species (ROS) and peroxynitrite (ONOO-) scavenging properties were evaluated, in which IC50 values ranged from 1.44-43.83 to 2.36-39.56 μM in ONOO- and ROS, respectively. The results derived from these computational and in vitro experiments provide additional scientific support for the anecdotal use of ginseng in traditional medicine to treat cardiovascular diseases such as hypertension.

Curcumin Scavenges Peroxynitrite in RAW 264.7 Macrophages through Phenolic Hydroxyl Groups

Background: Overproduction of free radicals is implicated in cell death and tissue injury. Peroxynitrite (PN) is a highly oxidizing and short-lived free radical that is formed by the interaction of nitric oxide (NO) with superoxide. Curcumin is a natural compound obtained from Curcuma longa and has high antioxidant and anti-inflammatory activities.
 Aim: We investigated the PN scavenging ability of curcumin to determine its potential as a therapeutic agent for chronic diseases caused by highly oxidative molecules.
 Methodology: We examined the PN scavenging ability of curcumin either by directly incubating lipopolysaccharide (LPS)+interferon (IFN)-γ-stimulated RAW 264.7 murinacrophages with PN or indirectly through incubation with a PN donor (the artificial substrate SIN-1). Student t-test and one-way ANOVA were used to determine the statistical significance of differences between the experimental and control groups. 
 Results: The results demonstrate that curcumin inhibits PN and the synthesis of PN from SIN-1. Curcumin also significantly improved the viability of LPS+IFN-γ-treated RAW 264.7 macrophages and inhibited NO production. In macrophages, curcumin inhibited cellular PN synthesis, as evidenced by the absence of 3-nitrotyrosine, a marker of PN-oxidized proteins. 
 Conclusion: Curcumin attenuates the immune responses that lead to cellular damage and cell death through suppression or scavenging of cytotoxic molecules such as NO and PN. The role of the phenolic hydroxyl of curcumin is critical in PN scavenging.

Discovery of Flazin, an Alkaloid Isolated from Cherry Tomato Juice, As a Novel Non-Enzymatic Protein Glycation Inhibitor via in Vitro and in Silico Studies.

Both overproduced reactive oxygen species/reactive nitrogen species and hyperglycemic conditions accompany a significant increase in protein glycation and nitration that contribute to the initiation and progression of diabetic complications and neuronal disorders. In this study, 19 compounds, including steroidal saponins, alkaloids, cerebroside, phenolic compounds, sterols, and nucleosides, were isolated from cherry tomato (Solanum lycopersicum var. cerasiforme) juice, of which flazin showed good inhibition on monosaccharide-induced non-enzymatic bovine pancreas insulin and bovine serum albumin (BSA) glycation. Molecular dynamics simulations revealed that flazin continuously interacts with Phe1, Val2, Tyr26, and Lys29 insulin residues, which play a key role in insulin glycation/dimerization. In addition, depending upon the flazin dose, this blocked the tyrosine nitration of BSA via scavenging peroxynitrite anions. Taken together, our novel findings suggest that flazin could be a lead compound for the treatment of diabetes and neuronal disorders via the inhibition of non-enzymatic protein glycation and the elimination of peroxynitrite.

Ballodiolic Acid A and B: Two New ROS, (•OH), (ONOO-) Scavenging and Potent Antimicrobial Constituents Isolated from Ballota pseudodictamnus (L.) Benth.

Bioassays guided phytochemical investigations on the ethyl acetate-soluble fraction of the root material of Ballota pseudodictamnus (L.) Benth. led to the isolation of two new compounds, ballodiolic acid A (1) and ballodiolic acid B (2), along with three known compounds ballodiolic acid (3), ballotenic acid (4), and β-amyrin (5), which were also isolated for the first time from this species by using multiple chromatographic techniques. The structures of the compounds (1–5) were determined by modern spectroscopic analysis including 1D and 2D NMR techniques and chemical studies. In three separate experiments, the isolated compounds (1–5) demonstrated potent antioxidant scavenging activity, with IC50 values ranging from 07.22–34.10 μM in the hydroxyl radical (•OH) inhibitory activity test, 58.10–148.55 μM in the total ROS (reactive oxygen species) inhibitory activity test, and 6.23–69.01 μM in the peroxynitrite (ONOO−) scavenging activity test. With IC50 values of (07.22 ± 0.03, 58.10 ± 0.07, 6.23 ± 0.04 μM) for •OH, total ROS, and scavenge ONOO−, respectively, ballodiolic acid B (2) showed the highest scavenging ability. Antibacterial and antifungal behaviors were also exposed to the pure compounds 1–5. In contrast to compounds 4 and 5, compounds 1–3 were active against all bacterial strains studied, with a good zone of inhibition proving these as a potent antibacterial agent. Similarly, compared to compounds 3–5, compounds 1 and 2 with a 47 percent and 45 percent respective inhibition zone were found to be more active against tested fungal strains.

Entacapone scavenges peroxynitrite and protects against kidney and liver injuries induced by renal ischemia/reperfusion in rats.

Acute kidney injury (AKI), secondary to renal ischemia/reperfusion (I/R), is a serious problem associated with high mortality. The pathophysiology of AKI after renal I/R involves peroxynitrite production; hence, scavenging this metabolite may rescue AKI. Entacapone is a catechol-O-methyl transferase (COMT) inhibitor which elicits antioxidant activity by scavenging peroxynitrite. Therefore, we hypothesized that the peroxynitrite scavenging activity of entacopone protects against AKI after renal I/R injury in rats. Male Wistar rats were given either entacapone or a well-known peroxynitrite scavenger (FeTPPS) daily for 10days before I/R procedures. I/R was induced by occluding both renal pedicles for 45min followed by reperfusion for 24h. Pre-treatment with either entacapone or FeTPPS improved renal function as indicated by a significant reduction in serum creatinine and urea when compared to I/R group (P < 0.05). I/R injury increased renal levels of NO (4-folds, P < 0.05), iNOS (4-folds, P < 0.05), and 3-nitrotyrosine (5-folds, P < 0.05) compared to sham control. These effects were abrogated in animals pre-treated with entacapone or FeTPPS before being subjected to I/R (P < 0.05). In addition, entacapone or FeTPPS significantly inhibited I/R-induced elevation in renal TNF-α levels (78% and 58%, respectively) and caspase-3 activity (72% and 56%, respectively) indicating the reduction of both inflammation and apoptosis in the kidney (P < 0.05). The two drugs also improved kidney and liver functions in rats with renal I/R injury. Our study showed that entacapone and FeTPPS protected against AKI and remote liver damage associated with renal I/R and this effect might be due to scavenging peroxynitrite and reducing nitrosative stress.

Peroxynitrite activates NLRP3 inflammasome and contributes to hemorrhagic transformation and poor outcome in ischemic stroke with hyperglycemia

This study aims to test the hypothesis that peroxynitrite-mediated inflammasome activation could be a crucial player in the blood-brain barrier (BBB) disruption, hemorrhagic transformation (HT) and poor outcome in ischemic stroke with hyperglycemia. We used an experimental rat stroke model subjected to 90 min of middle cerebral artery occlusion plus 24 h or 7 days of reperfusion with or without acute hyperglycemia. We detected the production of peroxynitrite, the expression of NADPH oxidase, iNOS, MMPs and NLRP3 inflammasome in the ischemic brains, and evaluated infarct volume, brain edema, HT, neurological deficit score and survival rates. Our results show that: (1) Hyperglycemia increased the expression of NADPH oxidase subunits p47phox and p67phox, and iNOS, and the production of peroxynitrite. (2) Hyperglycemia increased infarct volume, aggravated the BBB hyperpermeability, induced brain edema and HT, and worsened neurological outcomes. These brain damages and poor outcome were reversed by the treatments of FeTmPyP (a representative peroxynitrite decomposition catalyst, PDC), peroxynitrite scavenger uric acid, and iNOS inhibitor 1400W. Furthermore, the activations of MMPs and NLRP3 inflammasome including pro/active-caspase-1 and IL-1β were inhibited both PDC and 1400W, indicating the roles of peroxynitrite in the inductions of MMPs and NLRP3 inflammasome in the ischemic brains under hyperglycemia. (3) NLRP3 inflammasome inhibitor MCC950, caspase-1 inhibitor VX-765 and IL-1β inhibitor diacerein attenuated brain edema, minimized hemorrhagic transformation and improved neurological outcome, demonstrating the roles of NLRP3 inflammasome in the hyperglycemia-mediated HT and poor outcome in the ischemic stroke rats with acute hyperglycemia. In conclusion, peroxynitrite could mediate activations of MMPs and NLRP3 inflammasome, aggravate the BBB damage and HT, and induce poor outcome in ischemic stroke with hyperglycemia. Therefore, targeting peroxynitrite–mediated NLRP3 inflammasome could be a promising strategy for ischemic stroke with hyperglycemia.

How does ascorbate improve endothelial dysfunction? - A computational analysis

Low levels of ascorbate (Asc) are observed in cardiovascular and neurovascular diseases. Asc has therapeutic potential for the treatment of endothelial dysfunction, which is characterized by a reduction in nitric oxide (NO) bioavailability and increased oxidative stress in the vasculature. However, the potential mechanisms remain poorly understood for the Asc mitigation of endothelial dysfunction. In this study, we developed an endothelial cell based computational model integrating endothelial cell nitric oxide synthase (eNOS) biochemical pathway with downstream reactions and interactions of oxidative stress, tetrahydrobiopterin (BH4) synthesis and biopterin ratio ([BH4]/[TBP]), Asc and glutathione (GSH). We quantitatively analyzed three Asc mediated mechanisms that are reported to improve/maintain endothelial cell function. The mechanisms include the reduction of •BH3 to BH4, direct scavenging of superoxide (O2•−) and peroxynitrite (ONOO−) and increasing eNOS activity. The model predicted that Asc at 0.1–100 μM concentrations improved endothelial cell NO production, total biopterin and biopterin ratio in a dose dependent manner and the extent of cellular oxidative stress. Asc increased BH4 availability and restored eNOS coupling under oxidative stress conditions. Asc at concentrations of 1–10 mM reduced O2•− and ONOO− levels and could act as an antioxidant. We predicted that glutathione peroxidase and peroxiredoxin in combination with GSH and Asc can restore eNOS coupling and NO production under oxidative stress conditions. Asc supplementation may be used as an effective therapeutic strategy when BH4 levels are depleted. This study provides detailed understanding of the mechanism responsible and the optimal cellular Asc levels for improvement in endothelial dysfunction.