Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792) / Uso de antioxidantes e tempo de armazenamento a frio: efeitos sobre parâmetros de viabilidade e níveis enzimáticos no sêmen da truta arco-íris (Oncorhynchus mykiss, Walbaum, 1792)

The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Use of antioxidants and time of cold storage: effects over viability parameters and enzymatic levels in semen of rainbow trout (Oncorhynchus mykiss, Walbaum, 1792)

Abstract The cold storage of milt implies potentials alterations in its quality because the storage generates as main process, free radicals that produce spermatozoa membrane lipids damage with the consequent motility and fertilising capacity disruptions. To decrease the damage generated by free radicals the cells have antioxidant defences (proteins, enzymes, and low molecular weight substances). The objective of the present study evaluated the time storage effect and different antioxidants prepared in spermatic diluents on sperm viability of O. mykiss milt stored at 4°C. The two-way ANOVA denoted that the time storage and antioxidant influence have significant effects separated or combined on viability parameters (sperm motility and viability, proteins concentrations and superoxide dismutase enzymatic activity in seminal plasma). In contrast, only the storage time affected the fertilising capacity and catalase enzymatic activity in seminal plasma. The resulting analysis can conclude that the antioxidant presence improves the viability of cold stored milt, especially the transport conditions and the antioxidants allow the fecundity despite motility decrease.

Resumo O armazenamento a frio de leite implica potenciais alterações em sua qualidade, pois gera como processo principal radicais livres que provocam danos aos lipídios da membrana dos espermatozoides, com as consequentes alterações na motilidade e na capacidade de fertilização. Para diminuir os danos causados pelos radicais livres, as células têm defesas antioxidantes (proteínas, enzimas e substâncias de baixo peso molecular). O presente estudo avaliou o efeito do tempo de armazenamento e diferentes antioxidantes preparados em diluentes espermáticos no armazenamento de viabilidade de O. mykiss milt a 4°C. A ANOVA de duas vias denotou que o armazenamento no tempo e a influência antioxidante têm efeitos significativos separados ou combinados nos parâmetros de viabilidade (motilidade espermática, viabilidade espermática, concentrações de proteínas e atividade enzimática da superóxido dismutase no plasma seminal), enquanto apenas o tempo de armazenamento afetou a capacidade de fertilização e atividade enzimática da catalase no plasma seminal. A análise resultante pode concluir que a presença de antioxidante melhora a viabilidade do leite frio, especialmente as condições de transporte, e os antioxidantes permitem a fecundidade apesar da diminuição da motilidade.

Effects of vitamin D administration on nociception and spinal cord pro-oxidant and antioxidant markers in a rat model of neuropathic pain

Reactive oxygen species (ROS) are involved in neuropathic pain, a complicated condition after nerve tissue lesion. Vitamin D appears to improve symptoms of pain and exhibits antioxidant properties. We investigated the effects of oral administration of vitamin D3, the active form of vitamin D, on nociception, the sciatic functional index (SFI), and spinal cord pro-oxidant and antioxidant markers in rats with chronic constriction injury (CCI) of the sciatic nerve, a model of neuropathic pain. Vitamin D3 (500 IU/kg per day) attenuated the CCI-induced decrease in mechanical withdrawal threshold and thermal withdrawal latency (indicators of antinociception) and SFI. The vitamin prevented increased lipid hydroperoxide levels in injured sciatic nerve without change to total antioxidant capacity (TAC). Vitamin D3 prevented increased lipid hydroperoxide, superoxide anion generation (SAG), and hydrogen peroxide (H2O2) levels in the spinal cord, which were found in rats without treatment at 7 and 28 days post-CCI. A significant negative correlation was found between mechanical threshold and SAG and between mechanical threshold and H2O2 at day 7. Vitamin D3 also prevented decreased spinal cord total thiols content. There was an increase in TAC in the spinal cord of vitamin-treated CCI rats, compared to CCI rats without treatment only at 28 days. No significant changes were found in body weight and blood parameters of hepatic and renal function. These findings demonstrated, for first time, that vitamin D modulated pro-oxidant and antioxidant markers in the spinal cord. Since antinociception occurred in parallel with oxidative changes in the spinal cord, the oxidative changes may have contributed to vitamin D-induced antinociception.

Phagocytic activity of monocytes and neutrophils in patients with periodontitis, whether or not associated to type 2 diabetes / Atividade fagocítica de monócitos e neutrófilos em pacientes com periodontite, associada ou não ao diabetes tipo 2

ABSTRACT Phagocytic functions by neutrophils/ monocytes and biochemical parameters were assessed in peripheral blood of patients with periodontitis, whether or not associated to type 2 diabetes, or patients with type 2 diabetes, or systemically healthy people. Fifty-eight participants were divided into four groups: Control - systemically and periodontally healthy patients (C, n=16), Periodontitis (P, n=14), Type 2 Diabetes (DM, n=11) and Periodontitis associated with type 2 diabetes (DMP, n=17). Blood samples were used to analyze phagocytic activity and the production of superoxide anion using optical microscopy. Significantly lower phagocytic activity of neutrophils was observed in non-opsonized samples (p = 0.008, Kruskal- Wallis) of the periodontitis group and in opsonized samples (p = 0.029, Kruskal-Wallis) of the periodontitis associated with type 2 diabetes group when these groups were compared to the healthy individuals when a 20:1 yeast: phagocyte stimulus was used. Periodontitis patients, whether associated (p = 0.0007, sensitized; Kruskal-Wallis, 20:1) or not with diabetes (p = 0.018 and 0.0007, in the proportions 5:1 and 20:1 yeast: monocyte respectively in sensitized samples; Kruskal-Wallis) also showed lower phagocytic function of monocytes compared to the control group. There was no significant difference in the production of superoxide anion among the evaluated groups. Severe clinical attachment loss was associated with lower levels of HDL in periodontitis patients and a higher percentage of A1C in diabetes with periodontitis patients (p<0.05; Pearson and Spearman correlations, respectively). Patients with both associated diseases had higher levels of triglycerides and CRP (p<0.001, Kruskal-Wallis) compared to patients with diabetes only. The results of the present study suggest that periodontitis negatively interferes with the innate immune response and may represent a major risk of systemic complications such as cardiovascular disease in diabetic patients or even in healthy individuals.

RESUMO As funções fagocíticas de neutrófilos/monócitos e parâmetros bioquímicos foram avaliados no sangue periférico de pacientes com periodontite com ou sem diabetes do tipo 2, ou em pacientes com diabetes tipo 2, ou em pessoas saudáveis sistemicamente. 58 participantes foram divididos em quatro grupos: Controle - pacientes sistemicamente e periodontalmente saudáveis (C, n = 16), Periodontite (P, n = 14), Diabetes Tipo 2 (DM, n = 11) e Periodontite associada a diabetes tipo 2 (DMP, n = 17). Amostras de sangue foram usadas para analisar a atividade fagocítica e a produção de ânion superóxido por microscopia óptica. Observou-se menor atividade fagocítica dos neutrófilos em amostras não opsonizadas (p = 0,008, Kruskal-Wallis) do grupo periodontite e em amostras opsonizadas (p = 0,029, Kruskal-Wallis) do grupo periodontite associada ao diabetes tipo 2 quando esses grupos foram comparados aos indivíduos saudáveis sob um estímulo de levedura:monócito de 20:1. Pacientes com periodontite associada (p = 0,0007, sensibilizados; Kruskal-Wallis, 20: 1) ou não com diabetes (p = 0,018 e 0,0007, nas proporções 5: 1 e 20: 1 de levedura: monócito, respectivamente, em amostras sensibilizadas; Kruskal- Wallis) também demonstraram menor função fagocítica dos monócitos em comparação com o grupo controle. Não houve diferença significativa na produção de ânion superóxido entre os grupos avaliados. A perda de inserção clínica grave foi associada a níveis mais baixos de HDL na periodontite e maior percentual de A1C nos pacientes com periodontite associada ao diabetes (p<0,05; correlações de Person e Spearman, respectivamente). Os pacientes com ambas as doenças associadas apresentaram níveis mais altos de triglicerídeos e PCR (p<0,001, Kruskal- Wallis) em comparação aos pacientes com somente diabetes. Os resultados do presente estudo sugerem que a periodontite interfere negativamente na resposta imune inata e pode representar um risco maior para complicações sistêmicas, como a doença cardiovascular, em pacientes com diabetes ou mesmo em indivíduos saudáveis.

Ageing Induced Hyperproduction of Reactive Oxygen Species and Dysbalance in Enzymatic Link of Antioxidant Defense System of Skin and Therapeutic Efficacy of Artichoke Extract

Skin aging is multitarget persistence processing that immediately involve hyperproduction of free radicals under influence of intrinsic and extrinsic factors and deterioration in intimal antioxidant defense system. The goal of the study was the evaluation of the anti-oxidant potential of artichoke standartizated extracts, 2%, as a protective strategy against skin age-associated oxidative damage caused by D-galactose (D-gal) in rats. 58 female Wistar rats included in the experimental design. D-gal-induced aging was reproduced in 36 animals of main group, and 12 rats included in control group. All animals in main group were randomized for 3 groups: I – animals with skin aging reproduced model receive saline, II – animals with skin aging rats receive artichoke extracts (with content of chloroagenic acid 2.0%) in a dose of intradermal injection 0.13 mg/kg and main III group - animals with skin aging receive 1.3 mg/kg artichoke extract twice at weeks during 4 weeks. Influence of artichoke extracts restores skin relative weight and leads to decreasing the rate of generation of superoxide anion, hydrogen peroxide and lipid peroxidation (LPx), increasing activity of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and reverse ratio SOD/(catalase+GPx) to the production of H2O2 from superoxide dismutation coupling with the decrease ratio of generated O2-/H2O2. Low-dose of intradermally microinjection of artichoke extracts, 2%, activated the enzymatic link in innate antioxidant defense system in D-gal-induced skin aging model and could be recommended for applications in cosmetics as antiaging mesotherapy.

Age-related changes and effects of regular low-intensity exercise on gait, balance, and oxidative biomarkers in the spinal cord of Wistar rats

The present study aimed to analyze age-related changes to motor coordination, balance, spinal cord oxidative biomarkers in 3-, 6-, 18-, 24-, and 30-month-old rats. The effects of low-intensity exercise on these parameters were also analyzed in 6-, 18-, and 24-month-old rats. Body weight, blood glucose, total cholesterol, and high-density lipoprotein (HDL) cholesterol were assessed for all rats. The soleus muscle weight/body weight ratio was used to estimate skeletal muscle mass loss. Body weight increased until 24 months; only 30-month-old rats exhibited decreased blood glucose and increased total cholesterol and HDL cholesterol. The soleus muscle weight/body weight ratio increased until 18 months, followed by a small decrease in old rats. Exercise did not change any of these parameters. Stride length and step length increased from adult to middle age, but decreased at old age. Stride width increased while the sciatic functional index decreased in old rats. Performance in the balance beam test declined with age. While gait did not change, balance improved after exercise. Aging increased superoxide anion generation, hydrogen peroxide levels, total antioxidant capacity, and superoxide dismutase activity while total thiol decreased and lipid hydroperoxides did not change. Exercise did not significantly change this scenario. Thus, aging increased oxidative stress in the spinal cord, which may be associated with age-induced changes in gait and balance. Regular low-intensity exercise is a good alternative for improving age-induced changes in balance, while beneficial effects on gait and spinal cord oxidative biomarkers cannot be ruled out because of the small number of rats investigated (n=5 or 6/group).

Mitochondrial superoxide anions induced by exogenous oxidative stress determine tumor cell fate: an individual cell-based study / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Objective: Reactive oxygen species (ROS) are involved in a variety of biological phenomena and serve both deleterious and beneficial roles. ROS quantification and assessment of reaction networks are desirable but difficult because of their short half-life and high reactivity. Here, we describe a pro-oxidative model in a single human lung carcinoma SPC-A-1 cell that was created by application of extracellular H2O2 stimuli. Methods: Modified microfluidics and imaging techniques were used to determine O2•− levels and construct an O2•− reaction network. To elucidate the consequences of increased O2•− input, the mitochondria were given a central role in the oxidative stress mode, by manipulating mitochondria-interrelated cytosolic Ca2+ levels, mitochondrial Ca2+ uptake, auto-amplification of intra-cellular ROS and the intrinsic apoptotic pathway. Results and conclusions: Results from a modified microchip demonstrated that 1 mmol/L H2O2 induced a rapid increase in cellular O2•− levels (>27 vs. >406 amol in 20 min), leading to increased cellular oxidizing power (evaluated by ROS levels) and decreased reducing power (evaluated by glutathione (GSH) levels). In addition, we examined the dynamics of cytosolic Ca2+ and mitochondrial Ca2+ by confocal laser scanning microscopy and confirmed that Ca2+ stores in the endoplasmic reticulum were the primary source of H2O2-induced cytosolic Ca2+ bursts. It is clear that mitochondria have pivotal roles in determining how exogenous oxidative stress affects cell fate. The stress response involves the transfer of Ca2+ signals between organelles, ROS auto-amplification, mitochondrial dysfunction, and a caspase-dependent apoptotic pathway.

Anti-inflammatory and Immuno-modulatory Effects of Capparis spinosa Flower Bud Extract

Aims: Capparis spinosa L. is a plant widely used in traditional medicine for its different purpose including the anti-inflammatory properties. The aim of this study was to evaluate the anti-inflammatory properties of this plant and to define its possible mechanism of action by verifying its effect on the production of some inflammatory mediators. Methodology: The anti-inflammatory activity of Capparis spinosa bud methanolic extract was evaluated in vivo, using paw edema and air pouch inflammation models. In vitro, the ability of the extract to modulate the production of some pro and anti-inflammatory mediators such as TNF-α, IL-1β, IL-8 and IL-10 released from peripheral blood mononuclear cells stimulated by concanavalin A was evaluated. Moreover, the effect of the extract on LTB4 and superoxide anion released from neutrophils was tested. Results: Results showed that the oral administration of 200 and 400 mg/kg of Capparis spinosa methanolic extract reduced significantly carrageenan-induced paw edema. Above 2 h, both doses of the extract exerted a significant (P < 0.001) anti-edematous effect, with 52%-69%. In addition, this extract inhibited the neutrophil migration into the air pouch. The inhibition exerted by 1 mg/pouch of the extract (48.92%) was better than that exerted by indomethacin, used as reference. On the other hand, the extract inhibited significantly the production of TNF-α, IL-1β, LTB4 and superoxide anion generation. At 100 µg/mL, the inhibition values were 21.28%, 38.04%, 20.84% and 71.16%, respectively. In contrast, the extract did not show any significant effect on the release of IL-8 and IL-10. Conclusion: Capparis spinosa bud extract inhibited the inflammatory process by modulating the pro-inflammatory mediator release. Thus this extract can offer a new therapeutic strategy for the treatment of inflammatory disorders.

Treatment with ascorbic acid and alpha-tocopherol modulates oxidative-stress markers in the spinal cord of rats with neuropathic pain

Vitamin E (vit. E) and vitamin C (vit. C) are antioxidants that inhibit nociception. The effect of these vitamins on oxidative-stress markers in the spinal cord of rats with chronic constriction injury (CCI) of the sciatic nerve is unknown. This study investigated the effect of intraperitoneal administration of vit. E (15 mg·kg-1·day-1) and vit. C (30 mg·kg-1·day-1), given alone or in combination, on spinal cord oxidative-stress markers in CCI rats. Adult male Wistar rats weighing 200-250 g were divided equally into the following groups: Naive (rats did not undergo surgical manipulation); Sham (rats in which all surgical procedures involved in CCI were used except the ligature), and CCI (rats in which four ligatures were tied loosely around the right common sciatic nerve), which received injections of vitamins or vehicle (saline containing 1% Tween 80) for 3 or 10 days (n=6/each group). The vitamins prevented the reduction in total thiol content and the increase in superoxide-anion generation that were found in vehicle-treated CCI rats. While nitric-oxide metabolites increased in vehicle-treated CCI rats 3 days after surgery, these metabolites did not show significant changes in vitamin-treated CCI rats. In all rats, total antioxidant capacity and hydrogen-peroxide levels did not change significantly. Lipid hydroperoxides increased 25% only in vehicle-treated CCI rats. These changes may contribute to vit. C- and vit. E-induced antinociception, because scavenging reactive oxygen species seems to help normalize the spinal cord oxidative status altered by pain.

Optimization of subcritical aqueous extraction for polysaccharides from Punica granatum leaves and the in vitro antioxidant activity / 中成药

AIM To optimize the subcritical aqueous extraction for polysaccharides from the leaves of Punica granatum L.and to evaluate the in vitro antioxidant activity.METHODS With reaction pressure,solid-liquid ratio,extraction time and extraction temperature as influencing factors,yield of polysaccharides as an evalution index,the extraction was optimized by Box-Behnken method on the basis of single factor test.Then the scavenging effects of polysaccharides on hydroxyl free radical,superoxide anion and DPPH free radical were detected.RESULTS The optimal conditions were determined to be 5 MPa for reaction pressure,1 ∶ 27 for solid-liquid ratio,11 min for extraction time,and 155 ℃ for extraction temperature,the yield of polysaccharides was 1.809％.There was a dose-effect relationship between scavenging rate and polysaccharides' concentration.0.1 mg/mL Polysaccharides displayed the strongest scavenging effects on hydroxyl free radical,superoxide anion and DPPH free radical with the clearance rates of 57.36％,70.51％ and 58.02％,respectively.CONCLUSION This stable and reliable method can be used for the subcritical aqueous extraction for polysaccharides from P.granatum leaves with obvious in vitro antioxidant activity.

Recent Advances in Fluorescent Probes for Imaging of Superoxide Anion Radical / 分析化学

Superoxide anion radical ( O·-2 ) is the first generated reactive oxygen species ( ROS) and plays essential function in life processes. Normal level of O·-2 as important signaling molecular can regulate redox equilibrium, cellular proliferation and differentiation. However, abnormal level of O·-2 is closely associated with diseases, such as cancer, neurodegenerative diseases and diabetes. Hence it is significant to uncover diseases mechanism by exploring dynamic regulation of O·-2 . Considering the advantages of fluorescence imaging method, the key factor is to develop O·-2 probes with highly selective and sensitive properties for revealing the molecular mechanism of diseases. Recently, with the development of fluorescence microscopy, many fluorescent probes have been constructed and applied for imaging analysis of O·-2 . In this review, we mainly summarized the progress of O·-2 fluorescent probes according to the different probe structure and prospected the development directions of O·-2 probes.

Optimization of subcritical aqueous extraction for polysaccharides from Punica granatum leaves and the in vitro antioxidant activity / 中成药

AIM To optimize the subcritical aqueous extraction for polysaccharides from the leaves of Punica granatum L.and to evaluate the in vitro antioxidant activity.METHODS With reaction pressure,solid-liquid ratio,extraction time and extraction temperature as influencing factors,yield of polysaccharides as an evalution index,the extraction was optimized by Box-Behnken method on the basis of single factor test.Then the scavenging effects of polysaccharides on hydroxyl free radical,superoxide anion and DPPH free radical were detected.RESULTS The optimal conditions were determined to be 5 MPa for reaction pressure,1 ∶ 27 for solid-liquid ratio,11 min for extraction time,and 155 ℃ for extraction temperature,the yield of polysaccharides was 1.809％.There was a dose-effect relationship between scavenging rate and polysaccharides' concentration.0.1 mg/mL Polysaccharides displayed the strongest scavenging effects on hydroxyl free radical,superoxide anion and DPPH free radical with the clearance rates of 57.36％,70.51％ and 58.02％,respectively.CONCLUSION This stable and reliable method can be used for the subcritical aqueous extraction for polysaccharides from P.granatum leaves with obvious in vitro antioxidant activity.

The superoxide anion donor, potassium superoxide, induces pain and inflammation in mice through production of reactive oxygen species and cyclooxygenase-2

It is currently accepted that superoxide anion (O2•−) is an important mediator in pain and inflammation. The role of superoxide anion in pain and inflammation has been mainly determined indirectly by modulating its production and inactivation. Direct evidence using potassium superoxide (KO2), a superoxide anion donor, demonstrated that it induced thermal hyperalgesia, as assessed by the Hargreaves method. However, it remains to be determined whether KO2 is capable of inducing other inflammatory and nociceptive responses attributed to superoxide anion. Therefore, in the present study, we investigated the nociceptive and inflammatory effects of KO2. The KO2-induced inflammatory responses evaluated in mice were: mechanical hyperalgesia (electronic version of von Frey filaments), thermal hyperalgesia (hot plate), edema (caliper rule), myeloperoxidase activity (colorimetric assay), overt pain-like behaviors (flinches, time spent licking and writhing score), leukocyte recruitment, oxidative stress, and cyclooxygenase-2 mRNA expression (quantitative PCR). Administration of KO2 induced mechanical hyperalgesia, thermal hyperalgesia, paw edema, leukocyte recruitment, the writhing response, paw flinching, and paw licking in a dose-dependent manner. KO2 also induced time-dependent cyclooxygenase-2 mRNA expression in the paw skin. The nociceptive, inflammatory, and oxidative stress components of KO2-induced responses were responsive to morphine (analgesic opioid), quercetin (antioxidant flavonoid), and/or celecoxib (anti-inflammatory cyclooxygenase-2 inhibitor) treatment. In conclusion, the well-established superoxide anion donor KO2 is a valuable tool for studying the mechanisms and pharmacological susceptibilities of superoxide anion-triggered nociceptive and inflammatory responses ranging from mechanical and thermal hyperalgesia to overt pain-like behaviors, edema, and leukocyte recruitment.

Effects of enos uncoupling on the endothelial dysfunction of pulmonary artery in rats with decompression sickness / 解放军医学杂志

Objective To explore the effects of unsafe decompression on the endothelial function of pulmonary artery in rat and its possible related mechanism. Methods Sixty male SD rats (260±35g) were randomly divided into two groups (30 each): control group and decompression (DCS) group. Decompression sickness (DCS) model was reproduced by placing the rats in a compression chamber with air pressure of 600kPa for 60min, followed by decompression at a rate of 100kPa/min to normal pressure. The surviving rats in both control and DCS groups were sacrificed and their pulmonary artery was harvested. The endothelium dependent vasodilatation capacity of isolated pulmonary artery was assessed. The expression and uncoupling of endothelial nitric oxide synthetase (eNOS), as well as the nitration level of each kind of protein in the pulmonary artery tissue, were analyzed by Western blotting. The concentration of reactive oxygen species (ROS) in the pulmonary artery was determined with superoxide anion probe dihematoporphyrin ether (DHE) staining. Results Ten of 30 rats in DC group died of unsafe decompression, and the endothelium dependent vasodilatation capacity of excised pulmonary artery in survived rats was found to decline obviously (P0.05), but the ratio of eNOS monomer/dimer increased significantly in DC group than in control group (P<0.05). The tyrosine nitration level of each kind of protein in the pulmonary artery tissues was higher significantly in DC group than that in control group (P<0.05). DHE showed that the generated amount of DCS in pulmonary artery tissues was obviously higher in DC group than in control group (P<0.05). Conclusions Unsafe decompression may lead to uncoupling of eNOS dimers in the endothelium of pulmonary artery. Uncoupled eNOS monomers may inhibit the synthesis of NO, thereby affect the endothelium dependent vasodilatation function. On the other hand, the eNOS monomers may facilitate the anabolism of ONOO-, leading to an increase in tyrosine nitration level of each kind of protein in the pulmonary artery tissues, thereby cause the regulation disorder of cell information system. The eNOS monomers may also increase the production of ROC, there by mediate the peroxide injuries.

Role of ERK1/2 Kinase in the expression of iNOS by NDMA in human neutrophils.

Potential role of ERK1/2 kinase in conjunction with p38 in the regulation of inducible nitric oxide synthase (iNOS) expression and nitric oxide (NO) production, and superoxide anion generation by human neutrophils (PMNs) exposed to N-nitrosodimethylamine (NDMA) was determined. Increased synthesis of NO due to the involvement of iNOS in neutrophils exposed to NDMA was observed. In addition, intensified activation of ERK1/2 and p38 kinases was determined in these cells. Inhibition of kinase regulated by extracellular signals (ERK1/2) pathway, in contrast to p38 pathway, led to an increased production of NO and expression of iNOS in PMNs. Moreover, as a result of inhibition of ERK1/2 pathway, a decreased activation of p38 kinase was observed in neutrophils, while inhibition of p38 kinase did not affect activation of ERK1/2 pathway in these cells. An increased ability to release superoxide anion by the studied PMNs was observed, which decreased after ERK1/2 pathway inhibition. In conclusion, in human neutrophils, ERK1/2 kinase is not directly involved in the regulation of iNOS and NO production induced by NDMA; however, the kinase participates in superoxide anion production in these cells.

Antioxidative Action of Citrus limonum Essential Oil on Skin.

Aims: The purpose of this study was to investigate the action of Citrus limonum essential oil to control free radical-induced lipid peroxidation and preventing tissue damage in skin. Place and Duration of Study: Department of Internal Medicine (University of Roma “Tor Vergata) and A.R.P.A (Aging Research, Prevention and Therapy Association, www.antiaging. it), between January 2010 and June 2011. Methodology: The essential oil was subjected to GC-MS analysis. The superoxide anion scavenging activity of essential oil was evaluated by the enzymatic hypoxanthine/xanthine oxidase system. The same oil diluted in DMSO or grape-seed oil was spread on the face of human volunteers after UV exposition. A sample of skin lipids was collected and the presence of peroxyl radicals was detected based on the measurement of light emitted (chemiluminescence) when the excited carbonyl and singlet oxygen decay to ground state. Results: Our data demonstrate that the lemon essential oil is more active than - tocopherol against *O2 - and peroxide free radical inhibition at 1:100 dilution. A protocol for controlling free radical-induced lipid peroxidation in human skin was thus proposed. Conclusion: The scavenging action of lemon essential oil could have a practical application for treating human skin against oxidative damage.

Prevention and rehabilitation of Shengxue Pills on mice injured by low-dose radiation and their mechanisms / 中草药

Objective: To study the prevention and rehabilitation of Shengxue Pills on liver, spleen, bone marrow cells, peripheral blood lymphocyte of mice injured by low-dose radiation and their mechanisms. Methods: Mice were randomly divided into five groups: control, Shengxue Pills prevention, Shengxue Pills rehabilitation, model 1 (corresponding to rehabilitation), and model 2 (corresponding to prevention) groups. Using the ELISA method to detect the activity of hydroxyl radical, superoxide anion radical, and ornithine decarboxylase (ODC) in liver and spleen cells. The bone marrow cells and peripheral lymphocyte micronucleus rate were observed under high power lens. Results: Compared with control group, the amounts of hydroxyl radical and superoxide anion radical in the two model groups were obviously increased. Compared with the corresponding model groups, the above indexes in prevention group and rehabilitation group were significantly decreased. Compared with the control group, the activity of ODC in mice of the two model groups was obviously enhanced, but bone marrow cells and peripheral lymphocyte micronucleus rate were increased. Compared with the corresponding model groups, the activity of ODC in mice of prevention and rehabilitation groups was weakened, and bone marrow cells and peripheral lymphocyte micronucleus rate were decreased. But each index of spleen cells showed no significant difference. Conclusion: Shengxue Pills could effectively prevent and rehabilitate the damage in liver cells, peripheral blood lymphocytes, and bone marrow cells of mice injured by low-dose radiation.

Superoxide Anion Production by Human Neutrophils Activated by Trichomonas vaginalis

Neutrophils are the predominant inflammatory cells found in vaginal discharges of patients infected with Trichomonas vaginalis. In this study, we examined superoxide anion (O2(.-)) production by neutrophils activated by T. vaginalis. Human neutrophils produced superoxide anions when stimulated with either a lysate of T. vaginalis, its membrane component (MC), or excretory-secretory product (ESP). To assess the role of trichomonad protease in production of superoxide anions by neutrophils, T. vaginalis lysate, ESP, and MC were each pretreated with a protease inhibitor cocktail before incubation with neutrophils. Superoxide anion production was significantly decreased by this treatment. Trichomonad growth was inhibited by preincubation with supernatants of neutrophils incubated for 3 hr with T. vaginalis lysate. Furthermore, myeloperoxidase (MPO) production by neutrophils was stimulated by live trichomonads. These results indicate that the production of superoxide anions and MPO by neutrophils stimulated with T. vaginalis may be a part of defense mechanisms of neutrophils in trichomoniasis.

Niveles de la enzima superóxido dismutasa en niños normotensos, prehipertensos e hipertensos / Levels of superoxide dismutase enzyme in normotensive, prehypertensive and hypertensive children / Niveis da enzima superoxido dismutasa nas crianças normotensas, prehipertensas e hipertensas

La hipertensión arterial en los niños se ha convertido en un problema importante de salud, debido a la frecuencia con que se presenta en la actualidad. Las Especies Reactivas del Oxígeno son uno de los mecanismos involucrados en la fisiopatología de la hipertensión arterial. El objetivo del trabajo fue evaluar la actividad de la enzima superóxido dismutasa y las proteínas totales, por los métodos de Marklund y Lowry en 335 escolares de cuatro escuelas primarias de Santa Clara, según el sexo, color de piel, edad e índice de masa corporal. Para determinar las diferencias significativas entre los niños normotensos, prehipertensos e hipertensos, se utilizaron los tests de Student, Kruskal-Wallis y de Mann-Whinney, todos del software estadístico SPSS 15. Los prehipertensos e hipertensos tuvieron una disminución significativa de la actividad de la superóxido dismutasa. Algo similar ocurrió con los niños hipertensos de sexo masculino, de piel blanca, de 8 años de edad y con los normopesos. La afectación de esta enzima en los niños hipertensos puede favorecer la presencia de Especies Reactivas del Oxígeno y con ello de daños oxidativos.

High blood pressure in children has become a major health problem due to the frequency this disease is appearing today. Reactive oxygen species is one of the mechanisms involved in the pathophysiology of hypertension. The objective of this paper was to evaluate the activity of the superoxide dismutase enzyme and total proteins by the Lowry and Marklund methods in 335 students from four elementary schools in Santa Clara, according to sex, color, age and body mass index. To determine significant differences between normotensive, prehypertensive and hypertensive children, Student's tests, and Kruskal-Wallis and Mann-Whinney tests, which are part of the SPSS 15 statistical package, were used. Superoxide dismutase activity had a significant decrease in prehypertensive and hypertensive students. Something similar happened in male, white children, 8 years of age with high blood pressure, and with the ones of normal weight. When the activity of this enzyme is affected in children with high blood pressure, it can lead to the presence of reactive oxygen species and thus of oxidative damage.

A hipertensão arterial nas crianças tem se transformado em um problema importante de saúde devido à frequência com que atualmente se está apresentando. As Espécies Reativas do Oxigêno são um dos mecanismos envolvidos na fisiopatologia da hipertensão arterial. O objetivo do trabalho foi avaliar a atividade da enzima superóxido dismutase e das proteínas totais, pelos métodos de Marklund e Lowry em 335 crianças de 4 escolas primárias de Santa Clara, segundo o sexo, a cor da pele, a idade e o índice de massa corporal. Para determinar as diferenças significativas entre as crianças normotensas, pré-hipertensas e hipertensas foram utilizados os testes de Student, Kruskal-Wallis e de Mann-Whinney, todos do software estatístico SPSS 15. Os pré-hipertensos e hipertensos tiveram uma diminuição significativa da atividade da superóxido dismutase. Algo semelhante aconteceu com as crianças hipertensas de sexo masculino, de pele branca, de 8 anos de idade e com os normopesos. A afetação desta enzima nas crianças hipertensas pode favorecer a presença de Espécies Reativas do Oxigênio e com isso danos oxidativos.