Phosphorylated PKM2 regulates endothelium-dependent vasodilation in diabetes / 中南大学学报(医学版)

OBJECTIVES@#Endothelium-dependent vasodilation dysfunction is the pathological basis of diabetic macroangiopathy. The utilization and adaptation of endothelial cells to high glucose determine the functional status of endothelial cells. Glycolysis pathway is the major energy source for endothelial cells. Abnormal glycolysis plays an important role in endothelium-dependent vasodilation dysfunction induced by high glucose. Pyruvate kinase isozyme type M2 (PKM2) is one of key enzymes in glycolysis pathway, phosphorylation of PKM2 can reduce the activity of pyruvate kinase and affect the glycolysis process of glucose. TEPP-46 can stabilize PKM2 in its tetramer form, reducing its dimer formation and phosphorylation. Using TEPP-46 as a tool drug to inhibit PKM2 phosphorylation, this study aims to explore the impact and potential mechanism of phosphorylated PKM2 (p-PKM2) on endothelial dependent vasodilation function in high glucose, and to provide a theoretical basis for finding new intervention targets for diabetic macroangiopathy.@*METHODS@#The mice were divided into 3 groups: a wild-type (WT) group (a control group, C57BL/6 mice) and a db/db group (a diabetic group, db/db mice), which were treated with the sodium carboxymethyl cellulose solution (solvent) by gavage once a day, and a TEPP-46 group (a treatment group, db/db mice+TEPP-46), which was gavaged with TEPP-46 (30 mg/kg) and sodium carboxymethyl cellulose solution once a day. After 12 weeks of treatment, the levels of p-PKM2 and PKM2 protein in thoracic aortas, plasma nitric oxide (NO) level and endothelium-dependent vasodilation function of thoracic aortas were detected. High glucose (30 mmol/L) with or without TEPP-46 (10 μmol/L), mannitol incubating human umbilical vein endothelial cells (HUVECs) for 72 hours, respectively. The level of NO in supernatant, the content of NO in cells, and the levels of p-PKM2 and PKM2 protein were detected. Finally, the effect of TEPP-46 on endothelial nitric oxide synthase (eNOS) phosphorylation was detected at the cellular and animal levels.@*RESULTS@#Compared with the control group, the levels of p-PKM2 in thoracic aortas of the diabetic group increased (P<0.05). The responsiveness of thoracic aortas in the diabetic group to acetylcholine (ACh) was 47% lower than that in the control group (P<0.05), and that in TEPP-46 treatment group was 28% higher than that in the diabetic group (P<0.05), while there was no statistically significant difference in the responsiveness of thoracic aortas to sodium nitroprusside (SNP). Compared with the control group, the plasma NO level of mice decreased in the diabetic group, while compared with the diabetic group, the phosphorylation of PKM2 in thoracic aortas decreased and the plasma NO level increased in the TEPP-46 group (both P<0.05). High glucose instead of mannitol induced the increase of PKM2 phosphorylation in HUVECs and reduced the level of NO in supernatant (both P<0.05). HUVECs incubated with TEPP-46 and high glucose reversed the reduction of NO production and secretion induced by high glucose while inhibiting PKM2 phosphorylation (both P<0.05). At the cellular and animal levels, TEPP-46 reversed the decrease of eNOS (ser1177) phosphorylation induced by high glucose (both P<0.05).@*CONCLUSIONS@#p-PKM2 may be involved in the process of endothelium-dependent vasodilation dysfunction in Type 2 diabetes by inhibiting p-eNOS (ser1177)/NO pathway.

Effect of Physical Training on Nitric Oxide Levels in Patients with Arterial Hypertension: An Integrative Review

Abstract The regular practice of physical exercise as a non-pharmacological treatment of arterial hypertension (AH) has been encouraged due to causing a series of physiological responses in the cardiovascular system, such as the production of vasoactive substances, including nitric oxide (NO). NO is a relaxation factor released by the endothelium, and the decrease in its bioavailability is related to coronary and arterial diseases, such as AH. This study aimed to perform an integrative literature review to elucidate the effect of physical training on NO levels in patients with AH and to establish a relationship between these levels and blood pressure (BP) control. A literature review was was performed by searching PubMed / MEDLINE, Lilacs, Scielo, Cinahl and Embase databases. The search string used was ("arterial hypertension" OR hypertension) AND (exercise OR "physical exercise" OR "aerobic exercise" OR "exercise training" or "physical activity") AND ("nitric oxide"). We included fully available controlled and uncontrolled clinical trials published in English and Portuguese languages in the last 10 years. The review consisted of 16 articles, of which 13 reported an increase in NO production after the physical training intervention, and three studies found no change. In addition, 15 studies observed a reduction in BP after the intervention. In conclusion, regular practice of physical exercises, advocating moderate intensity, can improve NO bioavailability in pre-hypertensive and hypertensive individuals, which seems to be one of the mechanisms responsible for BP reduction.

Influence of electroacupuncture on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase signaling pathway in spontaneously hypertensive rats / 中西医结合学报

OBJECTIVE@#To investigate the influence of electroacupuncture (EA) on ghrelin and the phosphoinositide 3-kinase/protein kinase B/endothelial nitric oxide synthase (PI3K/Akt/eNOS) signaling pathway in spontaneously hypertensive rats (SHRs).@*METHODS@#Eight Wistar-Kyoto rats were used as the healthy blood pressure (BP) control (normal group), and 32 SHRs were randomized into model group, EA group, EA plus ghrelin group (EA + G group), and EA plus PF04628935 group (a potent ghrelin receptor blocker; EA + P group) using a random number table. Rats in the normal group and model group did not receive treatment, but were immobilized for 20 min per day, 5 times a week, for 4 continuous weeks. SHRs in the EA group, EA + G group and EA + P group were immobilized and given EA treatment in 20 min sessions, 5 times per week, for 4 weeks. Additionally, 1 h before EA, SHRs in the EA + G group and EA + P group were intraperitoneally injected with ghrelin or PF04628935, respectively, for 4 weeks. The tail-cuff method was used to measure BP. After the 4-week intervention, the rats were sacrificed by cervical dislocation, and pathological morphology of the abdominal aorta was observed using hematoxylin-eosin (HE) staining. Enzyme-linked immunosorbent assay (ELISA) was used to detect the levels of ghrelin, nitric oxide (NO), endothelin-1 (ET-1) and thromboxane A2 (TXA2) in the serum. Isolated thoracic aortic ring experiment was performed to evaluate vasorelaxation. Western blot was used to measure the expression of PI3K, Akt, phosphorylated Akt (p-Akt) and eNOS proteins in the abdominal aorta. Further, quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to measure the relative levels of mRNA expression for PI3K, Akt and eNOS in the abdominal aorta.@*RESULTS@#EA significantly reduced the systolic BP (SBP) and diastolic BP (DBP) (P < 0.05). HE staining showed that EA improved the morphology of the vascular endothelium to some extent. Results of ELISA indicated that higher concentrations of ghrelin and NO, and lower concentrations of ET-1 and TXA2 were presented in the EA group (P < 0.05). The isolated thoracic aortic ring experiment demonstrated that the vasodilation capacity of the thoracic aorta increased in the EA group. Results of Western blot and qRT-PCR showed that EA increased the abundance of PI3K, p-Akt/Akt and eNOS proteins, as well as expression levels of PI3K, Akt and eNOS mRNAs (P < 0.05). In the EA + G group, SBP and DBP decreased (P < 0.05), ghrelin concentrations increased (P < 0.05), and the concentrations of ET-1 and TXA2 decreased (P < 0.05), relative to the EA group. In addition, the levels of PI3K and eNOS proteins, the p-Akt/Akt ratio, and the expression of PI3K, Akt and eNOS mRNAs increased significantly in the EA + G group (P < 0.05), while PF04628935 reversed these effects.@*CONCLUSION@#EA effectively reduced BP and protected the vascular endothelium, and these effects may be linked to promoting the release of ghrelin and activation of the PI3K/Akt/eNOS signaling pathway.

Curcumin improves cardiac fibrosis by inhibiting endothelial-mesenchymal transition through NRF2-DDAH-ADMA-NO pathway / 中国中药杂志

The present study analyzed the correlations between curcumin(Cur), nuclear factor E2 related factor 2(NRF2)-dimethylarginine dimethylaminohydrolase(DDAH)-asymmetric dimethylarginine(ADMA)-nitric oxide(NO) pathway, and endothelial-mesenchymal transition(EndMT) based on SD rats with cardiac fibrosis, and explored the effect and mechanism of Cur in resisting cardiac fibrosis to provide an in-depth theoretical basis for its clinical application in the treatment of heart failure. The cardiac fibrosis model was induced by subcutaneous injection of isoprenaline(Iso) in rats. Thirty-two rats were randomly divided into a control group, a model group, a low-dose Cur group(100 mg·kg~(-1)·d~(-1)), and a high-dose Cur group(200 mg·kg~(-1)·d~(-1)), with eight in each group. After 21 days of treatment, cardiac function was detected by echocardiography, degree of cardiac fibrosis by Masson staining, expression of CD31 and α-SMA by pathological staining, expression of VE-cadherin, vimentin, NRF2, and DDAH by Western blot, and ADMA level by HPLC. Compared with the model group, the Cur groups showed alleviated cardiac fibrosis, accompanied by increased CD31 and VE-cadherin expression and decreased α-SMA and vimentin expression, indicating relieved EndMT. Additionally, DDAH and NRF2 levels were elevated and ADMA and NO expression declined. Cur improves cardiac fibrosis by inhibiting EndMT presumedly through the NRF2-DDAH-ADMA-NO pathway.

Normalization of the ratio of nitric oxide and peroxynitrite by promoting eNOS dimer activity is a new direction for diabetic nephropathy treatment / 生理学报

Diabetic nephropathy is a microvascular complication of diabetes. Its etiology involves metabolic disorder-induced endothelial dysfunction. Endothelium-derived nitric oxide (NO) plays an important role in a number of physiological processes, including glomerular filtration and endothelial protection. NO dysregulation is an important pathogenic basis of diabetic nephropathy. Hyperglycemia and dyslipidemia can lead to oxidative stress, chronic inflammation and insulin resistance, thus affecting NO homeostasis regulated by endothelial nitric oxide synthase (eNOS) and a conglomerate of related proteins and factors. The reaction of NO and superoxide (O2.-) to form peroxynitrite (ONOO-) is the most important pathological NO pathway in diabetic nephropathy. ONOO- is a hyper-reactive oxidant and nitrating agent in vivo which can cause the uncoupling of eNOS. The uncoupled eNOS does not produce NO but produces superoxide. Thus, eNOS uncoupling is a critical contributor of NO dysregulation. Understanding the regulatory mechanism of NO and the effects of various pathological conditions on it could reveal the pathophysiology of diabetic nephropathy, potential drug targets and mechanisms of action. We believe that increasing the stability and activity of eNOS dimers, promoting NO synthesis and increasing NO/ONOO- ratio could guide the development of drugs to treat diabetic nephropathy. We will illustrate these actions with some clinically used drugs as examples in the present review.

In vitro and in vivo antioxidant potentials of the methanolic crude extract from Inula glomerata Oliv. & Hiern (Asteraceae) and Salacia kraussii (Harv) Harv (Celastraceae) / Potenciales antioxidantes in vitro e in vivo del extracto crudo metanólico de Inula glomerata Oliv. & Hiern. (Asteraceae) y Salacia kraussii (Harv) Harv (Celastraceae)

Reactive oxygen species are implicated in multiple pathological conditions including erectile dysfunction. This study evaluated the in vitro and in vivo antioxidant potential of the methanolic extracts of Inula glomerata and Salacia kraussii. The plant materials were pulverized and extracted with methanol. The phytochemical analysis, ability of the crude extracts to scavenge free radicals (ABTS, DPPH, NO.) in vitroas well as the total phenolic and flavonoid contents was investigated. In vivo, antioxidant potentials of the crude extracts (50/250 mg/kg body weight) were determined in an erectile dysfunction rat model. The phytochemical analysis revealed that both plants contain flavonoids, tannins, terpenoids, and alkaloids. The crude extracts at varying degree of efficiency, scavenged ABTS and DPPH radicals. The crude extracts at low concentrations (50 mg/kg b.w) significantly (p<0.05) diminished the level of malondialdehyde, augmented catalase activities and elevated glutathione levels. However, SOD activities were significantly boosted in a dose-dependent manner by the crude extracts. Therefore, I. glomerataand S. kraussiipossess antioxidant properties, hence, can serve as a therapeutic modality in the treatment of oxidative stress-induced erectile dysfunction.

Las especies reactivas de oxígeno están implicadas en múltiples condiciones patológicas, incluyendo la disfunción eréctil. Este estudio evaluó el potencial antioxidante in vitro e in vivo de extractos metanólicos de Inula glomeratay Salacia kraussii. Los materiales vegetales fueron pulverizados y extraídos con metanol. A estos extractos crudos se les llevó a cabo el análisis fitoquímico junto con el contenido total de fenólicos y flavonoides, así como se les investigó la capacidad in vitro para atrapar radicales (ABTS, DPPH, NO.). Los potenciales antioxidantes in vivo de los extractos crudos (50/250 mg/kg de peso corporal) se determinaron en un modelo en ratas con disfunción eréctil. El análisis fitoquímico reveló que ambas plantas contuvieron flavonoides, taninos, terpenoides y alcaloides. Los extractos crudos con un grado variable de eficiencia, atraparon a los radicales ABTS y DPPH. Los extractos crudos a bajas concentraciones (50 mg/kg p.c) significativamente (p<0.05) disminuyeron el nivel de malondialdehído, aumentaron las actividades de catalasa y elevaron los niveles de glutatión. Sin embargo, las actividades de SOD por los extractos crudos fueron significativamente dosis-dependientes. Así, los extractos de I. glomeratay S. kraussii mostraron propiedades antioxidantes, y por lo tanto, podrían servir como una alternativa terapéutica en el tratamiento de disfunción eréctil inducida por estrés oxidativo.

Effects of vitamin D-induced supernatant of placental explants from preeclamptic women on oxidative stress and nitric oxide bioavailability in human umbilical vein endothelial cells

The study evaluated the effect of the supernatant of placental explants from preeclamptic (PE) and normotensive (NT) pregnant women after tissue treatment with or without vitamin D (VD) on oxidative stress and nitric oxide (NO) bioavailability in human umbilical vein endothelial cells (HUVEC). Placental explants were prepared from eight NT and eight PE women, and supernatants were obtained after incubation with or without hydrogen peroxide (H2O2) and/or VD. HUVEC were cultured for 24 h with supernatants, and the following parameters were analyzed in HUVEC cultures: NO, nitrate (NO3-), and nitrite (NO2-) levels, lipid peroxidation, and intracellular reactive oxygen species (ROS). Results showed that the production of NO3-, NO2-, malondialdehyde (MDA), and ROS were significantly higher in HUVEC treated with explant supernatant from PE compared to NT pregnant women, while the supernatant of PE explants treated with VD led to a decrease in these parameters. A significantly high production of NO was detected in HUVEC cultured with control supernatant of NT group, and in cultures treated with supernatant of PE explants treated with VD. Taken together, these results demonstrated that cultures of placental explants from PE women with VD treatment generated a supernatant that decreased oxidative stress and increased the bioavailability of NO in endothelial cells.

Rol de la fracción espirada de oxido nítrico en el niño con diagnóstico de asma bronquial / Role of the exhaled fraction of nitric oxide in children with diagnosis of bronchial asthma

El asma es la enfermedad respiratoria crónica pediátrica más frecuente. En la mayoría de los niños se caracteriza por inflamación de la vía aérea de tipo eosinofílica alérgica. La fracción espirada de óxido nítrico (FENO) es un biomarcador de inflamación eosinofílica de vía aérea, su medición es no invasiva y fácil de realizar y ha sido evaluado en los últimos años para su aplicación clínica en el diagnóstico y tratamiento del asma en niños y adultos. Esta revisión abordará el origen anatómico y bioquímico del FENO, aspectos prácticos de su medición, valores de referencia y su aplicación clínica en el diagnóstico y tratamiento del asma pediátrico.

Asthma is the most common pediatric chronic disease characterized in most children by allergic eosinophilic airway inflammation. The exhaled fraction of nitric oxide (FENO) is a biomarker of eosinophilic airway inflammation, constituting a non-invasive and easy-to-perform test that has been evaluated in recent years for its clinical application in the diagnosis and treatment of asthma in children and adults. This review will address the anatomical and biochemical origin of FENO, practical aspects of its measurement, reference values and its clinical application in the diagnosis and treatment of pediatric asthma.

Effects of music therapy on pain and oxidative stress in oocyte pick-up: A randomized clinical trial / Efeitos da musicoterapia sobre dor e estresse oxidativo na aspiração folicular: estudo clínico randomizado

Abstract Background and objective: The aim was to investigate the effects of Turkish classical music on pain and oxidative stress in patients undergoing oocyte pick-up. Methods: The study was a randomized, controlled trial. The groups included Group NM (Non-Music), control group; Group PM, which comprised patients who listened to music before the operation; and Group CM, which comprised patients who listened to music both before and during the operation. Blood was drawn prior to the operation to measure the oxidative stress values. Pain, hemodynamic parameters, oxidative stress values were assessed postoperatively. Results: The number of patients requiring additional propofol was higher in Group PM than in Groups NM and CM (p = 0.003). The postoperative Visual Analog Scale (VAS) score were lower in Groups PM and CM than in Group NM (p = 0.001, p = 0.007) in the 1st and 60th minutes. The postoperative VAS score was lower in Group CM than in Group NM (p = 0.045) in the 5th minute. The postoperative additional analgesic requirements were lower in Groups PM and CM than in Group NM (p = 0.045). The postoperative blood glutathione peroxidase values were significantly higher in Groups PM and CM than in Group NM (p = 0.001). The postoperative catalase values were significantly higher in Groups PM and CM than in Group NM (p = 0.008 and p < 0.001). The preoperative malondialdehyde values were significantly lower in Groups PM and CM than in Group NM. The preoperative nitric oxide values were higher in Groups PM and CM than in Group NM (p < 0.001), whereas the postoperative nitric oxide values were lower in Groups PM and CM than in Group NM (p < 0.001). Conclusion: Turkish classical music has beneficial effects on pain and oxidative stress in oocyte pick-up patients.

Resumo Justificativa e objetivo: O objetivo deste estudo foi investigar os efeitos da música clássica turca sobre a dor e o estresse oxidativo em pacientes submetidas a aspiração folicular. Método: Estudo randomizado controlado. Os grupos foram: grupo controle NM, sem música; Grupo PM, com pacientes que ouviram música antes da cirurgia; e Grupo CM, com pacientes que ouviram música antes e durante a cirurgia. Foi coletado sangue antes da cirurgia para avaliar os valores de estresse oxidativo. Dor, parâmetros hemodinâmicos e valores de estresse oxidativo foram avaliados após a cirurgia. Resultados: O número de pacientes que necessitaram de propofol adicional foi mais alto no Grupo PM do que nos grupos NM e CM (p = 0,003). A pontuação da Escala Visual Analógica (EVA) pós-operatória foi mais baixa nos Grupos PM e CM do que no Grupo NM (p = 0,001; p = 0,007), no 1° e 60° minutos. A pontuação da EVA pós-operatória foi mais baixa no Grupo CM do que no grupo NM (p = 0,045) no 5° minuto. A necessidade de analgesia pós-operatória adicional foi mais baixa nos Grupos PM e CM do que no Grupo NM (p = 0,045). Os valores pós-operatórios de glutationa peroxidase no sangue foram significantemente mais altos nos Grupos PM e CM do que no Grupo NM (p = 0,001). Os valores pós-operatórios de catalase foram significantemente mais altos nos Grupos PM e CM do que no Grupo NM (p = 0,008 e p≤ 0,001). Os valores pré-operatórios de malondialdeído foram significantemente mais baixos nos grupos PM e CM do que no Grupo NM. Os valores pré-operatórios de óxido nítrico foram mais altos nos grupos PM e CM do que no Grupo NM (p≤ 0,001), ao passo que valores pós-operatórios de óxido nítrico foram mais baixos nos grupos PM e CM do que no Grupo NM (p≤ 0,001). Conclusão: Música clássica turca exerce efeito benéfico sobre a dor e estresse oxidativo em pacientes na aspiração folicular.

Underexpression of endothelial nitric oxide synthase leads to more severe pulmonary complex vascular lesions associated with HIV patients / La baja expresión de oxido nítrico sintetasa provoca mayor severidad en las lesiones vasculares complejas asociadas al VIH

Abstract Background: Despite increase in survival of human immunodeficiency virus (HIV) patients due to highly active antiretroviral therapy, non-infectious complications are still prevalent such as presentation of lung vasculopathy, even in asymptomatic patients. Endothelial nitric oxide synthase (eNOS) is necessary to produce nitric oxide that causes pulmonary endothelial vasodilation. Participation of this protein in the pulmonary circulation in HIV patients has not been elucidated. This work studied the presence and expression of eNOS in pulmonary complex vascular lesions associated with HIV (PCVL/HIV). Methods: In lung tissues from patients who died from complications of HIV, we used immunohistochemistry and immune chemiluminescence (imageJ) to determine the different degrees of expression of eNOS in PCVL-HIV in comparison with non-PCVL/HIV. Reagents used were anti-eNOS and an automated system. All data are presented as mean and standard deviation. Differences were analyzed with Wilcoxon; p < 0.05 was accepted as statistically significant. Results: In 57 tissues, the histological evidence of pulmonary vasculopathy was showed as different types (proliferative, obliterative, and plexiform) and severe presentation of vasculopathy than non-PCVL/HIV. A statistically significant decrease of eNOS was observed in all PCVL/HIV tissue samples. Conclusion: eNOS has a relevant role in the pathogenesis of pulmonary vasculopathy in acquired immunodeficiency syndrome patients. It is necessary to determine in the future the participation of eNOS and other mechanisms involved in PCVL/HIV.

Resumen Antecedentes: A pesar del incremento en la sobrevivencia del paciente con virus de inmunodeficiencia humana (VIH) debido al uso del tratamiento antiretroviral altamente efectivo, las complicaciones no infecciosas siguen ocasionando vasculopatía pulmonar, aun en pacientes asintomáticos. La óxido nítrico sintetasa (ONSe) es necesaria para la producción de óxido nítrico la cual provoca vasodilatación pulmonar. La participación de esta proteína en la circulación pulmonar en los pacientes con VIH aún no se ha dilucidado. Este trabajo estudia la presencia y la expresión de ONSe en las lesiones vasculares pulmonares complejas asociadas al VIH (LVPC/VIH). Métodos: En tejidos pulmonares de pacientes que fallecieron por complicaciones del VIH, se utilizó inmunohistoquímica e inmunoquimioluminescencia (imageJ) para determinar los diferentes grados de expresión de la ONSe en LVPC/VIH. Los reactivos utilizados son anti-ONSe en sistema automatizado. Todos los datos son presentados en media y desviación estándar. Las diferencias son analizadas con la prueba de Wilcoxon; se aceptó como estadísticamente significativa una p < 0.05. Resultados: En 57 pacientes, la histología de la vasculopatía pulmonar mostró diferentes tipos (proliferativo, obliterativo y plexiforme) además de varias presentaciones de vasculopatía en tejidos no-LVPC/VIH. Se observó diferencia estadística en la disminución de ONSe en todos los tejidos LVPC/VIH. Conclusiones: La ONSe tiene un papel relevante en la patogénesis de la vasculopatía pulmonar en el VIH. Es necesario determinar en el futuro la participación de ONSe y otros mecanismos involucrados en LVPC/VIH.

Induction of selective liver hypothermia prevents significant ischemia/reperfusion injury in Wistar rats after 24 hours

Purpose To investigate the effects of induction of selective liver hypothermia in a rodent model. Methods Seven male Wistar rats were subjected to 90 minutes of partial 70% liver ischemia and topic liver 26°C hypothermia (H group). Other seven male Wistar rats were subjected to 90 minutes of partial 70% normothermic liver ischemia (N group). Five additional rats underwent a midline incision and section of liver ligaments under normothermic conditions and without any liver ischemia (sham group). All animals were sacrificed 24-h after reperfusion, and livers were sampled for analyses. Pathology sections were scored for sinusoidal congestion, ballooning, hepatocelllular necrosis and the presence of neutrophilic infiltrates. Results At the end of the experiment, liver tissue expressions of TNF-ɑ, IL-1β, iNOS and TNF-ɑ/IL-10 ratio were significantly reduced in the H group compared to N group, whereas IL-10 and eNOS were significantly increased in H group. Histopathological injury scores revealed a significant decrease in ischemia/reperfusion (I/R) injuries in H group. Conclusion Selective liver hypothermia prevented I/R injury by inhibiting the release of inflammatory cytokines, preserves microcirculation, prevents hepatocellular necrosis and leukocyte infiltration, allowing maintenance of the liver architecture.

Functional iron deficiency in patients on hemodialysis: prevalence, nutritional assessment, and biomarkers of oxidative stress and inflammation / Deficiência funcional de ferro em pacientes em hemodiálise: prevalência, avaliação nutricional e de biomarcadores de estresse oxidativo e de inflamação

Abstract Introduction: Anemic patients with chronic kidney disease (CKD) can be divided into anemic patients without or with functional iron deficiency (FID). The increase in the number of cases of hemosiderosis in patients on hemodialysis (HD) attributed to excessive intravenous iron replacement has called for the investigation of the factors involved in the genesis of FID. Objectives: This study aimed to describe the prevalence of FID in patients with CKD on HD, characterize the included individuals in terms of clinical and workup parameters, and assess their nutritional, oxidative stress, and inflammation statuses. This cross-sectional study assembled a convenience sample of 183 patients with CKD on HD treated in Southern Brazil. Patients meeting the inclusion and exclusion criteria were divided into two groups, one with anemic subjects with FID and one with anemic patients without FID. Participants answered a questionnaire probing into socio-epidemiological factors, underwent anthropometric measurements, and were tested for markers of anemia, oxidative stress, inflammation, and nutrition. Statistical analysis: The date sets were treated on software package GraphPad InStat version 3.1. Variables were tested with the Kolmogorov-Smirnov, chi-square, Student's t, and Mann-Whitney tests. Statistical significance was attributed to differences with a p < 0.05. Results: Markers of inflammation were not statistically different between the two groups. Markers of anemia and nutrition were significantly lower in patients with FID. Patients with FID were prescribed higher doses of parenteral iron (p < 0,05). Discussion: FID was associated with lower nutritional marker levels, but not to increased levels of markers of inflammation or oxidative stress, as reported in the literature. Additional studies on the subject are needed.

Resumo Introdução: A anemia na DRC pode ser dividida em anemia sem deficiência funcional de ferro e com deficiência funcional de ferro (ADFF). Diante do aumento dos casos de hemossiderose em pacientes em hemodiálise, atribuídos à reposição excessiva de ferro endovenoso, maiores conhecimentos sobre os fatores envolvidos na gênese da ADFF são importantes. Objetivos: documentar a prevalência de ADFF em renais crônicos em hemodiálise. Caracterizar clínica e laboratorialmente os portadores de ADFF em HD e avaliar o estado nutricional, estresse oxidativo e inflamatório. Estudo transversal, amostra de conveniência, envolvendo 183 renais crônicos em hemodiálise no sul do Brasil. Após aplicação dos critérios de exclusão, os pacientes foram separados em dois grupos: portadores de anemia com e sem deficiência funcional de ferro. Foram submetidos a questionário socioepidemiológico, à análise antropométrica e análise laboratorial dos marcadores de anemia, estresse oxidativo, inflamatórios e nutricionais. Análise estatística: programa GraphPad InStat versão 3.1. Foram aplicados os testes: Kolmogorov-Smirnov, qui-quadrado, t de Student e Mann-Whitney. Nível de significância adotado de 5%. Resultados: não houve diferença significativa nos marcadores inflamatórios entre os dois grupos. Houve diferença significativa nos marcadores de anemia e nutrição, significativamente menores nos pacientes com ADFF. Pacientes com ADFF receberam doses mais elevadas de ferro parenteral (p < 0,05). Discussão: ADFF esteve associada a menores valores de marcadores nutricionais, mas não esteve associada a marcadores inflamatórios ou de estresse oxidativo aumentados, como relatado na literatura. Estudos adicionais sobre o tema são necessários.

Inter-Individual Responses to Citrulline Malate Oral Supplementation on Post-Exercise Hypotension in Hypertensives: A 24-Hour Analysis / Respostas Interindividuais à Suplementação Oral de Citrulina Malato na Hipotensão Pós-Exercício em Hipertensos: Uma Análise de 24 Horas

Abstract Background: Studies have persuasively demonstrated that citrulline has a key role in the arginine-nitric oxide system, increasing nitric oxide bioavailability, an important mediator of peripheral vasodilation. Objective: To analyze the inter-individual post-exercise hypotension responsiveness following acute citrulline supplementation in hypertensives. Methods: Forty hypertensives were randomly assigned to one of the four experimental groups (control-placebo, control-citrulline, exercise-placebo, and exercise-citrulline). They ingested placebo or citrulline malate [CM] (6 grams). During the exercise session, individuals performed 40 minutes of walking/running on a treadmill at 60-70% of HR reserve. For the control session, the individuals remained seated at rest for 40 minutes. Office blood pressure (BP) was taken every 10 minutes until completing 60 minutes after the experimental session. The ambulatory BP device was programmed to take the readings every 20 minutes (awake time) and every 30 minutes (sleep time) over the course of 24 hours of monitoring. Statistical significance was defined as p < 0.05. Results: Unlike the other experimental groups, there were no "non-responders" in the exercise/citrulline (EC) for "awake" (systolic and diastolic BP) and "24 hours" (diastolic BP). The effect sizes were more consistent in the EC for systolic and diastolic ambulatorial BP response. The effects were "large" (> 0.8) for "awake", "asleep", and "24 hours" only in the EC for diastolic BP. Conclusion: CM supplementation can increase the post-exercise hypotensive effects in hypertensives. In addition, the prevalence of non-responders is lower when associated with aerobic exercise and CM supplementation.

Resumo Fundamento: Estudos demonstraram de maneira persuasiva que a citrulina tem um papel fundamental no sistema arginina-óxido nítrico, aumentando a biodisponibilidade do óxido nítrico, um importante mediador da vasodilatação periférica. Objetivo: Analisar a responsividade interindividual da hipotensão pós-exercício após suplementação aguda com citrulina em hipertensos. Métodos: Quarenta hipertensos foram aleatoriamente designados para um dos quatro grupos experimentais (controle-placebo, controle-citrulina, exercício-placebo e exercício-citrulina). Eles ingeriram placebo ou citrulina malato [CM] (6 gramas). Durante a sessão de exercício, os indivíduos realizaram 40 minutos de caminhada/corrida em esteira a 60-70% da FC de reserva. Para a sessão de controle, os indivíduos permaneceram sentados em repouso por 40 minutos. A medida da pressão arterial (PA) no consultório foi realizada a cada 10 minutos até completar 60 minutos após a sessão experimental. O dispositivo ambulatorial de PA foi programado para fazer as leituras a cada 20 minutos (tempo de vigília) e a cada 30 minutos (tempo de sono) ao longo de 24 horas de monitoramento. A significância estatística foi definida como p < 0,05. Resultados: Diferentemente de outros grupos experimentais, não houve "não respondedores" no exercício/citrulina (EC) para "acordado" (PA sistólica e diastólica) e "24 horas" (PA diastólica). Os tamanhos de efeito foram mais consistentes no EC para a resposta sistólica e diastólica da PA ambulatorial. Os efeitos foram "grandes" (> 0,8) para "acordado", "dormindo", e para "24 horas" apenas no EC para a PA diastólica. Conclusão: A suplementação com CM pode aumentar os efeitos hipotensivos pós-exercício em hipertensos. Além disso, a prevalência de "não respondedores" é menor quando associada ao exercício aeróbico e à suplementação com CM.

A influência do oxido nítrico na fisiopatologia da neuropatia glaucomatosa / The influence of nitric oxide on the pathophysiology of glaucomatous neuropathy

Resumo O oxido nitrico (NO) é um fator relaxante derivado do endotélio e um potente vasodilatador que impacta em vários sistemas em todo o corpo. Estudos comprovam que o fluxo sanguíneo ocular basal é regulado pelo NO, sendo um importante regulador da homeostase, especialmente dentro dos tecidos uveais. A disfunção da produção de NO seria associado ao glaucoma através da alteração da perfusão da cabeça do nervo óptico associado ao aumento da pressão intraocular devido um sistema de drenagem trabecular deficiente. O NO tornou-se uma molécula atraente para o tratamento do glaucoma devido a possibilidade de modulação da drenagem trabecular, abaixando a pressão intraocular e ação neuroprotetora melhorando a perfusão sanguínea na cabeça do nervo óptico.

Abstract Nitric Oxide (NO) is a relaxing endothelium-derived factor and a potent vasodilator that impacts various systems throughout the body. Proven studies of basal ocular blood flow are regulated by NO, being an important regulator of homeostasis, especially within the uveal tissues. The dysfunction of the production associated with glaucoma due to alteration of the optic nerve head associated to the increase of the intraocular pressure by a deficient trabecular meshwork. NO became an attractive molecule for the treatment of glaucoma due to a modulation of the trabecular meshwork, lowering the neuroprotective intra and ocular pressure for a blood surgery in the head of the optic nerve.

Pharmacological characterization of the calcium influx pathways involved in nitric oxide production by endothelial cells / Caracterização farmacológica das vias de influxo de cálcio envolvidas na produção de óxido nítrico por células endoteliais

ABSTRACT Objective: To characterize the calcium influx pathways implicated in the sustained elevation of endothelial intracellular calcium concentration, required for the synthesis and release of relaxing factors. Methods: We evaluated the effect of the newly synthesized pyrazole derivatives, described as selective inhibitors for ORAI (BTP2/Pyr2 and Pyr6) and TRPC3 (Pyr3 and Pyr10) channels, upon endothelium- and extracellular calcium-dependent relaxations stimulated by acetylcholine and thapsigargin, in pre-constricted rat thoracic aortic rings. Results: Acetylcholine and thapsigargin responses were completely reverted by Pyr2 and Pyr6 (1 to 3μM). Pyr3 (0.3 to 3μM) caused a rapid reversal of acetylcholine (6.2±0.08mg.s−1) and thapsigargin (3.9±0.25mg.s−1) relaxations, whereas the more selective TRPC3 blocker Pyr10 (1 to 3μM) had no effect. The recently described TRPC4/5 selective blocker, ML204 (1 to 3μM), reverted completely acetylcholine relaxations, but minimally thapsigargin induced ones. Noteworthy, relaxations elicited by GSK1016790A (TRPV4 agonist) were unaffected by pyrazole compounds or ML204. After Pyr2 and Pyr6 pre-incubation, acetylcholine and thapsigargin evoked transient relaxations similar in magnitude and kinetics to those observed in the absence of extracellular calcium. Sodium nitroprusside relaxations as well as phenylephrine-induced contractions (denuded aorta) were not affected by any of pyrazole compounds (1 to 3μM). Conclusion: These observations revealed a previously unrecognized complexity in rat aorta endothelial calcium influx pathways, which result in production and release of nitric oxide. Pharmacologically distinguishable pathways mediate acetylcholine (ORAI/TRPC other than TRPC3/TRPC4 calcium-permeable channels) and thapsigargin (TRPC4 not required) induced calcium influx.

RESUMO Objetivo: Caracterizar as vias do influxo de cálcio envolvidas no aumento sustentado da concentração intracelular de cálcio na célula endotelial, essencial para a síntese e a liberação de fatores relaxantes. Métodos: Analisamos o efeito de derivados pirazólicos sintetizados recentemente, descritos como inibidores seletivos para canais ORAI (BTP2/Pyr2 e Pyr6) e TRPC3 (Pyr3 e Pyr10), nos relaxamentos dependentes de endotélio e cálcio extracelular, produzidos por acetilcolina e tapsigargina, em anéis pré-contraídos da aorta torácica de rato. Resultados: As respostas de acetilcolina e tapsigargina foram completamente revertidas por Pyr2 e Pyr6 (1 a 3μM). Pyr3 (0,3 a 3μM) produziu reversão rápida dos relaxamentos de acetilcolina (6,2±0,08mg.s−1) e tapsigargina (3,9±0,25mg.s−1), enquanto o bloqueador mais seletivo para TRPC3, Pyr10 (1 a 3μM), não apresentou efeito. ML204 (1 a 3μM), bloqueador seletivo de TRPC4, descrito há pouco tempo, reverteu os relaxamentos induzidos por acetilcolina de forma completa, mas afetou minimamente aqueles produzidos por tapsigargina. Os derivados pirazólicos ou ML204 não afetaram os relaxamentos estimulados com GSK1016790A (TRPV4-agonista). Ainda, após pré-incubação com Pyr2 e Pyr6, acetilcolina e tapsigargina provocaram relaxamentos transitórios semelhantes em magnitude e cinética àqueles observados na ausência de cálcio extracelular. Os relaxamentos do nitroprussiato de sódio e as contrações induzidas pela fenilefrina (aorta sem endotélio) não foram afetados pelos compostos pirazólicos (1 a 3μM). Conclusão: Essas observações revelaram uma complexidade desconhecida das vias de influxo de cálcio no endotélio da aorta de rato, que resultam na produção e na liberação de óxido nítrico. Vias distinguíveis farmacologicamente medeiam o influxo estimulado por acetilcolina (ORAI TRPC, diferentes de TRPC3 TRPC4) e tapsigargina (TRPC4 não requerido).

Dental pulp fibroblasts response after stimulation with hema and adhesive system

Abstract This study evaluated in vitro cell viability and metabolism, nitric oxide release and production of chemokines by cultured human dental pulp fibroblasts (DPF) under contact with HEMA and Single Bond. Cultures of DPF were established by means of an explant technique. Once plated, cells were kept under contact with increasing concentrations of HEMA (10, 100 and 1000 nM) or Single Bond (SB) [10-fold serially diluted in culture medium (10-4, 10-3 and 10-2 v/v)] and also with polymerized SB components. Cytotoxicity was assessed by Trypan Blue exclusion method and MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. Nitric oxide release on cell supernatant was detected by Griess Method whereas chemokines (CXCL12 and CXCL8) were detected by ELISA. RT-qPCR was employed for chemokines gene expression analysis. Cytotoxic tests showed significant differences for SB 10-2. None of the tested materials significantly altered NO levels. Protein levels of CXCL12 were significantly decreased only by HEMA. On the other hand, while CXCL12 mRNA remained unaltered, gene expression of CXCL8 had significant decrease with all materials, except for polymerized SB. In conclusion, Single Bond and HEMA at various concentrations, decreased expression and production of molecules involved in inflammatory processes and, therefore, the use of adhesive systems such as pulp capping materials must be viewed with caution due to its large cytotoxic effect when in close contact with the pulp.

Resumo Este estudo avaliou in vitro a viabilidade e metabolismo celular, liberação de óxido nítrico e produção de quimiocinas em cultura de fibroblastos de polpa dental humana (DPF) em contato com HEMA e Single Bond. Culturas de DPF foram estabelecidas por meio de uma técnica de explante. Uma vez plaqueadas, as células foram mantidas em contato com concentrações crescentes de HEMA (10, 100 e 1000 nM) ou Single Bond (SB) [10 vezes diluídas em série em meio de cultura (10-4, 10-3 e 10-2 v/v)] e também com SB polimerizado. A citotoxicidade foi avaliada pelo método de exclusão de Trypan Blue e pelo ensaio de 3-(4,5-dimetiltiazol-2-il)-2,5-difeniltetrazólio brometo (MTT). A liberação de óxido nítrico no sobrenadante celular foi detectada pelo método de Griess, enquanto as quimiocinas (CXCL12 e CXCL8) foram detectadas por ELISA. RT-qPCR foi empregada para análise de expressão gênica de quimiocinas. Testes citotóxicos mostraram diferenças significativas para SB 10-2. Nenhum dos materiais testados alterou significativamente os níveis de NO. Os níveis de proteína de CXCL12 foram significativamente diminuídos apenas pelo HEMA. Por outro lado, enquanto o RNAm de CXCL12 permaneceu inalterado, a expressão gênica de CXCL8 teve redução significativa com todos os materiais, com exceção do SB polimerizado. Em conclusão, Single Bond e HEMA, em várias concentrações, diminuíram a expressão e produção de moléculas envolvidas em processos inflamatórios e, portanto, o uso de sistemas adesivos, como o material protetor da polpa, deve ser visto com cautela devido ao seu grande efeito citotóxico quando em contato com a polpa.

Liver ischemia and reperfusion injury. Pathophysiology and new horizons in preconditioning and therapy

Abstract It is well known that during hepatic operative procedures, it is often critical that the irrigation is interrupted to avoid possible bleeding, blood transfusions, variable intensities, and their short and long-term consequences. It was believed in the past that the flow interruption should not exceed 20 minutes, which limited the use of this maneuver. However, it has been postulated that ischemia could be maintained for more than 60 minutes in healthy livers. The present paper review includes: 1) A brief introduction to justify the rationale of the review design; 2) Aspects of the pathophysiology of the three stages of the liver ischemia-reperfusion injury; 3) The innate and acquired immunity; 4) Oxidative stress; 5) Apoptosis and autophagy, Some essential biomarkers (Tumor Necrosis Factor-α, nitric oxide, metalloproteinases); and, finally; 6) Preventive ("cheating") strategies, non-pharmacological and pharmacological options to treat the liver IR injury.

Vasorelaxant activity of Euphorbia furcillata Kunth mainly by activation of NO/cGMP pathway and calcium channel blockade / Actividad vasorrelajante de Euphorbia furcillata Kunth principalmente por activación de la vía NO/GMPc y bloqueo de canales de calcio]

The aim of current study was to determinate ex vivo and chromatographic fingerprint by HPLC of four extracts of Euphorbia furcillata K. Ethyl acetate extract of Euphorbia furcillata (EaEEf) was the most effective and potent extract (Emax=98.69±1.24%) and its effect was partially endothelium-dependent. Functional vasorelaxant mechanism of action of EaEEf was determinate, EaEEf showed efficient relaxation of KCl [80 mM]-induced contraction and norepinephrine and CaCl2 contraction curves showed diminution of maximal contraction in the presence of EAEEf and EaEEf-relaxation curve was shifted to the right in the presence of L-NAME (nitric oxide synthase inhibitor) and ODQ (guanylate cyclase inhibitor). Chromatographic fingerprints analysis suggests presence of diterpenoid such as abietane, tigliane, and ingenane skeletons. Our experiments suggest the EaEEf vasorelaxant activity could be attributed to diterpenoid molecules whose mechanism involves nitric oxide production and calcium channel blockade.

Se determinó el efecto vasorrelajante ex vivo y los perfiles cromatográficos mediante HPLC de cuatro extractos de Euphorbia furcillata K.. El extracto de acetato de etilo de E. furcillata (EaEEf) fue el más eficaz y potente en la contracción inducida por norepinefrina (Emax=98.69±1.24%) y el efecto fue parcialmente dependiente del endotelio vascular. Se determinó el mecanismo de acción vasorrelajante para EaEEf, este mostró ser eficaz sobre la contracción inducida por KCl [80 mM] y la curva de contracción en respuesta a norepinefrina y CaCl2 en presencia de EaEEf mostró disminución en la contracción máxima, mientras que la curva de relajación de EaEEf en presencia de L-NAME (inhibidor de óxido nítrico sintasa) y ODQ (inhibidor de guanilato ciclasa) se desplazó hacia la derecha. El análisis cromatográfico de EaEEf sugiere la presencia de moléculas diterpenoides como abietano, tigliano y esqueletos de ingenano. Nuestros resultados sugieren que el efecto vasorrelajante de EaEEf podría atribuirse a moléculas diterpenoides, cuyo mecanismo de acción involucra la producción de óxido nítrico y bloqueo de canales de calcio.