Genotoxicity, cytotoxicity and chemical profile from Inga laurina (Fabaceae).

This study aimed to evaluate the cytotoxicity and genotoxicity from Inga laurina leaves extracts and fractions and obtain their chemical profile. The chemical profile of the crude extract from I. laurina leaves and its fractions was investigated through 1H NMR, RP-HPLC-PDA by co-injection with authentic standards and HPLC-MS. The quinone reductase induction as a biomarker for cancer chemoprevention was evaluated in murine hepatocellular carcinoma line, whereas the cytotoxicity was evaluated by sulforhodamine B assay (SRB) using HepG2 cell line and genotoxicity was evaluated by comet assay. The phytochemical analysis of the leaves crude extract and its fractions showed the presence of 2-hydroxyethyl-dodecanoate and the phenolic compounds: gallic acid, methyl gallate, p-coumaric acid, cinnamic acid, myricetin-3-O-(2″-O-galoyl)-α-rhamnopyranoside, proanthocyanidin A-2 and myricetrin. All the fractions tested were not considered cytotoxic against the selected human cancer cell lines, they did not cause genotoxic in some concentrations damage and induced the enzyme quinone reductase.

Oxidative status and intestinal health of gilthead sea bream (Sparus aurata) juveniles fed diets with different ARA/EPA/DHA ratios.

The present work assessed the effects of dietary ratios of essential fatty acids, arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic acid (DHA), on liver and intestine oxidative status, intestinal histomorphology and gut microbiota of gilthead sea bream. Four isoproteic and isolipidic plant-based diets were formulated containing a vegetable oil blend as the main lipid source. Diets were supplemented with ARA/EPA/DHA levels (%DM) equivalent to: 2%:0.2%:0.1% (Diet A); 1.0%:0.4%:0.4% (Diet B); 0%:0.6%:0.6% (Diet C); 0%:0.3%:1.5% (Diet D) and tested in triplicate groups for 56 days. Lipid peroxidation was higher in fish fed diets C and D while no differences were reported between diets regarding total, oxidized, and reduced glutathione, and oxidative stress index. Glutathione reductase was higher in fish fed diet A than diets C and D. No histological alterations were observed in the distal intestine. Lower microbiota diversity was observed in intestinal mucosa of fish fed diet C than A, while diets C and D enabled the proliferation of health-promoting bacteria from Bacteroidetes phylum (Asinibacterium sp.) and the absence of pathogenic species like Edwardsiella tarda. Overall, results suggest that a balance between dietary ARA/EPA + DHA promotes gilthead sea bream juveniles' health however higher dietary content of n-3 LC-PUFA might limited the presence of microbial pathogens in intestinal mucosa.

Hypotensive effect induced by microinjection of Alamandine, a derivative of angiotensin-(1-7), into caudal ventrolateral medulla of 2K1C hypertensive rats.

In the present study we evaluated the cardiovascular effects produced by microinjection of the new component of the renin-angiotensin system, alamandine, into caudal ventrolateral medulla of urethane-anesthetized normotensive and hypertensive 2K1C rats. The participation of different angiotensin receptors in the effects of alamandine was also evaluated. Microinjection of angiotensin-(1-7) was used for comparison. The microinjection of 4, 40 and 140pmol of alamandine or angiotensin-(1-7) into caudal ventrolateral medulla induced similar hypotensive effects in Sham-operated rats. However, contrasting with angiotensin-(1-7), in 2K1C rats the MAP response to the highest dose of alamandine was similar to that observed with saline. The microinjection of A-779, a selective Mas receptor antagonist, blunted the angiotensin-(1-7) effects but did not block the hypotensive effect of alamandine in Sham or in 2K1C rats. However, microinjection of D-Pro7-angiotensin-(1-7), a Mas/MrgD receptor antagonist, blocked the hypotensive effect induced by both peptides. Furthermore, microinjection of PD123319, a putative AT2 receptor antagonist blocked the hypotensive effect of alamandine, but not of angiotensin-(1-7), in Sham and 2K1C rats. Microinjection of the AT1 receptor antagonist, losartan, did not alter the hypotensive effect of angiotensin-(1-7) or alamandine in both groups. These results provide new insights about the differential mechanisms participating in the central cardiovascular effects of alamandine and angiotensin-(1-7) in normotensive and 2K1C hypertensive rats.

Influence of antihypertensive drugs on aortic and coronary effects of Ang-(1-7) in pressure-overloaded rats.

This study investigated the influence of antihypertensive drugs, such as angiotensin-converting enzyme inhibitors (ACEIs), AT1 receptor blockers (ARBs), voltage-gated L-type calcium channel blockers, and mineralocorticoid receptor antagonists (MRAs), on the effects of angiotensin-(1-7) [Ang-(1-7)] on aorta and coronary arteries from pressure-overloaded rats. Pressure overload was induced by abdominal aortic banding (AB). To evaluate the role of antihypertensive drugs on the effect of Ang-(1-7), AB male Wistar rats weighing 250-300 g were treated with vehicle or low doses (5 mg·kg-1·day-1, gavage) of losartan, captopril, amlodipine, or spironolactone. Isolated aortic rings and isolated perfused hearts under constant flow were used to evaluate the effect of Ang-(1-7) in thoracic aorta and coronary arteries, respectively. Ang-(1-7) induced a significant relaxation in the aorta of sham animals, but this effect was reduced in the aortas of AB rats. Chronic treatments with losartan, captopril or amlodipine, but not with spironolactone, restored the Ang-(1-7)-induced aorta relaxation in AB rats. The coronary vasodilatation evoked by Ang-(1-7) in sham rats was blunted in hypertrophic rats. Only the treatment with losartan restored the coronary vasodilatory effect of Ang-(1-7) in AB rat hearts. These data support a beneficial vascular effect of an association of Ang-(1-7) and some antihypertensive drugs. Thus, this association may have potential as a new therapeutic strategy for cardiovascular diseases.

Effects of lipoic acid on growth and biochemical responses of common carp fed with carbohydrate diets.

Lipoic acid (LA) is an antioxidant that also favors glucose uptake in mammals. Until now, there are no studies evaluating the potential effect of this molecule on glycemic control in fish. It was evaluated LA effects on glucose uptake in common carp Cyprinus carpio fed with carbohydrate diets from two carbohydrate sources: glucose (GLU) and starch (STA), and supplemented or not with LA, being the diets: +GLU/-LA (GLU); +GLU/+LA (GLU + LA); +STA/-LA (STA); and +STA/+LA (STA + LA). Carp juveniles (6.5 ± 0.1 g) were fed with each diet ad libitum 4 times a day, during 68 days. Muscle glycogen concentration was higher (p < 0.05) in GLU and GLU + LA than in STA and STA + LA groups. On fish fed with starch, muscle cholesterol and triglyceride concentrations were higher (p < 0.05) in fish fed diets supplemented with LA. Muscle protein levels were higher in fish fed with LA, independent of the diet carbohydrate source. Lipid peroxidation was significantly reduced (p < 0.05) in fish muscle on fish fed the STA + LA diets when compared with the STA diet. Our findings indicate that LA modulates lipid, proteins and carbohydrate metabolism together with the well-known antioxidant effect. Also, LA showed to enhance starch utilization taking into account muscle cholesterol and triglyceride levels.

Comportamento ingestivo de bovinos leiteiros alimentados com farelo de crambe / Ingestive behavior of dairy catle fed crambe bran

O objetivo deste trabalho foi estudar os efeitos da utilização de farelo de crambe em substituição ao farelo de soja sobre o comportamento ingestivo de bovinos leiteiros. Foram utilizados quatro machos castrados Holandês x Zebu, fistulados no rúmen, com peso vivo médio de 664kg, distribuídos em delineamento quadrado latino 4 x 4. Os tratamentos consistiram em quatro dietas isoproteicas e isoenergéticas, formuladas com relação volumoso:concentrado 60:40 com base na matéria seca (MS). O volumoso foi composto de silagem de milho (51% MS) e feno de Tifton (49% MS), e o concentrado formulado com níveis crescentes de substituição do farelo de soja pelo farelo de crambe em 0%, 2,8%, 6,4% e 11,0% na MS da dieta. O comportamento ingestivo foi avaliado por meio do método direto de avaliação visual, em intervalos de 10 minutos, durante períodos de 24 horas. Registraram-se a frequência de alimentação, a ruminação e o ócio, bem como a posição do animal (em pé ou em decúbito). As variáveis em pé e em decúbito não diferiram entre os tratamentos, assim como os tempos gastos em alimentação, ruminação e ócio. O consumo de matéria seca e de FDN expressos em g/dia e gFDN/dia, respectivamente, a eficiência de ruminação expressa em gMS/min, a eficiência de ruminação expressa em gFDN/dia e o tempo de mastigação total não diferiram significativamente. No entanto, a eficiência de alimentação (gMS/min) variou de forma linear decrescente com a inclusão do farelo de crambe. Os períodos do dia influenciaram todas as atividades. O maior tempo de alimentação foi observado nos períodos após o fornecimento da dieta, e a maior atividade de ruminação foi verificada no período noturno. A substituição de farelo de soja por farelo de crambe não afetou o comportamento ingestivo, exceto para o parâmetro eficiência de alimentação. Nesse sentido, considerando o comportamento ingestivo, recomenda-se a substituição do farelo de soja por farelo de crambe para alimentação de bovinos leiteiros.

The aim of this work was to study the effects of the use of crambe meal replacing soy bean meal on the ingestive behavior of dairy cattle. Four male Holstein x Zebu rumen, with average live weight of 664 kg distributed in a 4x4 latin square design. The treatments consisted of four isonitrogenous and isocaloric diets, formulated with a roughage:concentrate ratio of 60:40 based on dry matter (DM). The forage was corn silage (51% DM) and Tifton grass hay (49% DM), and the concentrate was formulated with increasing levels of substitution of soy bean meal by crambe meal at 0%, 2.8%, 6.4% and 11.0% of the diet DM. The feeding behavior was evaluated using the direct method of visual assessment in 10 minute intervals during a 24 hour period. The frequency of feeding, idling time and the animal's position (standing or supine) were recorded. The variables standing and supine did not differ between treatments as well as time spent feeding, ruminating and idling times. The consumption of dry matter and NDF expressed in g/day and gNDF/day respectively, the efficiency of rumination expressed in gDM/min, the efficiency of rumination expressed in gNDF/day and the total chewing time did not differ significantly. However, the efficiency of power (gDM/min) decreased linearly with the inclusion of crambe meal. Periods of the day influenced all the activities. The longer feeding time was observed in the periods after the diet intake and greater rumination activity was observed at night. The replacement of soy bean meal by meal crambe did not affect feeding behavior, except for the feeding efficiency parameter. In this sense, considering the feeding behavior, the replacement of soy bean meal by crambe meal to feed dairy cattle is recommended.

Effect of l-glutamic acid supplementation on performance and nitrogen balance of broilers fed low protein diets.

The aim of this study was to evaluate the effect of protein reduction and supplementation of l-glutamic acid in male broiler diets. A total of 648 chicks of the Cobb 500 strain were distributed in a completely randomized design with six treatments and six replications with eighteen birds per experimental unit. The study comprised pre-starter (1-7 days), starter (8-21 days), growth (22-35 days) and final (36-45 days) phases. The first treatment consisted of a control diet formulated according to the requirements of essential amino acids for each rearing phase. The second and third treatments had crude protein (CP) reduced by 1.8 and 3.6 percentage points (pp) in relation to the control diet respectively. In the fourth treatment, l-glutamic acid was added to provide the same glutamate level as the control diet, and in the last two treatments, the broilers were supplemented with 1 and 2 pp of glutamate above that of the control diet respectively. The reduction in CP decreased the performance of broilers and the supplementation of l-glutamic acid did not influence performance when supplied in the diets with excess of glutamate. The lowest excreted nitrogen values were observed in the control diet, and treatments 2 and 3, respectively, in comparison with treatments with the use of l-glutamic acid (5 and 6). Retention efficiency of nitrogen was better in the control diet and in the treatment with a reduction of 1.8 pp of CP. It was verified that the serum uric acid level decreased with the CP reduction. A reduction in CP levels of up to 21.3%, 18.8%, 18.32% and 17.57% is recommended in phases from 1 to 7, 8 to 21, 22 to 35 and at 36 to 42 days, respectively, with a level of glutamate at 5.32%, 4.73%, 4.57%, 4.38%, also in these phases.

Basic life support knowledge of first-year university students from Brazil

We aimed to evaluate knowledge of first aid among new undergraduates and whether it is affected by their chosen course. A questionnaire was developed to assess knowledge of how to activate the Mobile Emergency Attendance Service - MEAS (Serviço de Atendimento Móvel de Urgência; SAMU), recognize a pre-hospital emergency situation and the first aid required for cardiac arrest. The students were also asked about enrolling in a first aid course. Responses were received from 1038 of 1365 (76.04%) new undergraduates. The questionnaires were completed in a 2-week period 1 month after the beginning of classes. Of the 1038 respondents (59.5% studying biological sciences, 11.6% physical sciences, and 28.6% humanities), 58.5% knew how to activate the MEAS/SAMU (54.3% non-biological vs 61.4% biological, P=0.02), with an odds ratio (OR)=1.39 (95%CI=1.07-1.81) regardless of age, sex, origin, having a previous degree or having a relative with cardiac disease. The majority could distinguish emergency from non-emergency situations. When faced with a possible cardiac arrest, 17.7% of the students would perform chest compressions (15.5% non-biological vs 19.1% biological first-year university students, P=0.16) and 65.2% would enroll in a first aid course (51.1% non-biological vs 74.7% biological, P<0.01), with an OR=2.61 (95%CI=1.98-3.44) adjusted for the same confounders. Even though a high percentage of the students recognized emergency situations, a significant proportion did not know the MEAS/SAMU number and only a minority had sufficient basic life support skills to help with cardiac arrest. A significant proportion would not enroll in a first aid course. Biological first-year university students were more prone to enroll in a basic life support course.

Time-course effects of aerobic exercise training on cardiovascular and renal parameters in 2K1C renovascular hypertensive rats

Exercise training (Ex) has been recommended for its beneficial effects in hypertensive states. The present study evaluated the time-course effects of Ex without workload on mean arterial pressure (MAP), reflex bradycardia, cardiac and renal histology, and oxidative stress in two-kidney, one-clip (2K1C) hypertensive rats. Male Fischer rats (10 weeks old; 150–180 g) underwent surgery (2K1C or SHAM) and were subsequently divided into a sedentary (SED) group and Ex group (swimming 1 h/day, 5 days/week for 2, 4, 6, 8, or 10 weeks). Until week 4, Ex decreased MAP, increased reflex bradycardia, prevented concentric hypertrophy, reduced collagen deposition in the myocardium and kidneys, decreased the level of thiobarbituric acid-reactive substances (TBARS) in the left ventricle, and increased the catalase (CAT) activity in the left ventricle and both kidneys. From week 6 to week 10, however, MAP and reflex bradycardia in 2K1C Ex rats became similar to those in 2K1C SED rats. Ex effectively reduced heart rate and prevented collagen deposition in the heart and both kidneys up to week 10, and restored the level of TBARS in the left ventricle and clipped kidney and the CAT activity in both kidneys until week 8. Ex without workload for 10 weeks in 2K1C rats provided distinct beneficial effects. The early effects of Ex on cardiovascular function included reversing MAP and reflex bradycardia. The later effects of Ex included preventing structural alterations in the heart and kidney by decreasing oxidative stress and reducing injuries in these organs during hypertension.

Basic life support knowledge of first-year university students from Brazil.

We aimed to evaluate knowledge of first aid among new undergraduates and whether it is affected by their chosen course. A questionnaire was developed to assess knowledge of how to activate the Mobile Emergency Attendance Service - MEAS (Serviço de Atendimento Móvel de Urgência; SAMU), recognize a pre-hospital emergency situation and the first aid required for cardiac arrest. The students were also asked about enrolling in a first aid course. Responses were received from 1038 of 1365 (76.04%) new undergraduates. The questionnaires were completed in a 2-week period 1 month after the beginning of classes. Of the 1038 respondents (59.5% studying biological sciences, 11.6% physical sciences, and 28.6% humanities), 58.5% knew how to activate the MEAS/SAMU (54.3% non-biological vs 61.4% biological, P=0.02), with an odds ratio (OR)=1.39 (95%CI=1.07-1.81) regardless of age, sex, origin, having a previous degree or having a relative with cardiac disease. The majority could distinguish emergency from non-emergency situations. When faced with a possible cardiac arrest, 17.7% of the students would perform chest compressions (15.5% non-biological vs 19.1% biological first-year university students, P=0.16) and 65.2% would enroll in a first aid course (51.1% non-biological vs 74.7% biological, P<0.01), with an OR=2.61 (95%CI=1.98-3.44) adjusted for the same confounders. Even though a high percentage of the students recognized emergency situations, a significant proportion did not know the MEAS/SAMU number and only a minority had sufficient basic life support skills to help with cardiac arrest. A significant proportion would not enroll in a first aid course. Biological first-year university students were more prone to enroll in a basic life support course.

Time-course effects of aerobic exercise training on cardiovascular and renal parameters in 2K1C renovascular hypertensive rats.

Exercise training (Ex) has been recommended for its beneficial effects in hypertensive states. The present study evaluated the time-course effects of Ex without workload on mean arterial pressure (MAP), reflex bradycardia, cardiac and renal histology, and oxidative stress in two-kidney, one-clip (2K1C) hypertensive rats. Male Fischer rats (10 weeks old; 150-180 g) underwent surgery (2K1C or SHAM) and were subsequently divided into a sedentary (SED) group and Ex group (swimming 1 h/day, 5 days/week for 2, 4, 6, 8, or 10 weeks). Until week 4, Ex decreased MAP, increased reflex bradycardia, prevented concentric hypertrophy, reduced collagen deposition in the myocardium and kidneys, decreased the level of thiobarbituric acid-reactive substances (TBARS) in the left ventricle, and increased the catalase (CAT) activity in the left ventricle and both kidneys. From week 6 to week 10, however, MAP and reflex bradycardia in 2K1C Ex rats became similar to those in 2K1C SED rats. Ex effectively reduced heart rate and prevented collagen deposition in the heart and both kidneys up to week 10, and restored the level of TBARS in the left ventricle and clipped kidney and the CAT activity in both kidneys until week 8. Ex without workload for 10 weeks in 2K1C rats provided distinct beneficial effects. The early effects of Ex on cardiovascular function included reversing MAP and reflex bradycardia. The later effects of Ex included preventing structural alterations in the heart and kidney by decreasing oxidative stress and reducing injuries in these organs during hypertension.

Exercise training restores oxidative stress and nitric oxide synthases in the rostral ventrolateral medulla of renovascular hypertensive rats.

We hypothesize that exercise training (EX) reverses the level of nitric oxide (NO) and oxidative stress into rostral ventrolateral medulla (RVLM) of renovascular hypertensive rats (two kidneys, one clip - 2K1C). Microinjections of L-arginine (5 nmol), L-NAME (10 nmol), or saline (100 nl) were made into RVLM of 2K1C and normotensive (SHAM) rats sedentary (SED) or subjected to swimming for 4 weeks. mRNA expression (by qRT-PCR) of nitric oxide synthases isoforms (nNOS, eNOS, and iNOS), manganese superoxide dismutase (MnSOD), copper and zinc superoxide (Cu/ZnSOD), catalase (CAT), NADPH oxidase subunit p22(phox), concentration of thiobarbituric acid-reactive substances (TBARS), and CAT activity into RVLM were evaluated. The mean arterial pressure was reduced in 2K1C EX compared with that in 2K1C SED rats. L-arginine into RVLM induced hypertensive effect in 2K1C and SHAM SED rats, while L-NAME prevented hypertensive effect only in SHAM-SED. EX reduced hypertensive effect of L-arginine in SHAM and 2K1C rats. mRNA expression of NOS isoforms, p22(phox), and concentration of TBARS were increased while CAT and Cu/ZnSOD expression and CAT activity decreased into RVLM of 2K1C-SED compared with SHAM-SED rats. Additionally, EX reversed mRNA expression of CAT and NOS isoforms, concentration of TBARS, and CAT activity into RVLM of 2K1C-EX rats. These data suggest that the levels of NOS and oxidative stress into RVLM are important to determine the level of hypertension. Furthermore, EX can restore the blood pressure by reversing the levels of NOS and CAT expression, and reducing TBARS concentration into RVLM for the physiological state.

Silagem de capim-elefante adicionada de casca de maracujá / Elephant grass silage added to passion fruit's peel

Avaliaram-se-se as características fermentativas e químico-bromatológicas da silagem de capim-elefante contendo diferentes níveis de inclusão do coproduto da extração da polpa do maracujá, a casca de maracujá (CM). Utilizaram-se níveis crescentes de adição desse coproduto (0,0%; 12,5%; 25,0%; 37,5% e 50,0%) em relação à matéria natural do capim-elefante durante a ensilagem. As silagens foram obtidas a partir do corte da forrageira aos 150 dias de idade. O material foi ensilado em silos laboratoriais e, após 180 dias, os silos foram abertos e coletaram-se amostras para a determinação dos teores de matéria seca (MS); carboidratos solúveis (CHOS); potencial hidrogeniônico (pH); nitrogênio amoniacal em porcentagem do nitrogênio total (NNH3/NT); extrato etéreo (EE); proteína bruta (PB); proteína insolúvel em detergente neutro em porcentagem do nitrogênio total (PIDN/NT); proteína insolúvel em detergente ácido em porcentagem do nitrogênio total (PIDA/NT); fibra em detergente neutro (FDN); fibra em detergente ácido (FDA); celulose (CEL); lignina (LIG); nitrogênio insolúvel em detergente neutro (NIDN); nitrogênio insolúvel em detergente ácido (NIDA) e digestibilidade in vitro da matéria seca (DIVMS). Para a avaliação do efeito dos tratamentos, utilizou-se delineamento experimental inteiramente ao acaso, com quatro repetições, executando-se o estudo de regressão para cada variável analisada (P<0,05). De acordo com as equações de regressão, para as características fermentativas, as silagens podem ser consideradas de muito boa qualidade. Quanto às frações fibrosas da silagem, a inclusão de níveis crescentes desse coproduto contribuiu para diminuí-las, o que resultou em maior DIVMS. Os parâmetros analisados permitem concluir que a inclusão da CM in natura durante a ensilagem do capim-elefante é recomendada em todos os níveis avaliados.

We evaluated the fermentative, chemical and qualitative characteristics of elephant grass silage containing different levels of inclusion of coproduct extraction of the passion fruit pulp in nature, passion fruit peel (CM). We used increasing levels of addition of this coproduct (0.0, 12.5, 25.0, 37.5 and 50.0%) in relation to natural field of elephant grass during ensiling. The silages were obtained from the cut forage at 150 days of age. The material was ensiled in laboratory silos, and after 180 days, the silos were opened and samples were collected for determination of dry matter (DM); soluble carbohydrates (CHOS); hydrogen potential (pH); ammonia nitrogen in percentage of total nitrogen (NNH3/NT); ether extract (EE); crude protein (CP); neutral detergent insoluble protein as a percentage of total nitrogen (NDIP/NT); acid detergent insoluble protein as a percentage of total nitrogen (PIDA/NT); neutral detergent fiber (NDF); acid detergent fiber (ADF); cellulose (CEL); lignin (LIG); neutral detergent insoluble nitrogen (NDIN); acid detergent insoluble nitrogen (ADIN) and in vitro digestibility of dry matter (IVDDM). To assess the effect of the treatments we used a completely randomized design with four replications, running the regression analysis for each variable analyzed (P<0.05). According to the regression equations for the fermentative characteristics, silage can be considered very good quality. As for fiber digestibility of silage, the increasing levels of this coproduct contributed to decrease it, which resulted in higher IVDDM. The parameters analyzed showed that the inclusion of CM in nature with elephant grass silage is recommended at all levels evaluated.

Angiotensin-(1-7) attenuates airway remodelling and hyperresponsiveness in a model of chronic allergic lung inflammation.

BACKGROUND AND PURPOSE: A long-term imbalance between pro- and anti-inflammatory mediators leads to airway remodelling, which is strongly correlated to most of the symptoms, severity and progression of chronic lung inflammation. The Angiotensin-(1-7) [Ang-(1-7)]/Mas receptor axis of the renin-angiotensin system is associated with attenuation of acute and chronic inflammatory processes. In this study, we investigated the effects of Ang-(1-7) treatment in a model of chronic allergic lung inflammation. EXPERIMENTAL APPROACH: Mice were sensitized to ovalbumin (OVA; 4 injections over 42 days, 14 days apart) and were challenged three times per week (days 21-46). These mice received Ang-(1-7) (1 µg·h(-1) , s.c.) by osmotic mini-pumps, for the last 28 days. Histology and morphometric analysis were performed in left lung and right ventricle. Airway responsiveness to methacholine, analysis of Ang-(1-7) levels (RIA), collagen I and III (qRT-PCR), ERK1/2 and JNK (Western blotting), IgE (elisa), cytokines and chemokines (elisa multiplex), and immunohistochemistry for Mas receptors were performed. KEY RESULTS: Infusion of Ang-(1-7) in OVA-sensitized and challenged mice decreased inflammatory cell infiltration and collagen deposition in the airways and lung parenchyma, and prevented bronchial hyperresponsiveness. These effects were accompanied by decreased IgE and ERK1/2 phosphorylation, and decreased pro-inflammatory cytokines. Mas receptors were detected in the epithelium and bronchial smooth muscle, suggesting a site in the lung for the beneficial actions of Ang-(1-7). CONCLUSIONS AND IMPLICATIONS: Ang-(1-7) exerted beneficial attenuation of three major features of chronic asthma: lung inflammation, airway remodelling and hyperresponsiveness. Our results support an important protective role of Ang-(1-7) in lung inflammation.

Cardiovascular and behavioral effects produced by administration of liposome-entrapped GABA into the rat central nervous system.

Liposomes are nanosystems that allow a sustained release of entrapped substances. Gamma-aminobutyric acid (GABA) is the most prevalent inhibitory neurotransmitter of the central nervous system (CNS). We developed a liposomal formulation of GABA for application in long-term CNS functional studies. Two days after liposome-entrapped GABA was injected intracerebroventricularly (ICV), Wistar rats were submitted to the following evaluations: (1) changes in mean arterial pressure (MAP), heart rate (HR) and renal sympathetic nerve activity (RSNA) to ICV injection of bicuculline methiodide (BMI) in anesthetized rats; (2) changes in cardiovascular reactivity to air jet stress in conscious rats; and (3) anxiety-like behavior in conscious rats. GABA and saline-containing pegylated liposomes were prepared with a mean diameter of 200 nm. Rats with implanted cannulas targeted to lateral cerebral ventricle (n = 5-8/group) received either GABA solution (GS), empty liposomes (EL) or GABA-containing liposomes (GL). Following (48 h) central microinjection (2 µL, 0.09 M and 99 g/L) of liposomes, animals were submitted to the different protocols. Animals that received GL demonstrated attenuated response of RSNA to BMI microinjection (GS 48 ± 9, EL 43 ± 9, GL 11 ± 8%; P < 0.05), blunted tachycardia in the stress trial (ΔHR: GS 115 ± 14, EL 117 ± 10, GL 74 ± 9 bpm; P<0.05) and spent more time in the open arms of elevated plus maze (EL 6 ± 2 vs. GL 18 ± 5%; P = 0.028) compared with GS and EL groups. These results indicate that liposome-entrapped GABA can be a potential tool for exploring the chronic effects of GABA in specific regions and pathways of the central nervous system.

Toll-like receptor 4 inhibition reduces vascular inflammation in spontaneously hypertensive rats.

AIMS: Hypertension is associated with increased levels of circulating cytokines and recent studies have shown that innate immunity contributes to hypertension. The mechanisms which hypertension stimulates immune response remain unclear, but may involve formation of neo-antigens that activate the immune system. Toll like receptor 4 (TLR4) is an innate immune receptor that binds a wide spectrum of exogenous (lipopolysaccharide) and endogenous ligands. TLR4 signaling leads to activation of nuclear factor kappa B (NFκB) and transcription of genes involved in inflammatory response. We previously demonstrated that TLR4 blockade reduces blood pressure and the augmented vascular contractility in spontaneously hypertensive rats (SHR). Here we hypothesized that inhibition of TLR4 ameliorates the vascular inflammatory process by a NFκB signaling pathway. MAIN METHODS: SHR and Wistar rats were treated with anti-TLR4 antibody (1µg/day) or unspecific IgG for 15days (i.p.). KEY FINDINGS: Anti-TLR4 treatment decreased production of reactive oxygen species and expression of IL-6 cytokine in mesenteric resistance arteries from SHR, when compared with IgG-treated SHR. Anti-TLR4 treatment also abolished the increased vascular reactivity to noradrenaline observed in IgG-treated SHR, as described before, and inhibition of NFκB decreased noradrenaline responses only in IgG-treated SHR. Mesenteric arteries from SHR treated with anti-TLR4 displayed decreased expression of MyD88, but not TRIF, key molecules in TLR4 signaling. Phosphorylation of p38 and NF-κB p65 were decreased in arteries from anti-TLR4-treated SHR versus IgG-treated SHR. SIGNIFICANCE: Together, these results suggest that TLR4 is a key player in hypertension and vascular inflammatory process by a NFκB signaling pathway.

Butyrate impairs atherogenesis by reducing plaque inflammation and vulnerability and decreasing NFκB activation.

BACKGROUND & AIMS: Butyrate is a four-carbon fatty acid that presents anti-inflammatory, anti-oxidative and apoptotic properties in colon and several cell lines. Because atherosclerosis has important oxidative and inflammatory components, butyrate could reduce oxidation and inflammation, impairing atherogenesis. We evaluated the effects of butyrate supplementation of butyrate on atherosclerosis and its mechanisms of action. METHODS AND RESULTS: ApoE knockout mice were fed on chow diet or 1% butyrate-supplemented chow diet (Butyrate) for 10 weeks to assess atherosclerosis lesions area and inflammatory status. Macrophage and endothelial cells were also pretreated with butyrate (0.5 mM) for 2 h before oxLDL stimulation to study oxLDL uptake and pro and anti-inflammatory cytokine production. Butyrate reduced atherosclerosis in the aorta by 50%. In the aortic valve, butyrate reduced CCL2, VCAM1 and MMP2 productions in the lesion site, resulting in a lower migration of macrophage and increased collagen depositions in the lesion and plaque stability. When EA.hy926 cells were pretreated with butyrate, oxLDL uptake, CD36, VCAM1, CCL2 TNF, IL1ß and IL6 productions were reduced, whereas IL10 production was increased. These effects were accompanied by a lower activation of NFκB due to a lower nuclear translocation of the p65 subunit. CONCLUSION: Oral butyrate is able to slow the progression of atherosclerosis by reducing adhesion and migration of macrophages and increasing plaque stability. These actions are linked to the reduction of CD36 in macrophages and endothelial cells, decreased pro-inflammatory cytokines and lower activation of NFκB all of these data support a possible role for butyrate as an atheroprotective agent.

AVE 0991, a non-peptide mimic of angiotensin-(1-7) effects, attenuates pulmonary remodelling in a model of chronic asthma.

BACKGROUND AND PURPOSE: AVE 0991 (AVE) is a non-peptide compound, mimic of the angiotensin (Ang)-(1-7) actions in many tissues and pathophysiological states. Here, we have investigated the effect of AVE on pulmonary remodelling in a murine model of ovalbumin (OVA)-induced chronic allergic lung inflammation. EXPERIMENTAL APPROACH: We used BALB/c mice (6-8 weeks old) and induced chronic allergic lung inflammation by OVA sensitization (20 µg·mouse(-1) , i.p., four times, 14 days apart) and OVA challenge (1%, nebulised during 30 min, three times per·week, for 4 weeks). Control and AVE groups were given saline i.p and challenged with saline. AVE treatment (1 mg·kg(-1) ·per day, s.c.) or saline (100 µL·kg(-1) ·per day, s.c.) was given during the challenge period. Mice were anaesthetized 72 h after the last challenge and blood and lungs collected. In some animals, primary bronchi were isolated to test contractile responses. Cytokines were evaluated in bronchoalveolar lavage (BAL) and lung homogenates. KEY RESULTS: Treatment with AVE of OVA sensitised and challenged mice attenuated the altered contractile response to carbachol in bronchial rings and reversed the increased airway wall and pulmonary vasculature thickness and right ventricular hypertrophy. Furthermore, AVE reduced IL-5 and increased IL-10 levels in the BAL, accompanied by decreased Ang II levels in lungs. CONCLUSIONS AND IMPLICATIONS: AVE treatment prevented pulmonary remodelling, inflammation and right ventricular hypertrophy in OVA mice, suggesting that Ang-(1-7) receptor agonists are a new possibility for the treatment of pulmonary remodelling induced by chronic asthma.

HLA alleles and HLA-B27 haplotypes associated with susceptibility and severity of ankylosing spondylitis in a Portuguese population.

Human leukocyte antigen (HLA)-B27 is the mostly known major histocompatibility complex (MHC) gene associated with ankylosing spondylitis (AS). Nonetheless, there is substantial evidence that other MHC genes appear to be associated with the disease, although it has not yet been established whether these associations are driven by direct associations or by linkage disequilibrium (LD) mechanisms. We aimed to investigate the contributions of HLA class I and II alleles and B27-haplotypes for AS in a case-control study. A total of 188 HLA-B27 AS cases and 189 HLA-B27 healthy controls were selected and typed for HLA class I and II by the Luminex polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) method. Allelic and haplotypic distributions were estimated by maximum likelihood method using Arlequin v3.11 and statistical analysis were performed by Stata10.1. No associations were found between non-HLA-B27 loci and AS susceptibility, but several associations were observed for phenotypic features of the disease. DRB1*08 was identified as a risk factor for uveitis and DQB1*04 seems to provide protection for AS severity (functional, metrological and radiological indexes). A*02/B27/C*02/DRB1*01/DQB1*05 [P<0.0001; odds ratio (OR) = 39.06; 95% confidence interval (CI) (2.34-651)] is the only haplotype that seems to confer susceptibility to AS. Moreover, the haplotype A*02/B27/C*01/DRB1*08/DQB1*04 seems to provide protection for disease functional and radiological repercussions. Our findings are compatible with the hypothesis that other genes within the HLA region besides HLA-B27 might play some role in AS susceptibility and severity.

Effects of short-term administration of estradiol on reperfusion arrhythmias in rats of different ages

Little is known about age-related differences in short-term effects of estradiol on ischemia-reperfusion (I/R) insults. The present study was designed to evaluate the effects of short-term treatment with estradiol on reperfusion arrhythmias in isolated hearts of 6-7-week-old and 12-14-month-old female rats. Wistar rats were sham-operated, ovariectomized and treated with vehicle or ovariectomized and treated with 17β-estradiol (E2; 5 µg·100 g-1·day-1) for 4 days. Hearts were perfused by the Langendorff technique. Reperfusion arrhythmias, i.e., ventricular tachycardia and/or ventricular fibrillation, were induced by 15 min of left coronary artery ligation and 30 min of reperfusion. The duration and incidence of I/R arrhythmias were significantly higher in young rats compared to middle-aged rats (arrhythmia severity index: 9.4 ± 1.0 vs 3.0 ± 0.3 arbitrary units, respectively, P < 0.05). In addition, middle-aged rats showed lower heart rate, systolic tension and coronary flow. Four-day E2 treatment caused an increase in uterine weight. Although E2 administration had no significant effect on the duration of I/R arrhythmias in middle-aged rats, it induced a marked reduction in the rhythm disturbances of young rats accompanied by a decrease in heart rate of isolated hearts. Also, this reduction was associated with an increase in QT interval. No significant changes were observed in the QT interval of middle-aged E2-treated rats. These data demonstrate that short-term estradiol treatment protects against I/R arrhythmias in hearts of young female rats. The anti-arrhythmogenic effect of estradiol might be related to a lengthening of the QT interval.