Clinical validation of a liquid chromatography-tandem mass spectrometry method for the quantification of calcineurin and mTOR inhibitors in dried matrix on paper discs.

Introduction: Advances in liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) have enabled the quantification of immunosuppressants using microsampling techniques. In this context, dried matrix on paper discs (DMPD) could be a useful alternative to conventional venipuncture. Although analytical validation is necessary to establish the suitability of method performance, it is not sufficient to proceed with its implementation into routine clinical practice. Also necessary is that equivalence between sampling methods be demonstrated in a clinical validation study. Objetives: To clinically validate a LC-MS/MS method for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A using DMPD. Methods: According to the recommendations of international guidelines, at least 40 whole blood (WB) and DMPD paired samples for each analyte were collected by skilled technicians and analyzed using LC-MS/MS. Results were evaluated in terms of statistical agreement and bias values at medical decision points. Results: For all analytes, Passing-Bablok regression analysis revealed that confidence intervals (CIs) for slopes and intercepts included 1 and 0, respectively. It also showed that biases at medical decision points were not clinically relevant. No statistically significant differences between DMPD and WB were found using difference plots and agreement analysis. In this regard, CIs for bias estimators included 0, and more than 95% of the results fell within the limits of agreement. Conclusion: The feasibility of the clinical application of simultaneous quantification of tacrolimus, sirolimus, everolimus and cyclosporin A in DMPD was demonstrated. Results showed that this microsampling technique is interchangeable with conventional WB sampling when specimens are collected by trained personnel.

Efeitos do Óleo de Copaíba em Marcadores Periféricos de Estresse Oxidativo em um Modelo de Cor Pulmonale em Ratos / Effects of Copaiba Oil in Peripheral Markers of Oxidative Stress in a Model of Cor Pulmonale in Rats

Resumo Fundamento Até o presente momento, os efeitos sistêmicos do óleo de copaíba jamais foram documentados no Cor pulmonale induzido por monocrotalina. Objetivos Investigar os efeitos do óleo de copaíba nos marcadores periféricos de stress oxidativo em ratos com Cor pulmonale. Métodos Ratos Wistar machos (170±20g, n=7/grupo) foram divididos em quatro grupos: controle (CO), monocrotalina (MCT), óleo de copaíba (O), e monocrotalina + óleo de copaíba (MCT-O). Foi administrada a MCT (60 mg/kg i.p.) e, depois de uma semana, foi iniciado o tratamento com óleo de copaíba (400 mg/kg/day-gavagem-14 dias). Foi realizado o ecocardiograma e, depois disso, foi coletado sangue do tronco para a realização de avaliações de stress oxidativo. Análise estatística: ANOVA de duas vias com teste Student-Newman-Keuls post hoc. P-valores <0,05 foram considerados significativos. Resultados O óleo de copaíba reduziu a resistência vascular pulmonar e a hipertrofia do ventrículo direito (VD) hipertrofia (Índice de Fulton (mg/mg)): MCT-O= 0,39±0,03; MCT= 0,49±0,01), e função sistólica melhorada (fração de encurtamento do VD, %) no grupo MCT-O (17,8±8,2) em comparação com o grupo de MCT (9,4±3,1; p<0,05). Além disso, no grupo MCT-O, espécies reativas do oxigênio e os níveis de carbonila foram reduzidos, e os parâmetros antioxidantes aumentaram no sangue periférico (p <0,05). Conclusões Os resultados deste estudo sugerem que o óleo de copaíba tem um efeito antioxidante sistêmico interessante, que se reflete na melhoria da função e na morfometria do VD nesse modelo de Cor pulmonale . A atenuação do Cor pulmonale promovida pelo óleo de copaíba coincidiu com uma redução no stress oxidativo sistêmico.

Abstract Background To date, copaiba oil's systemic effects have never documented in Cor pulmonale induced by monocrotaline. Objectives To investigate copaiba oil's effects in peripheral markers of oxidative stress in rats with Cor pulmonale. Methods Male Wistar rats (170±20g, n=7/group) were divided into four groups: control (CO), monocrotaline (MCT), copaiba oil (O), and monocrotaline+copaiba oil (MCT-O). MCT (60 mg/kg i.p.) was administered, and after one week, treatment with copaiba oil (400 mg/kg/day-gavage-14 days) was begun. Echocardiography was performed and, later, trunk blood collection was performed for oxidative stress evaluations. Statistical analysis: two-way ANOVA with Student-Newman-Keuls post-hoc test. P values<0.05 were considered significant. Results Copaiba oil reduced pulmonary vascular resistance and right ventricle (RV) hypertrophy (Fulton index (mg/mg): MCT-O=0.39±0.03; MCT=0.49±0.01), and improved RV systolic function (RV shortening fraction, %) in the MCT-O group (17.8±8.2) as compared to the MCT group (9.4±3.1; p<0.05). Moreover, in the MCT-O group, reactive oxygen species and carbonyl levels were reduced, and antioxidant parameters were increased in the peripheral blood (p<0.05). Conclusions: Our results suggest that copaiba oil has an interesting systemic antioxidant effect, which is reflected in the improvements in function and RV morphometry in this Cor pulmonale model. Cor pulmonale attenuation promoted by copaiba oil coincided with a reduction in systemic oxidative stress.

Validation of a simple liquid chromatography coupled to tandem mass spectrometry method for the simultaneous determination of tacrolimus, sirolimus, everolimus and cyclosporin A in dried matrix on paper discs.

INTRODUCTION: Due to its high specificity and sensitivity, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the gold standard method for immunosuppressant quantification in therapeutic drug monitoring. In this context, dried blood spots (DBS) have become a promising strategy as a sample collection procedure. Although the advantages of DBS over venipuncture are well known, this approach has limitations that strongly influence the acceptance of analytical results. Among them, the most important is hematocrit (Ht). The easiest way of overcoming this problem is by analyzing complete spots. In this strategy, called dried matrix on paper discs (DMPD), blood is volumetrically applied on pre-punched discs. OBJECTIVES: To validate an LC-MS/MS method for the quantification of tacrolimus, sirolimus, everolimus and cyclosporin A using DMPD. METHODS: The procedure was validated according to international guidelines using a commercial kit. The following performance parameters were evaluated: selectivity, carryover, linearity, accuracy, precision, lower limit of quantitation, relative recovery, commutability and stability. In addition, a method comparison study was performed to evaluate the clinical influence of Ht on the results. RESULTS: All performance parameters were within acceptance criteria and, hence, it was determined that the validated method is fit for the intended purpose. Likewise, calculated bias values on medical decision levels showed that there was no clinical influence of Ht on the results. CONCLUSION: Unlike other similar methodologies that have been published, here, a simple method has been fully validated. This is the first LC-MS/MS methodology adapting a commercial kit to use DMPD as a sampling strategy.

Effects of Copaiba Oil in Peripheral Markers of Oxidative Stress in a Model of Cor Pulmonale in Rats. / Efeitos do Óleo de Copaíba em Marcadores Periféricos de Estresse Oxidativo em um Modelo de Cor Pulmonale em Ratos.

BACKGROUND: To date, copaiba oil's systemic effects have never documented in Cor pulmonale induced by monocrotaline. OBJECTIVES: To investigate copaiba oil's effects in peripheral markers of oxidative stress in rats with Cor pulmonale. METHODS: Male Wistar rats (170±20g, n=7/group) were divided into four groups: control (CO), monocrotaline (MCT), copaiba oil (O), and monocrotaline+copaiba oil (MCT-O). MCT (60 mg/kg i.p.) was administered, and after one week, treatment with copaiba oil (400 mg/kg/day-gavage-14 days) was begun. Echocardiography was performed and, later, trunk blood collection was performed for oxidative stress evaluations. Statistical analysis: two-way ANOVA with Student-Newman-Keuls post-hoc test. P values<0.05 were considered significant. RESULTS: Copaiba oil reduced pulmonary vascular resistance and right ventricle (RV) hypertrophy (Fulton index (mg/mg): MCT-O=0.39±0.03; MCT=0.49±0.01), and improved RV systolic function (RV shortening fraction, %) in the MCT-O group (17.8±8.2) as compared to the MCT group (9.4±3.1; p<0.05). Moreover, in the MCT-O group, reactive oxygen species and carbonyl levels were reduced, and antioxidant parameters were increased in the peripheral blood (p<0.05). Conclusions: Our results suggest that copaiba oil has an interesting systemic antioxidant effect, which is reflected in the improvements in function and RV morphometry in this Cor pulmonale model. Cor pulmonale attenuation promoted by copaiba oil coincided with a reduction in systemic oxidative stress.

FUNDAMENTO: Até o presente momento, os efeitos sistêmicos do óleo de copaíba jamais foram documentados no Cor pulmonale induzido por monocrotalina. OBJETIVOS: Investigar os efeitos do óleo de copaíba nos marcadores periféricos de stress oxidativo em ratos com Cor pulmonale. MÉTODOS: Ratos Wistar machos (170±20g, n=7/grupo) foram divididos em quatro grupos: controle (CO), monocrotalina (MCT), óleo de copaíba (O), e monocrotalina + óleo de copaíba (MCT-O). Foi administrada a MCT (60 mg/kg i.p.) e, depois de uma semana, foi iniciado o tratamento com óleo de copaíba (400 mg/kg/day-gavagem-14 dias). Foi realizado o ecocardiograma e, depois disso, foi coletado sangue do tronco para a realização de avaliações de stress oxidativo. Análise estatística: ANOVA de duas vias com teste Student-Newman-Keuls post hoc. P-valores <0,05 foram considerados significativos. RESULTADOS: O óleo de copaíba reduziu a resistência vascular pulmonar e a hipertrofia do ventrículo direito (VD) hipertrofia (Índice de Fulton (mg/mg)): MCT-O= 0,39±0,03; MCT= 0,49±0,01), e função sistólica melhorada (fração de encurtamento do VD, %) no grupo MCT-O (17,8±8,2) em comparação com o grupo de MCT (9,4±3,1; p<0,05). Além disso, no grupo MCT-O, espécies reativas do oxigênio e os níveis de carbonila foram reduzidos, e os parâmetros antioxidantes aumentaram no sangue periférico (p <0,05). CONCLUSÕES: Os resultados deste estudo sugerem que o óleo de copaíba tem um efeito antioxidante sistêmico interessante, que se reflete na melhoria da função e na morfometria do VD nesse modelo de Cor pulmonale . A atenuação do Cor pulmonale promovida pelo óleo de copaíba coincidiu com uma redução no stress oxidativo sistêmico.

Correction: Impact of combined exercise training on the development of cardiometabolic and neuroimmune complications induced by fructose consumption in hypertensive rats.

[This corrects the article DOI: 10.1371/journal.pone.0233785.].

Impact of combined exercise training on the development of cardiometabolic and neuroimmune complications induced by fructose consumption in hypertensive rats.

This study evaluated the impact of combined exercise training on the development of cardiovascular and neuroimmune complications induced by fructose consumption (10% in the drinking water) in hypertensive rats (SHR). After weaning, SHR were divided into 3 groups: SHR (H), SHR+fructose (HF) and SHR+fructose+combined exercise training (treadmill+ladder, 40-60% of maximum capacity) (HFTC). Metabolic, hemodynamic, autonomic, inflammatory and oxidative stress parameters were evaluated in the subgroups (n = 6 group/time) at 7, 15, 30 and 60 days of protocol. Fructose consumption (H vs. HF groups) decreased spontaneous baroreflex sensitivity and total variance of pulse interval at day 7 (7 to 60); increased IL-6 and TNFα in the heart (at day 15, 30 and 60) and NADPH oxidase activity and cardiac lipoperoxidation (LPO) (day 60); increased white adipose tissue weight, reduced insulin sensitivity and increased triglycerides (day 60); induced an additional increase in mean arterial pressure (MAP) (days 30 and 60). Combined exercise training prevented such dysfunctions and sustained increased cardiac IL-10 (day 7) and glutathione redox balance (GSH/GSSG) for the entire protocol. In conclusion, combined exercise training performed simultaneously with exacerbated fructose consumption prevented early cardiovascular autonomic dysfunction, probably trigging positive changes in inflammation and oxidative stress, resulting in a better cardiometabolic profile in rats genetically predisposed to hypertension.

Ovarian status modulates cardiovascular autonomic control and oxidative stress in target organs.

Studies have presented conflicting findings regarding the association between both fluctuation and deprivation of ovarian hormones and cardiovascular autonomic modulation and oxidative stress and their potential impact on resting arterial pressure (AP) and cardiovascular risk. This study aimed to assess cardiovascular autonomic modulation, baroreflex sensitivity (BRS), and oxidative stress in male rats (M) and in female rats during ovulatory (FOV) and non-ovulatory phases (FNOV) of the estrous cycle and after deprivation of ovarian hormones (FO). Direct AP was recorded, and BRS was assessed by using increasing doses of phenylephrine and sodium nitroprusside. AP and heart rate variability were assessed by spectral analysis. Oxidative stress profile was evaluated in cardiac, renal, and muscle tissues. In females, the ovulatory phase and ovarian hormone deprivation induced an increase in AP (FOV and FO ~ 9 mmHg) when compared to the non-ovulatory phase. Ovariectomy promoted increased cardiac sympathovagal balance (~ 17-37%) when compared to other groups. Both FOV and FO groups presented impaired BRS, associated with higher AP variability. In general, antioxidant capacity was higher in the FNOV than in the M group. Ovarian hormone deprivation induced a decrease in catalase activity in cardiac and renal tissues and an increase in lipid peroxidation in all tissues analyzed. Positive correlations (p < 0.05) were found between vascular sympathetic modulation and lipid peroxidation in cardiac (r = 0.60), renal (r = 0.60), and muscle (r = 0.57) tissues. In conclusion, both oscillation and deprivation of ovarian hormones play an important role in cardiovascular autonomic control and oxidative stress profile in target organs, which is reflected in AP changes.

Investigación traslacional en microbioma (InTraMic): un área de interés común y creciente en el IMTIB, Hospital Italiano e Instituto Universitario / Translational research in microbiome (InTraMic): an area of common and growing interest at IMTIB, Hospital Italiano and Instituto Universitario

Se estima que aproximadamente 100 trillones de microorganismos (incluidos bacterias, virus y hongos) residen en el intestino humano adulto y que el total del material genético del microbioma es 100 veces superior al del genoma humano. Esta comunidad, conocida como microbioma se adquiere al momento del nacimiento a través de la flora comensal de la piel, vagina y heces de la madre y se mantiene relativamente estable a partir de los dos años desempeñando un papel crítico tanto en el estado de salud como en la enfermedad. El desarrollo de nuevas tecnologías, como los secuenciadores de próxima generación (NGS), permiten actualmente realizar un estudio mucho más preciso de ella que en décadas pasadas cuando se limitaba a su cultivo. Si bien esto ha llevado a un crecimiento exponencial en las publicaciones, los datos sobre las poblaciones Latinoamérica son casi inexistentes. La investigación traslacional en microbioma (InTraMic) es una de las líneas que se desarrollan en el Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). Esta se inició en 2018 con la línea de cáncer colorrectal (CCR) en una colaboración con el Colorectal Cancer Research Group del Leeds Institute of Medical Research en el proyecto Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents. A fines de 2019 se cumplió el objetivo de comprobar la factibilidad de la recolección, envío y análisis de muestras de MBF en 5 continentes, incluyendo muestras provenientes de la Argentina, Chile, India y Vietnam. Luego de haber participado de capacitaciones en Inglaterra, se ha cumplido con el objetivo de la etapa piloto, logrando efectivizar la recolección, envío y análisis metagenómico a partir de la secuenciación de la región V4 del ARNr 16S. En 2019, la línea de enfermedad de hígado graso no alcohólico se sumó a la InTraMic iniciando una caracterización piloto en el marco de una colaboración con el laboratorio Novartis. Los resultados de ese estudio, así como el de cáncer colorrectal, están siendo enviados a publicación. En 2020, con la incorporación de la línea de trasplante alogénico de células progenitoras hematopoyéticas, fue presentado un proyecto para un subsidio del CONICET que ha superado la primera etapa de evaluación. En el presente artículo se brinda una actualización sobre la caracterización taxonómica de microbioma y se describen las líneas de investigación en curso. (AU)

It is estimated that approximately 100 trillion microorganisms (including bacteria, viruses, and fungi) reside in the adult human intestine, and that the total genetic material of the microbiome is 100 times greater than that of the human genome. This community, known as the microbiome, is acquired at birth through the commensal flora of the mother's skin, vagina, and feces and remains relatively stable after two years, playing a critical role in both the state of health and in disease. The development of new technologies, such as next-generation sequencers (NGS), currently allow for a much more precise study of it than in past decades when it was limited to cultivation. Although this has led to exponential growth in publications, data on Latin American populations is almost non-existent. Translational research in microbiome (InTraMic) is one of the lines developed at the Instituto de Medicina Traslacional e Ingeniería Biomédica (IMTIB). This started in 2018 with the Colorectal Cancer Line (CRC) in a collaboration with the Colorectal Cancer Research Group of the Leeds Institute of Medical Research in the project "Large bowel microbiome disease network: Creation of a proof of principle exemplar in colorectal cancer across three continents". At the end of 2019, the objective of verifying the feasibility of collecting, sending and analyzing MBF samples on 5 continents, including samples from Argentina, Chile, India and Vietnam, was met. After having participated in training in England, the objective of the pilot stage has been met, achieving the collection, delivery and metagenomic analysis from the sequencing of the V4 region of the 16S rRNA. In 2019, the non-alcoholic fatty liver disease line joined InTraMic, initiating a pilot characterization in the framework of a collaboration with the Novartis laboratory. The results of that study, as well as that of colorectal cancer, are being published. In 2020, with the incorporation of the allogeneic hematopoietic stem cell transplantation line, a project was presented for a grant from the CONICET that has passed the first stage of evaluation. This article provides an update on the taxonomic characterization of the microbiome and describes the lines of ongoing research. (AU)

Urban air pollution induces redox imbalance and epithelium hyperplasia in mice cornea.

The aim of the study was to evaluate the time course of the effects of urban air pollutants on the ocular surface, focusing on the morphological changes, the redox balance, and the inflammatory response of the cornea. 8-week-old mice were exposed to urban or filtered air (UA-group and FA-group, respectively) in exposure chambers for 1, 2, 4, and 12 weeks. After each time, the eyes were enucleated and the corneas were isolated for biochemical analysis. UA-group corneas exhibited a continuous increase in NADPH oxidase-4 levels throughout the exposure time, suggesting an increased production of reactive oxygen species (ROS). After 1 week, an early adaptive response to ROS was observed as an increase in antioxidant enzymes. After 4 weeks, the enzymatic antioxidants were decreased, meanwhile an increase of the glutathione was shown, as a later compensatory antioxidant response. However, redox imbalance took place, evidenced by the increased oxidized proteins, which persisted up to 12 weeks. At this time point, corneal epithelium hyperplasia was also observed. The inflammatory response was modulated by the increase in IL-10 levels after 1 week, which early regulates the release of TNF-α and IL-6. These results suggest that air pollution alters the ocular surface, supported by the observed cellular hyperplasia. The redox imbalance and the inflammatory response modulated by IL-10 play a key role in the response triggered by air pollutants on the cornea. Taking into account this time course study, the ocular surface should also be considered as a relevant target of urban air pollutants.

Imbalance between nitric oxide and superoxide anion induced by uncoupled nitric oxide synthase contributes to human melanoma development.

Melanoma is the most aggressive type of cutaneous tumors due to its metastatic potential and high mortality. Increased levels of reactive oxygen species, including superoxide anion (O2-), and the consequent installation of a pro-oxidant environment are associated with melanoma development. The enzyme nitric oxide synthase (NOS), responsible for the production of nitric oxide (NO), when uncoupled is as a source of O2-, for example by the absence of its cofactor tetrahydrobiopterin (BH4). Western blot analysis showed increased expression of endothelial and inducible NOS in human melanoma cells, altering the stoichiometry between NOS levels and BH4 concentration and together with decreased BH4:BH2 ratio are contributing to NOS uncoupling. The treatment of melanoma cells with exogenous BH4 increased NO concentration and decreased O2- levels, leading to NOS coupling, which in turn reduced cell viability, cell proliferation and the ability of melanoma cells to form melanoma spheroids. Moreover, BH4 level restoration rendered melanoma cells more sensitive to apoptosis, demonstrating the role of dysfunctional NOS in melanoma genesis.

Exercise training initiated at old stage of lifespan attenuates aging-and ovariectomy-induced cardiac and renal oxidative stress: Role of baroreflex.

BACKGROUND: The association of aging and menopause is a potent risk factor for cardiometabolic disease. We studied the impact of aerobic exercise training (ET) initiated in the old stage of lifespan in hemodynamics, metabolic, autonomic and oxidative stress. METHODS: Aged (18 months old) female Wistar rats were divided into: ovariectomized and untrained (AG-OVX), and ovariectomized and trained (AG-OVXt, ET for 8 weeks). Intact aged (AG) and young female rats (3 months old; Y) were also studied. Blood pressure and metabolic parameters were measured. Baroreflex sensitivity (BRS) was studied by bradycardic (BR) and tachycardic (TR) responses to vasoactive drugs. Cardiac and renal lipid peroxidation (LPO), catalase (CAT), superoxide dismutase (SOD) and gluthatione peroxidase (GPx), and gluthatione redox balance (GSH/GSSG) were analyzed. RESULTS: AG-OVXt group increased aerobic performance in 35%, decreased adipose tissue and triglycerides in 36% and 27%, respectively, and improved insulin tolerance in 50% in comparison to AG-OVX. AG-OVX presented hypertensive levels of blood pressure (systolic: 155 ±â€¯5, diastolic: 111 ±â€¯3 mmHg). In contrast, AG-OVXt presented blood pressure values similar to Y rats (systolic: 129 ±â€¯3, diastolic: 112 ±â€¯3 mmHg). TR and BR were reduced by 70% and 46%, respectively, in AG-OVX vs. Y. Once more, AG-OVXt presented similar results to Y. ET decreased LPO in the heart and kidney. In the latter, renal CAT and SOD were corrected by ET, while cardiac redox balance was partially recovered. Improved BRS was correlated with improved oxidative stress markers. CONCLUSIONS: Even when initiated after aging and ovariectomy deleterious effects, ET is able to normalize BRS and highly improve cardiac and renal oxidative stress.

Combined Aerobic and Resistance Exercise Training Improve Hypertension Associated With Menopause.

The prevalence of hypertension sharply increases in menopausal women. Recent studies have demonstrated that aerobic or resistance training may help control hypertension. In this study, we report that combining aerobic and resistance training may provide an effective therapeutic approach for hypertension control, attenuating inflammation and oxidative stress in ovariectomized rats. Female Wistar and spontaneous hypertensive rats (SHR) were distributed into four groups: sedentary control (C), sedentary hypertensive (HR), sedentary hypertensive ovariectomized (HR-O), and combined trained hypertensive ovariectomized (T-HR-O). Combined exercise training was performed on a motor treadmill (aerobic training) and on a ladder adapted to rats (resistance training), in alternate days for 8 weeks. Direct arterial pressure was recorded and oxidative stress and inflammation were evaluated in cardiac and renal tissue. Ovariectomy increases increased mean arterial blood pressure, sympathetic modulation, and oxidative stress in SHR. Combining aerobic and resistance training reduced mean arterial blood pressure (12% vs. HR-O), heart rate (8% vs. HR-O), vascular sympathetic modulation (40% vs. HR-O), and improved baroreflex sensitivity. Combined training reduced cardiac inflammation (TNF and IL-6) and cardiac and renal lipoperoxidation (59% and 57%, respectively vs. HR-O). It also enhanced cardiac (71%) and renal (76%) total antioxidant capacity when compared to HR-O group. In conclusion, combining aerobic and resistance training improves mean arterial blood pressure, cardiovascular autonomic control, preventing cardiac and renal oxidative stress and inflammation in an experimental hypertension model with surgical menopause induced with ovariectomy.

Copaiba Oil Attenuates Right Ventricular Remodeling by Decreasing Myocardial Apoptotic Signaling in Monocrotaline-Induced Rats.

There is an increase in oxidative stress and apoptosis signaling during the transition from hypertrophy to right ventricular (RV) failure caused by pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT). In this study, it was evaluated the action of copaiba oil on the modulation of proteins involved in RV apoptosis signaling in rats with PAH. Male Wistar rats (±170 g, n = 7/group) were divided into 4 groups: control, MCT, copaiba oil, and MCT + copaiba oil. PAH was induced by MCT (60 mg/kg intraperitoneally) and, 7 days later, treatment with copaiba oil (400 mg/kg by gavage) was given for 14 days. Echocardiographic and hemodynamic measurements were performed, and the RV was collected for morphometric evaluations, oxidative stress, apoptosis, and cell survival signaling, and eNOS protein expression. Copaiba oil reduced RV hypertrophy (24%), improved RV systolic function, and reduced RV end-diastolic pressure, increased total sulfhydryl levels and eNOS protein expression, reduced lipid and protein oxidation, and the expression of proteins involved in apoptosis signaling in the RV of MCT + copaiba oil as compared to MCT group. In conclusion, copaiba oil reduced oxidative stress, and apoptosis signaling in RV of rats with PAH, which may be associated with an improvement in cardiac function caused by this compound.

Combined aerobic and resistance exercise training attenuates cardiac dysfunctions in a model of diabetes and menopause.

The study aimed at evaluating the effects of combined aerobic and resistance exercise training on cardiac morphometry and function, oxidative stress and inflammatory parameters in diabetic ovariectomized rats. For this, female Wistar rats (10 weeks-old) were divided into 4 groups (n = 8): euglycemic (E), diabetic (streptozotocin, 50 mg/kg, iv) (D), diabetic ovariectomized (DO) and trained diabetic ovariectomized (TDO). The combined exercise training was performed on a treadmill and in a ladder adapted to rats (8 weeks, at 40-60% of maximal capacity). The left ventricle (LV) morphometry and function were evaluated by echocardiography. Oxidative stress and inflammatory markers were measured on ventricles tissue. The sedentary diabetic animals (D and DO) showed impaired systolic and diastolic functions, as well as increased cardiac overload, evaluated by myocardial performance index (MPI- D: 0.32 ± 0.05; DO: 0.39 ± 0.13 vs. E: 0.25 ± 0.07), in relation to E group. Systolic and MPI dysfunctions were exacerbated in DO when compared to D group. The DO group presented higher protein oxidation and TNF-α/IL-10 ratio than D groups. Glutathione redox ratio (GSH/GSSG) and IL-10 were decreased in both D and DO groups when compared to E group. Exercise training improved exercise capacity, systolic and diastolic functions and MPI (0.18±0.11). The TDO group showed reduced protein oxidation and TNF-α/IL-10 ratio and increased GSH/GSSG and IL-10 in relation to the DO group. These results showed that combined exercise training was able to attenuate the cardiac dysfunctions, probably by reducing inflammation and oxidative stress in an experimental model of diabetes and menopause.

Volcanic ash from Puyehue-Cordón Caulle Volcanic Complex and Calbuco promote a differential response of pro-inflammatory and oxidative stress mediators on human conjunctival epithelial cells.

Volcanic ash could pose a hazard to the ocular surface as it is constantly exposed to environmental particles. We exposed conjunctival cells to Puyehue-Cordón Caulle volcanic complex (PCCVC) or Calbuco ash particles and evaluated proliferation, viability, apoptosis, MUC1 expression, pro-inflammatory cytokines, and oxidative stress markers. Ash particles from these volcanoes vary in size, composition, and morphology. Our results demonstrate that PCCVC but not Calbuco ash particles induce cytotoxicity on human conjunctival epithelial cells viewed as a decrease in cell proliferation and the transmembrane mucin MUC1 expression; a pro-inflammatory response mediated by IL-6 and IL-8; and an imbalance of the redox environment leading to protein oxidative damage. This is the first in vitro study that assesses the biological effect of volcanic ash particles on human conjunctival epithelial cells and the involvement of inflammatory mediators and oxidative stress as the mechanisms of damage. Our results could provide a better understanding of the ocular symptoms manifested by people living near volcanic areas.

Baroreflex Impairment Precedes Cardiometabolic Dysfunction in an Experimental Model of Metabolic Syndrome: Role of Inflammation and Oxidative Stress.

This study analyzes whether autonomic dysfunction precedes cardiometabolic alterations in spontaneously hypertensive rats (SHR) with fructose overload. Animals were randomly distributed into three groups: control, hypertensive and hypertensive with fructose overload. Fructose overload (100 g/L) was initiated at 30 days old, and the animals (n = 6/group/time) were evaluated after 7, 15, 30 and 60 days of fructose consumption. Fructose consumption reduced baroreflex sensitivity by day 7, and still induced a progressive reduction in baroreflex sensitivity over the time. Fructose consumption also increased TNFα and IL-6 levels in the adipose tissue and IL-1ß levels in the spleen at days 15 and 30. Fructose consumption also reduced plasmatic nitrites (day 15 and 30) and superoxide dismutase activity (day 15 and 60), but increased hydrogen peroxide (day 30 and 60), lipid peroxidation and protein oxidation (day 60). Fructose consumption increased arterial pressure at day 30 (8%) and 60 (11%). Fructose consumption also induced a late insulin resistance at day 60, but did not affect glucose levels. In conclusion, the results show that baroreflex sensitivity impairment precedes inflammatory and oxidative stress disorders, probably by inducing hemodynamic and metabolic dysfunctions observed in metabolic syndrome.

Diesel exhaust particles (DEP) induce an early redox imbalance followed by an IL-6 mediated inflammatory response on human conjunctival epithelial cells.

The aim of this study was to evaluate the time course of oxidative stress markers and inflammatory mediators in human conjunctival epithelial cells (IOBA-NHC) exposed to diesel exhaust particles (DEP) for 1, 3, and 24 h. Reactive oxygen species (ROS) production, lipid and protein oxidation, Nrf2 pathway activation, enzymatic antioxidants, glutathione (GSH) levels and synthesis, as well as cytokine release and cell proliferation were analyzed. Cells exposed to DEP showed an increase in ROS at all time points. The induction of NADPH oxidase-4 appeared later than mitochondrial superoxide anion production, when the cell also underwent a proinflammatory response mediated by IL-6. DEP exposure triggered the activation of Nrf2 in IOBA-NHC, as a strategy for increasing cellular antioxidant capacity. Antioxidant enzyme activities were significantly increased at early stages except for glutathione reductase (GR) that showed a significant decrease after a 3-h-incubation. GSH levels were found increased after 1 and 3 h of incubation with DEP, despite the increase in its consumption by the antioxidant enzymes as it works as a cofactor. GSH recycling and the de novo synthesis were responsible for the maintenance of its content at these time points, respectively. After 24 h, the decrease in GR and glutamate cysteine ligase as wells as the enhanced activity of glutathione peroxidase and glutathione S-transferase produced a depletion in the GSH pool. Lipid-peroxidation was found increased in cells exposed to DEP after 1-h-incubation, whereas protein oxidation was found increased in cells exposed to DEP after a 3-h-incubation that persisted after a longer exposure. Furthermore, DEP lead IOBA-NHC cells to hyperplasia after 1 and 3 h of incubation, but a decrease in cell proliferation was found after longer exposure. ROS production seems to be an earlier event triggered by DEP on IOBA-NHC, comparing to the proinflammatory response mediated by IL-6. Despite the fact that under short periods of exposure to DEP lipids and then proteins are targets of oxidative damage, the viability of the cells is not affected at early stages, since cell hyperplasia was detected as compensatory mechanism. Although after 24 h Nrf2 pathway is still enhanced, the epithelial cell capacity to maintain redox balance is exceeded. The antioxidant enzymes activation and the depleted GSH pool are not capable of counteracting the increased ROS production, leading to oxidative damage.

Hypertension induces additional cardiometabolic impairments and attenuates aerobic exercise training adaptations in fructose-fed ovariectomized rats.

We tested whether hypertension favors the development of additional cardiometabolic changes in fructose-fed ovariectomized rats and how it affects aerobic exercise training (ET) effects. All rats received fructose in drinking water (10%) beginning at weaning, were ovariectomized at 10 weeks of age and divided into the normotensive sedentary (NFOS) and trained (NFOT) and hypertensive sedentary (HFOS) and trained (HFOT) groups. ET was performed on a treadmill. Arterial pressure (AP) was directly recorded; heart rate and AP variabilities were analyzed. Lipoperoxidation (LPO) and antioxidant enzyme levels were measured in the left ventricle. In addition to increased AP levels, when compared with the NFOS group, the hypertensive groups had resting tachycardia, a reduction of 29% in the pulse interval variance (VAR-PI), 19% in RMSSD (root mean square of successive differences, a cardiac parasympathetic index) and 53% in the α-index (spontaneous baroreflex), while the systolic AP variance (VAR-SAP) and its low-frequency band (LF-SAP) were sharply increased. ET did not alter AP levels. Even in the presence of hypertension, ET induced resting bradycardia, decreases of 33% in VAR-SAP and 49% in LF-SAP, and an increase of more than 60% in VAR-PI and the α-index. However, some of these parameters were still impaired relative to those of normotensive rats. LPO was reduced and catalase was increased in both trained groups, with no difference between the normotensive and hypertensive groups. Negative correlations were obtained between LPO and RMSSD (r=-0.60, P<0.05) and α-index (r=-0.63, P<0.05). In conclusion, hypertension augmented the dysfunctions in fructose-fed ovariectomized rats and attenuated metabolic aerobic ET benefits. These changes may be related to cardiovascular autonomic and oxidative stress alterations.

Cholinergic Stimulation Improves Oxidative Stress and Inflammation in Experimental Myocardial Infarction.

We previously reported that cholinergic stimulation with pyridostigmine (PY) induces anti-inflammatory cell recruitment soon after myocardial infarction (MI). In this study, we evaluated the anti-inflammatory effects of PY during the proliferative phase of cardiac repair by analyzing the infiltration of macrophages, Treg lymphocytes, oxidative stress and inflammatory cytokines. Wistar rats underwent control sham surgery or ligation of the left coronary artery and were randomly allocated to remain untreated (untreated infarcted group, I) or to receive PY (30 mg·kg(-1)·day(-1)) in the supplied water (infarcted treated group, I + PY). Blood pressure and heart rate variability were registered at day 5 post-MI. The animals were euthanized 7 days after thoracotomy, when the hearts were removed and processed for immunohistochemistry (CD68, CD206, FOXP3), cytokines (IL-1ß, IL-6, IL-10, TNF-α) and oxidative stress (superoxide dismutase, catalase, glutathione peroxidase, lipidic and protein peroxidation). PY treatment increased parasympathetic modulation, M2 macrophages and the anti-oxidant enzyme activity but reduced protein oxidation (carbonyls) and the concentration of IL-1ß, IL-6, TNF-α and IL-10. Cholinergic stimulation induces parasympathetic neuro-immune modulation and anti-inflammatory cell enrollment as well as prevents oxidative stress and cytokine production after MI.

llex paraguariensis decreases oxidative stress in bone and mitigates the damage in rats during perimenopause.

During perimenopause, oxidative stress increases, which may result in disruption of bone turnover, and consequently in osteoporosis. The use of antioxidants may be an effective nutritional approach to reducing osteoporosis in this period of life. Mate tea (MT) (Ilex paraguariensis), a typical and inexpensive beverage consumed in the Brazilian south-east, Argentina and Uruguay, increases antioxidant defense. Our hypothesis was that MT would decrease oxidative stress and mitigate bone deterioration. To test this, we analyzed oxidative stress markers of bone turnover, and local and systemic markers of bone metabolism of rats during natural perimenopause. Female Wistar rats (aged 16months) in proven perimenopause period received 20mg/kgBW/day of mate tea, by gavage (PM+MT Group, n=10) or water (PM Group, n=10). Female rats aged 4months (AD Group, n=10) received water. The treatment period was four weeks. MT minimized the deterioration of rat microarchitecture, characterized by increase in the bone trabecular area, number of osteocytes and areal bone mineral density. These results were accompanied by a lower level of malondialdehyde, an oxidative stress marker, in femoral tissue homogenate. Plasmatic tartrate-resistant acid phosphatase, a typical osteoclastic function marker, decreases after treatment, indicating a decrease in osteoclastic function. MT also modified the immunostaining pattern of bone metabolism markers, decreasing the receptor activator of nuclear factor kappa-B ligant (RANKL), superoxide dismutase isoform 2 (SOD2) and increasing osteoprotegerin (OPG), a decoy receptor for the RANKL, which positively modulates bone mass. These results suggested MT was capable of decreasing bone resorption by inhibiting the osteoclastogenesis in a RANKL-dependent signaling pathway activated by oxidative stress. Taken together, the results indicated that MT minimized bone loss in perimenopause and this effect is at least partly due to the decrease in oxidative stress, confirming our hypothesis.