Cerebrolysin alleviates early brain injury after traumatic brain injury by inhibiting neuroinflammation and apoptosis via TLR signaling pathway

Purpose: Traumatic brain injury (TBI) is a major cause of death and disability. Cerebrolysin (CBL) has been reported to be anti-inflammatory by reducing reactive oxygen species (ROS) production. However, the neuroprotection of CBL in TBI and the potential mechanism are unclear. We aimed to investigate the neuroprotection and mechanisms of CBL in TBI. Methods: The TBI model was established in strict accordance with the Feeney weight-drop model of focal injury. The neurological score, brain water content, neuroinflammatory cytokine levels, and neuronal damage were evaluated. The involvement of the early brain injury modulatory pathway was also investigated. Results: Following TBI, the results showed that CBL administration increased neurological scores and decreased brain edema by alleviating blood­brain barrier (BBB) permeability, upregulating tight junction protein (ZO­1) levels, and decreasing the levels of the inflammatory cytokines tumor necrosis factor­α (TNF­α), interleukin­1ß (IL­1ß), IL­6, and NF­κB. The TUNEL assay showed that CBL decreased hippocampal neuronal apoptosis after TBI and decreased the protein expression levels of caspase­3 and Bax, increasing the levels of Bcl­2. The levels of Toll­like receptor 2 (TLR2) and TLR4 were significantly decreased after CBL treatment. In TBI patients, CBL can also decrease TNF­α, IL­1ß, IL­6, and NF­κB levels. This result indicates that CBL­mediated inhibition of neuroinflammation and apoptosis ameliorated neuronal death after TBI. The neuroprotective capacity of CBL is partly dependent on the TLR signaling pathway. Conclusions: Taken together, the results of this study indicate that CBL can improve neurological outcomes and reduce neuronal death against neuroinflammation and apoptosis via the TLR signaling pathway in mice.

Perillyl alcohol has antibacterial effects and reduces ROS production in macrophages

Abstract Natural products have emerged as a rich source of bioactive compounds for adjunctive treatments of many infectious and inflammatory conditions, including periodontitis. Among the monoterpenes with significant biological properties, there is the perillyl alcohol (POH), which can be found in several essential oils and has shown immunomodulatory properties in recent studies, which may be interesting in the treatment of non-neoplastic inflammatory disorders. Objective To determine the antibacterial and immune modulatory activities of the POH. Methodology The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of the POH for two significant Gram-negative periodontal pathogens were determined by macrodilution and subculture, respectively. Cell proliferation and cytotoxicity in RAW 264.7 macrophages were determined by Trypan Blue and mitochondrial enzymatic activity assay. The modulation of reactive oxygen species (ROS) was analyzed by flow cytometry and expression of TNF and arginase-1 by real-time PCR. Results The POH was effective against P. gingivalis (ATCC 33277) and F. nucleatum (ATCC 25586) with MIC= MBC=1600 μM. No cytotoxicity up to 100 µM was observed on macrophages. The cell proliferation was inhibited from 48 hours at 100 μM (p<0.05) and 250 μM (p<0.01). The POH increased ROS production at both 10 μM and 100 μM (p<0.05) in unstimulated cells. The PMA-induced ROS production was not affected by POH, whereas 100 μM significantly reduced lipopolysaccharide-induced (LPS-induced) ROS. The expression of TNF was not affected by POH in unstimulated cells or in cells polarized to M1 phenotype, whereas both concentrations of POH reduced (p<0.05) the expression of arginase-1 in M2-polarized macrophages. Conclusion The POH has antibacterial activity against periodontal pathogens and reduced proliferation of murine macrophages without significant cytotoxicity at concentrations up to 100 μM. In addition, the POH reduced the LPS-induced ROS and the expression of arginase-1 in M2-polarized macrophages.

Reduction of oxidative stress improves insulin signaling in cardiac tissue of obese mice / Redução do estresse oxidativo melhora a sinalização da insulina em tecido cardíaco de camundongos obesos

ABSTRACT Objective To evaluate the effects of oxidative stress on insulin signaling in cardiac tissue of obese mice. Methods Thirty Swiss mice were equally divided (n=10) into three groups: Control Group, Obese Group, and Obese Group Treated with N-acetylcysteine. After obesity and insulin resistance were established, the obese mice were treated with N-acetylcysteine at a dose of 50mg/kg daily for 15 days via oral gavage. Results Higher blood glucose levels and nitrite and carbonyl contents, and lower protein levels of glutathione peroxidase and phosphorylated protein kinase B were observed in the obese group when compared with their respective control. On the other hand, treatment with N-acetylcysteine was effective in reducing blood glucose levels and nitrite and carbonyl contents, and significantly increased protein levels of glutathione peroxidase and phosphorylated protein kinase B compared to the Obese Group. Conclusion Obesity and/or a high-lipid diet may result in oxidative stress and insulin resistance in the heart tissue of obese mice, and the use of N-acetylcysteine as a methodological and therapeutic strategy suggested there is a relation between them.

RESUMO Objetivo Avaliar os efeitos do estresse oxidativo sobre a sinalização da insulina em tecido cardíaco de camundongos obesos. Métodos Utilizaram-se 30 camundongos Swiss subdivididos igualmente (n=10) em três grupos: Grupo Controle, Grupo Obeso e Grupo Obeso Tratado com N-acetilcisteína. Após estabelecidas a obesidade e a resistência à insulina, os camundongos obesos foram tratados diariamente, durante 15 dias, via gavagem oral, com N-acetilcisteína na dose de 50mg/kg. Resultados Observaram-se maiores níveis de glicose sanguínea, conteúdos de nitrito e carbonil, e menores níveis proteicos de glutationa peroxidase e proteína quinase B fosforilada no Grupo Obeso quando comparado a seu respectivo controle. Por outro lado, o tratamento com N-acetilcisteína se mostrou eficiente em diminuir os níveis glicêmicos, os conteúdos de nitrito e carbonil, e aumentar significativamente os níveis proteicos de glutationa peroxidase e proteína quinase B fosforilada, quando comparados ao Grupo Obeso. Conclusão Obesidade e/ou dieta hiperlipídica levam a estresse oxidativo e à resistência à insulina no tecido cardíaco de camundongos obesos, e o uso da N-acetilcisteína como estratégia metodológica e terapêutica sugeriu haver relação entre ambos.

Evaluation of the therapeutic effect of methylene blue on the liver of rats submitted to ischemia and reperfusion

Abstract Purpose: To analyze the effect of methylene blue (MB) therapy during the liver ischemia-reperfusion injury (I/R) process. Methods: Thirty-five male Wistar rats were used, (70%) submitted to partial ischemia (IR) or not (NIR) (30%) were obtained from the same animal. These animals were divided into six groups: 1) Sham (SH), 2) Sham with MB (SH-MB); 3) I/R, submitted to 60 minutes of partial ischemia and 15 minutes of reperfusion; 4) NI/R, without I/R obtained from the same animal of group I/R; 5) I/R-MB submitted to I/R and MB and 6) NI/R-MB, without I/R. Mitochondrial function was evaluated. Osmotic swelling of mitochondria as well as the determination of malondialdehyde (MDA) was evaluated. Serum (ALT/AST) dosages were also performed. MB was used at the concentration of 15mg/kg, 15 minutes before hepatic reperfusion. Statistical analysis was done by the Mann Whitney test at 5%. Results: State 3 shows inhibition in all ischemic groups. State 4 was increased in all groups, except the I/R-MB and NI/R-MB groups. RCR showed a decrease in all I/R and NI/R groups. Mitochondrial osmotic swelling showed an increase in all I/R NI/R groups in the presence or absence of MB. About MDA, there was a decrease in SH values in the presence of MB and this decrease was maintained in the I/R group. AST levels were increased in all ischemic with or without MB. Conclusions: The methylene blue was not able to restore the mitochondrial parameters studied. Also, it was able to decrease lipid peroxidation, preventing the formation of reactive oxygen species.

Intracellular reactive oxygen species production and phagocytosis of Staphylococcus aureus by milk neutrophils as tool to diagnose mastitis and identify susceptible dairy cows / Produção intracelular de espécies reativas de oxigênio e fagocitose de Staphylococcus aureus por neutrófilos lácteos como ferramentas para o diagnóstico da mastite e identificação de vacas leiteiras susceptíveis à mastite

The immune response capacity of the mammary gland plays a major role to determine if mastitis will or not be established. Thus, we hypothesize that a better understanding of polymorphonuclear neutrophil leukocyte (PMN) function will elucidate mechanisms that will improve our knowledge of how we could avoid an inflammatory process by increasing the immune capacity of the cow, and even further, to search for a tool to diagnose mastitis or a possible way to select and identify non-susceptible animals. The present study utilized 112 quarters from 28 Holstein dairy cows that were divided into quarters milk samples with somatic cell count (SCC) <2×105 cells mL-1 (n=72) and SCC >2×105 cells mL-1 (n=40). The percentages of milk PMNs and the levels of intracellular reactive oxygen species (ROS) and the phagocytosis of Staphylococcus aureus by milk neutrophils were evaluated by flow cytometry. Our results showed a higher percentage of neutrophils in quarter milk samples with high SCC (P=0.0003), and this group also had a significantly higher percentage of neutrophils that produced ROS (P=0.008). On the other hand, the phagocytosis intensity of S. aureus by milk neutrophils was higher in quarters with low SCC (P=0.003), suggesting a better mammary gland immunity against invading pathogens. Analyzing the results of the predictive values of the measured PMN functions, they cannot be used isolated as a good diagnosis test since none of them had a satisfactory sensitivity and specificity values, which was also confirmed by the Youden index values being far from one. In conclusion, the assessment of milk bovine neutrophil functions could improve our understanding of the cellular basis of mastitis. Although, the intracellular ROS production and S. aureus phagocytosis by milk neutrophil did not have high predictive values to detect intramammary infections, our results strengthen the idea that that poor bovine mammary gland neutrophil phagocytic ability may be associated with high SCC, and might be considered to identify susceptible dairy cows to mastitis.(AU)

A resposta imune da glândula mamária desempenha um papel importante ao determinar o estabelecimento da infecção. Desta forma, a melhor compreensão da função dos neutrófilos irá nos subsidiar conhecimentos, pelo qual podemos evitar o processo inflamatório pela otimização da resposta imune de bovinos leiteiros, e fornece ferramentas para diagnosticar a mastite ou um possível instrumento para identificar e selecionar animais resistentes à infecção intramamária, aumentando a produtividade do rebanho. O presente estudo utilizou 112 amostras provenientes de quartos mamários de 28 vacas Holandesas que foram divididos em amostras de leite com baixa (n=72; <2×105 células mL-1) ou alta (n=40; 2×105 células mL-1) contagem de células somáticas (CCS). A porcentagem de neutrófilos no leite, a produção intracelular de espécies reativas de oxigênio (ERO) e a fagocitose de Staphylococcus aureus pelos neutrófilos do leite foram avaliadas por citometria de fluxo. Os resultados do presente estudo demonstraram maior percentagem de neutrófilos (CH138+; P=0,0003) e percentagem de neutrófilos que produziram ERO (P=0,008) em amostras de leite com alta CCS. Por outro lado, a intensidade de fagocitose de S. aureus por neutrófilos em amostras de leite com baixa CCS (P=0,003), que demonstra maior atividade funcional destas células neste grupo. As funções neutrofílicas para o diagnóstico da mastite não apresentaram valores de sensibilidade e especificidade altos, que foram confirmados pelo índice Youden. Desta forma, conclui-se que a produção intracelular de ERO e fagocitose de S. aureus pelos neutrófilos do leite não apresentaram valores preditivos altos para detecção de mastite. Além disto, os resultados do presente estudo reforçam a ideia de neutrófilos do leite com menor capacidade fagocítica podem ser associados à alta CCS, e pode ser considerado como uma ferramenta para identificar animais mais susceptíveis à infecções intramamárias.(AU)

Cryotherapy: biochemical alterations involved in reduction of damage induced by exhaustive exercise

When exercises are done in intense or exhaustive modes, several acute biochemical mechanisms are triggered. The use of cryotherapy as cold-water immersion is largely used to accelerate the process of muscular recovery based on its anti-inflammatory and analgesic properties. The present study aimed to study the biochemical effects of cold-water immersion treatment in mice submitted to exercise-induced exhaustion. Swiss albino mice were divided into 4 treatment groups: control, cold-water immersion (CWI), swimming exhaustive protocol (SEP), and SEP+CWI. Treatment groups were subdivided into times of analysis: 0, 1, 3, and 5 days. Exhaustion groups were submitted to one SEP session, and the CWI groups submitted to one immersion session (12 min at 12°C) every 24 h. Reactive species production, inflammatory, cell viability, and antioxidant status were assessed. The SEP+CWI group showed a decrease in inflammatory damage biomarkers, and reactive species production, and presented increased cell viability compared to the SEP group. Furthermore, CWI increased acetylcholinesterase activity in the first two sessions. The present study showed that CWI was an effective treatment after exercise-induced muscle damage. It enhanced anti-inflammatory response, decreased reactive species production, increased cell viability, and promoted redox balance, which could decrease the time for the recovery process.

Avaliação do efeito antiproliferativo da apocinina e diapocinina em células de osteossarcoma humano / Evaluation of the antiproliferative effect of apocynin and diapocynin on human osteosarcoma cells

O osteossarcoma (OS) é o tumor maligno primário mais comum do tecido ósseo, caracterizado pela formação de osteócitos anormais. Apesar do avanço nas terapias convencionais (quimioterapia e retirada do tumor), essas não conseguem eliminar totalmente as células tumorais e impedir a progressão da doença. Recentemente, agentes derivados de fontes naturais ganharam considerável atenção por causa de sua segurança, eficácia e disponibilidade imediata. Nesse sentido, a apocinina, inibidor do complexo NADPH-oxidase, vem sendo estudada como agente antitumoral em alguns tipos de câncer como: pâncreas, próstata, pulmão e mama. Apocinina é um pró-fármaco e sua ação parece estar relacionada à sua conversão produzindo a diapocinina, a qual se mostrou mais efetiva do que a apocinina. Portanto, o objetivo desse estudo é avaliar, in vitro, o potencial antitumoral da apocinina e diapocinina em células de osteossarcoma humano. Para isso, foram utilizados osteoblastos humanos normais (HOb) e osteossarcoma humano imortalizadas (SaOS-2) tratados ou não com apocinina e diapocinina em diversas concentrações. Foram realizados os ensaios de viabilidade celular, alterações morfológicas, apoptose celular, produção de espécies reativas de oxigênio (EROs), formação de colônias, migração, invasão e expressão do fator indutor de hipóxia-1alfa (HIF-1). Também foram conduzidos ensaios para verificar a atividade de metaloproteinase de matriz (MMP) 2 e 9. Os resultados em SaOS-2 mostraram que o tratamento com apocinina nas concentrações de 1,5 e 3 mM; e diapocinina nas concentrações de 0,75 e 1,5 mM reduziram a viabilidade; aumentaram o número de células em apoptose e diminuíram a produção de EROs; sem causar danos às células HOb. Além disso, essas mesmas concentrações inibiram a migração e invasão celular; diminuíram a expressão de HIF-1; e reduziram a atividade de MMP-2 em SaOS-2. Considerando os resultados obtidos, concluímos que a apocinina e diapocinina podem atuar como possíveis moduladores de células tumorais, sendo que a diapocinina mostrou ser mais efetiva nos parâmetros testados.(AU)

Osteosarcoma (OS) is the most common primary malignant tumor of bone tissue, characterized by the formation of abnormal osteocytes. Despite advances in conventional therapies (chemotherapy and surgery) they cannot completely eliminate tumor cells and prevent the progression of the disease. Recently, agents derived from natural sources have achieved considerable attention because of their safety, efficacy and immediate availability of therapies. In this way, apocynin, an inhibitor of the NADPH-oxidase complex, has been studied as an antitumor agent in some types of cancer, such as pancreas, prostate, lung and breast. Apocynin is a prodrug and its action indicate to be related to its conversion to diapocynin, which has been shown to be more efficient than apocynin itself. Thus, the aim of this study is to evaluate, in vitro, the antitumor potential of apocynin and diapocynin in human osteosarcoma cells. For this, normal human osteoblasts (HOb) and immortalized human osteosarcoma cells (SaOS-2) were treated or no-treated with apocynin and diapocynin in various concentrations. Cell viability assay, morphological alterations, cellular apoptosis, reactive oxygen species (ROS) production, colony formation, migration, invasion and expression of hypoxia-inducible factor-1 alpha (HIF-1) were performed. We also performed assays to verify the activity of matrix metalloproteinase (MMP) 2 and 9. The results in SaOS-2 showed that treatment with apocynin at concentrations of 1,5 e 3 mM; and diapocynin at concentrations of 0,75 e 1,5 mM reduced cell viability; increased the number of cells in apoptosis and decreased the production of ROS; without damaging HOb cells. Moreover, these same concentrations inhibited cell migration and invasion; decreased HIF-1 expression; and reduced MMP 2 activity in SaOS-2. Considering the results, we suggest that apocynin and diapocynin may act as possible modulators of tumor cells, and diapocynin has been shown to be more effective.(AU)

Reactive oxygen species generation mediated by NADPH oxidase and PI3K/Akt pathways contribute to invasion of Streptococcus agalactiae in human endothelial cells

BACKGROUND Streptococcus agalactiae can causes sepsis, pneumonia, and meningitis in neonates, the elderly, and immunocompromised patients. Although the virulence properties of S. agalactiae have been partially elucidated, the molecular mechanisms related to reactive oxygen species (ROS) generation in infected human endothelial cells need further investigation. OBJECTIVES This study aimed to evaluate the influence of oxidative stress in human umbilical vein endothelial cells (HUVECs) during S. agalactiae infection. METHODS ROS production during S. agalactiae-HUVEC infection was detected using the probe CM-H2DCFDA. Microfilaments labelled with phalloidin-FITC and p47phox-Alexa 546 conjugated were analysed by immunofluorescence. mRNA levels of p47phox (NADPH oxidase subunit) were assessed using Real Time qRT-PCR. The adherence and intracellular viability of S. agalactiae in HUVECs with or without pre-treatment of DPI, apocynin (NADPH oxidase inhibitors), and LY294002 (PI3K inhibitor) were evaluated by penicillin/gentamicin exclusion. Phosphorylation of p47phox and Akt activation by S. agalactiae were evaluated by immunoblotting analysis. FINDINGS Data showed increased ROS production 15 min after HUVEC infection. Real-Time qRT-PCR and western blotting performed in HUVEC infected with S. agalactiae detected alterations in mRNA levels and activation of p47phox. Pre-treatment of endothelial cells with NADPH oxidase (DPI and apocynin) and PI3K/Akt pathway (LY294002) inhibitors reduced ROS production, bacterial intracellular viability, and generation of actin stress fibres in HUVECs infected with S. agalactiae. CONCLUSIONS ROS generation via the NADPH oxidase pathway contributes to invasion of S. agalactiae in human endothelial cells accompanied by cytoskeletal reorganisation through the PI3K/Akt pathway, which provides novel evidence for the involvement of oxidative stress in S. agalactiae pathogenesis.

Adiponectin is protective against endoplasmic reticulum stress-induced apoptosis of endothelial cells in sepsis

Endoplasmic reticulum (ER) stress is a critical molecular mechanism involved in the pathogenesis of sepsis. Hence, strategies for alleviating this stress may be essential for preventing cardiovascular injuries under sepsis. Adiponectin is secreted by adipocytes and its levels are decreased in sepsis. The purpose of this study was to investigate the protective effects of adiponectin treatment on endothelial cells and its mechanism. Male Wistar rats underwent cecal ligation and puncture (CLP) before being treated with adiponectin (72 and 120 μg/kg). The levels of malondialdehyde (MDA) in plasma, histological structure, and apoptosis of endothelial cells were evaluated. In vitro, human umbilical vein endothelial cells (HUVECs) were treated with adiponectin at 10 and 20 μg/mL for 24 h after stimulation by lipopolysaccharide (LPS). The levels of reactive oxygen species (ROS), ultrastructure, rate of apoptosis, the expression of inositol-requiring enzyme 1α (IRE1α) protein, and its downstream molecules (78 kDa glucose-regulated protein (GRP78), C/EBP homologous protein (CHOP), and caspase-12) were detected. The results showed that the levels of MDA and ROS induced by CLP or LPS stimulation were increased. Furthermore, endothelial cell apoptosis was increased under sepsis. The IRE1α pathway was initiated, as evidenced by activated IRE1α, increased GRP78, and up-regulated CHOP and caspase-12 in HUVECs. Following treatment with adiponectin, the number of apoptotic endothelial cells was markedly decreased. These findings demonstrated that treatment with adiponectin decreased apoptosis of endothelial cells caused by sepsis by attenuating the ER stress IRE1α pathway activated by oxidative stress.

CIRBP protects H9C2 cells against myocardial ischemia through inhibition of NF-kappaB pathway

Myocardial ischemia is a major cause of death and remains a disease with extremely deficient clinical therapies and a major problem worldwide. Cold inducible RNA-binding protein (CIRBP) is reported to be involved in multiple pathological processes, including myocardial ischemia. However, the molecular mechanisms of myocardial ischemia remain elusive. Here, we first overexpressed CIRBP by transfection of pc-CIRBP (pcDNA3.1 containing coding sequenced for CIRBP) and silenced CIRBP by transfection of small interfering RNA targeting CIRBP (siCIRBP). pcDNA3.1 and the negative control of siCIRBP (siNC) were transfected into H9C2 cells to act as controls. We then constructed a cell model of myocardial ischemia through culturing cells in serum-free medium with hypoxia in H9C2 cells. Subsequently, AlamarBlue assay, flow cytometry and western blot analysis were used, respectively, to assess cell viability, reactive oxygen species (ROS) level and apoptosis, and expression levels of IκBα, p65 and Bcl-3. We demonstrated that CIRBP overexpression promoted cell proliferation (P<0.001), inhibited cell apoptosis (P<0.05), reduced ROS level (P<0.001), down-regulated phosphorylated levels of IκBα and p65 (P<0.01 or P<0.001), and up-regulated expression of Bcl-3 (P<0.001) in H9C2 cells with myocardial ischemia. The influence of CIRBP knockdown yielded opposite results. Our study revealed that CIRBP could protect H9C2 cells against myocardial ischemia through inhibition of NF-κB pathway.

Protective effects of deferasirox and N-acetyl-L-cysteine on iron overload-injured bone marrow

Using an iron overload mouse model, we explored the protective effect of deferasirox (DFX) and N-acetyl-L-cysteine (NAC) on injured bone marrow hematopoietic stem/progenitor cells (HSPC) induced by iron overload. Mice were intraperitoneally injected with 25 mg iron dextran every 3 days for 4 weeks to establish an iron overload (Fe) model. DFX or NAC were co-administered with iron dextran in two groups of mice (Fe+DFX and Fe+NAC), and the function of HSPCs was then examined. Iron overload markedly decreased the number of murine HSPCs in bone marrow. Subsequent colony-forming cell assays showed that iron overload also decreased the colony forming capacity of HSPCs, the effect of which could be reversed by DFX and NAC. The bone marrow hematopoiesis damage caused by iron overload could be alleviated by DFX and NAC.

Marcadores salivares para a periodontite crônica no diabético tipo 2 / Salivar markers for chronic periodontitis in diabetic type 2

Introdução: a periodontite crônica é a sexta doença infecciosa mais prevalente no mundo, seus fatores de risco são o aumento da idade, tabaco, fatores genéticos, obesidade e distúrbios sistêmicos como o diabetes. O diabetes é uma pandemia tanto em países desenvolvidos como em desenvolvimento, é uma doença complexa com graus variados de complicações sistêmicas e orais. Assim, é urgente a necessidade de um método fácil para detectá-la permitindo a intervenção antes da progressão da doença periodontal. Á análise de metabólitos da saliva tem sido proposta como uma ferramenta efetiva para o diagnóstico e tratamento periodontal. Objetivo: discorrer sobre alternativas de diagnóstico de periodontite crônica em pacientes diabéticos tipo 2 utilizando marcadores salivares. Metodologia: Foi realizada uma busca eletrônica no Portal de Periódicos da Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), para a realização do referido levantamento, utilizou-se os descritores "Type 2 diabetes mellitus, chronic periodontitis, Biomarkers, Saliva". Resultados: a detecção precoce da periodontite crônica não é apenas vital para reduzir a sua gravidade e prevenir complicações, mas também crítico para aumentar a taxa de sucesso da terapia. Conclusão: a utilização de biomarcadores na medicina é uma realidade difundida e tem relação direta com a modernização dos meios de saúde, na odontologia vem se pleiteando seu uso, mas ainda necessita de maiores estudos e aperfeiçoamentos das técnicas já empregadas com o intuito de diminuir seus custos, aumentar especificidade e acesso dos profissionais de saúde.(AU)

Introduction: Chronic periodontitis is the sixth most prevalent infectious disease in the world. Its risk factors are increased age, tobacco, genetic factors, obesity, and systemic disorders such as diabetes. Diabetes is a pandemic in both developed and developing countries, it is a complex disease with varying degrees of systemic and oral complications. Thus, there is an urgent need for an easy method to detect it allowing intervention before the progression of periodontal disease. The analysis of saliva metabolites has been proposed as an effective tool for periodontal diagnosis and treatment. Objective: To discuss alternatives for diagnosis of chronic periodontitis in type 2 diabetic patients using salivary markers. Methodology: An electronic search was performed in the Portal of Periodicals of the Coordination of Improvement of Higher Level Personnel (CAPES). The descriptors "Type 2 diabetes mellitus, chronic periodontitis, Biomarkers, Saliva" were used to perform this survey. Results: early detection of chronic periodontitis is not only vital to reduce its severity and prevent complications, but also critical to increase the success rate of therapy. Conclusion: the use of biomarkers in medicine is a widespread reality and has a direct relationship with the modernization of health facilities. In dentistry, its use has been sought, but it still needs further studies and improvements of techniques already employed with the intention of reducing its Increase the specificity and access of health professionals.(AU)

Effect of ß-mercaptoetanol and cysteine on post-thawing quality and oxidative activity of ram sperm and on the viability of vitrified sheep embryos / Efeito do ß-mercaptoetanol e da cisteína sobre a qualidade e a atividade oxidativa do sêmen ovino após o descongelamento e sobre a viabilidade de embriões ovinos vitrificados

The effects of ß-mercaptoethanol (BME) and cysteine on the viability and oxidative activity of ram sperm after thawing and on development in vitro and viability of vitrified sheep embryos were evaluated. Ejaculates from four rams were pooled and extended, composing six treatments: no antioxidants; 2mM BME; 5mM BME; 2mM BME and 5mM cysteine; 5mM BME and 5mM cysteine; and 5mM cysteine. Sperm motility, membrane and acrosome integrity, mitochondrial functionality, production of reactive oxygen species and total antioxidant capacity were similar across treatments (P>0.05). A medium with no antioxidant presented cleavage and blastocyst development rates (60.3% and 33.6%, respectively) similar (P>0.05) to those of a medium with 50µM BME and 600µM cysteine (64.3% and 36.6%, respectively). Post-thawing viability of vitrified embryos was similar between media (P>0.05). Cysteine and BME had no influence on the post-thawing viability and oxidative activity of ram sperm and on the viability of vitrified sheep embryos.(AU)

Foram avaliados os efeitos do ß-mercaptoetanol (BME) e da cisteína sobre a viabilidade e a atividade oxidativa após o descongelamento do sêmen ovino e sobre o desenvolvimento in vitro e a viabilidade de embriões ovinos vitrificados. Ejaculados de quatro carneiros foram agrupados e diluídos, compondo seis tratamentos: sem antioxidantes; com BME 2mM; com BME 5mM; com BME 2mM e cisteína 5mM; com BME 5mM e cisteína 5mM; e com cisteína 5mM. Motilidade, integridade da membrana e do acrossoma, função mitocondrial, produção de espécies reativas de oxigênio e capacidade antioxidante total foram semelhantes entre os tratamentos (P>0,05). Em um meio sem antioxidantes, as taxas de clivagem e de desenvolvimento embrionário até blastocisto (60,3%, e 33,6%, respectivamente) foram semelhantes (P>0,05) às obtidas em um meio com BME 50µM e cisteína 600µM (64,3% e 36,6%, respectivamente). A viabilidade pós-descongelamento dos embriões vitrificados não diferiu entre os meios (P>0,05). O BME e a cisteína não influenciaram a viabilidade e a atividade oxidativa do sêmen ovino após o descongelamento e a viabilidade de embriões ovinos vitrificados.(AU)

Oxidative stress in carotid arteries of patients submitted to carotid endarterectomy. The role of aging process

ABSTRACT PURPOSE: To evaluated the role of oxidative stress on aging process in patients submitted to carotid endarterectomy. METHODS: Twenty patients were divided into two groups: older group (≥ 70 years old); and the younger group (< 70 years old). We evaluated the reactive oxygen species (ROS) concentration, nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase, superoxide dismutase (SOD) and catalase (CAT) activities as so as nitrite levels in fragments of carotid arteries harvested during carotid endarterectomy for treatment of high grade carotid stenosis. RESULTS: We observed a higher levels of ROS and NADPH oxidase activity in the older group (p<0.05). Furthermore, the nitrite concentration was lower in the older group (14.55 ± 5.61 x 10-3 versus 26.42 ± 8.14 x 10-3 µM; p=0.0123). However, the activities of antioxidant enzymes (CAT and SOD) were similar in both the groups. CONCLUSIONS : Arterial aging is associated with increased concentrations of oxygen species and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity as so as nitrite reduction in human carotid artery specimens. Maybe therapies that block NADPH oxidase activity and enhance nitrite stores would be a good strategy to reduce the effect of oxidative stress in arterial aging.

Efeito de diferentes concentrações de melatonina em espermatozoides de carneiros sobre estresse oxidativo após criopreservação / Effect of different concentrations of melatonin to ram spermatozoa on oxidative stress after cryopreservation

Objetivou-se avaliar o efeito da adição de diferentes concentrações de melatonina no sêmen diluído de carneiros após criopreservação. Foram coletados 10 ejaculados de três carneiros adultos (n=30), por meio de vagina artificial para ovinos. Os ejaculados coletados foram diluídos em Tris-Gema de ovo, para a concentração final de 200x106 sptz/mL, mantidos em banho maria a 32°C, e a melatonina adicionada conforme os tratamentos: Controle; 100pM; 100nM; 100µM e 1mM de melatonina. Então, as amostras foram resfriadas em câmara fria a 5°C por duas horas, envasadas em palhetas de 0,5 mL e lacradas. Logo após, foram acondicionadas sob vapores do nitrogênio liquido, por 15 minutos, a 8cm da lâmina líquida e congeladas com nitrogênio líquido. As amostras foram analisadas quanto à motilidade espermática, integridade da membrana plasmática, membrana acrossomal, atividade mitocondrial, quantificação do estresse oxidativo e a capacidade de ligação. As variáveis foram submetidas à análise de variância e as médias foram comparadas pelo teste de Tukey a 5% de probabilidade. A motilidade total e progressiva dos espermatozoides descongelados foi maior nas amostras tratadas com 100pM de melatonina (62,99 e 45,07% respectivamente; P<0,05) quando comparado aos demais tratamentos. A adição das diferentes concentrações de melatonina no sêmen diluído, com exceção da concentração de 1 mM, apresentou maior percentual de células com membrana plasmática íntegra, quando comparadas com o controle (P<0,05). O percentual de espermatozoides com integridade da membrana do acrossoma foi maior no sêmen tratado com 100 pM de melatonina (P<0,05) do que nos demais tratamentos. A alta atividade mitocondrial foi maior nos espermatozoides tratados com 100 pM de melatonina (69,30%; P<0,05). A adição de 100 nM de melatonina reduziu a quantidade de TBARS após a criopreservação (2,84; P<0,05) quando comparado aos demais tratamentos. Após o descongelamento, o número de espermatozoides que se ligaram à membrana perivitelina foi maior nos tratados com 100 pM de melatonina (155,73; P<0,05). Portanto, a adição de melatonina no sêmen diluído pode ser útil para aperfeiçoar a criopreservação do sêmen de ovinos, melhorando as taxas de fertilização por meio da inseminação artificial.(AU)

The aim was to evaluate the effect of adding different concentrations of melatonin in ram semen diluted after cryopreservation. Ten ejaculates were collected0 from three adult ram (n=30) by means of artificial vagina for sheep. The collected samples were diluted in Tris-egg yolk, to a final concentration 200x106 sptz/mL kept in water bath at 32°C, and melatonin added as treatments: control; 100pM; 100nM; 100µM and 1mM melatonin. Then, the samples were cooled in a cold chamber at 5°C for two hours, in straws of 0.5mL and sealed. They were stored under the liquid nitrogen vapor for 15 minutes to 8cm of liquid blade and frozen with liquid nitrogen. Samples were analyzed for sperm motility, membrane integrity, acrosomal membrane, mitochondrial activity, oxidative stress and quantification of the binding capacity. The variables were subjected to analysis of variance and the means were compared by Tukey test at 5% probability. The total and progressive motility of thawed sperm were higher in samples treated with 100pM melatonin (62.99 and 45.07%, respectively; P<0.05) when compared to other treatments. The addition of different concentrations of melatonin in semen diluted with the exception of 1mM concentration, a higher percentage of cells with intact plasma membrane, as compared with the control (P<0.05). The percentage of sperm with acrosome membrane integrity was higher in the semen with 100pM melatonin (P<0.05) than the other treatments. The high mitochondrial activity was higher in spermatozoa treated with 100pM melatonin (69.30%; P<0.05). Addition of 100nM melatonin reduced the amount of TBARS after cryopreservation (2.84, P<0.05) when compared with the other treatments. After thawing, the number of sperm which bind to the perivitelline membrane was higher in the melatonin treated with 100pM (155,73; P<0.05). Therefore, melatonin addition the semen diluted can be useful to enhance the cryopreservation of sheep semen, improving fertilization rates through artificial insemination.(AU)

Influência da suplementação de vitaminas A, D e E na função imune de bezerros alimentados com dieta á base de feno capim tifton (cynodon spp) / Influence of A, D and E vitamin supplements on immune function of calves fed diet tifton (cynodon spp) hay

As vitaminas são compostos orgânicos necessários em poucas quantidades no organismo, todavia indispensáveis para as funções metabólicas. Elas se inserem em inúmeras reações metabólicas, fisiológicas e imunes das células, necessárias para a manutenção da saúde animal, além de atuarem como imunoestimulante. Embora a dieta rica em folhagens verdes frescas forneça quantidades suficientes de vitaminas A, D e E a suplementação intensiva com alimentos conservados na forma de feno ou silagem pode reduzir em até 50 % dos teores destas vitaminas no alimento. Diante disso, a proposta do trabalho foi verificar se a administração parenteral de vitaminas A, D e E age como imunoestimulante em garrotes estabulados e alimentados exclusivamente com feno de tifton. Para tanto 14 bovinos foram divididos em dois grupos homogêneos, sendo o grupo S, suplementado com vitamina A, D e E em dose única de 30 mL por via intramuscular; e o grupo C, sem suplementação. Ambos os grupos foram alojados em baias parcialmente privadas de sol, e alimentados com feno por um período de três meses. A avaliação imune foi realizada por hemogramas e ensaio de função leucocitária (metabolismo oxidativo e fagocitose) nos momentos antes do tratamento, três e dez dias após os tratamentos. Tendo em vista que a suplementação com polivitamínicos A, D e E aumentou a porcentagem da atividade de células granulocítica e a intensidade da atividade de células mononucleares, além de intensificar o efeito antioxidante prolongando a sobrevida de hemácias e neutrófilos, conclui-se que esta suplementação promoveu efeito benéfico na resposta imune de bezerros da Raça Holandesa, apesar dos efeitos deletérios da alimentação exclusiva com feno e da privação parcial da incidência solar direta.

Vitamins are organic compounds which are required in small quantities in the body, however essential for the metabolic functions. They participate in numerous metabolic reactions, physiological and immune cells, needed to maintain animal health, as well as act as immunostimulants. Although the diet rich in fresh green foliage provides sufficient amounts of vitamin A, D and E, intensive supplementation with food stored in the form of hay or silage can reduce up to 50% of the levels of these vitamins in food. Given this, the proposal of this study was to verify how the parenteral administration of vitamins ADE acts as immunostimulant in steers fed exclusively with hay of tifton. For that, 14 cattle were divided into two homogeneous groups: Group S, supplemented with vitamin A, D e E given in a single intramuscular dose of 30mL, and Group C without supplementation. Both groups were housed in private stalls and fed with hay for a period of three months. Immune evaluation was performed by blood count and testing of leukocyte function (oxidative metabolism and phagocytosis) in the moments before treatment, three and ten days after the treatments. Considering that supplementation with vitamin A, D e E increased the percentage of granulocytic cell activity and the intensity of the activity of mononuclear cells, as well as intensified the antioxidant effect prolonging the survival of red blood cells and neutrophils, it can be concluded that this treatment had a beneficial effect on the immune response of Holstein calves, despite the damaging effects of exclusive feeding hay, and the partially deprivation of solar incidence.

Antioxidant effect of Legalon(r) SIL in ischemia-reperfusion injury of rat skeletal muscle

PURPOSE: To evaluated the potential antioxidant agent Legalon (r) SIL (silibinin-C-2',3-bis(hydrogensuccinat)) in the skeletal muscle of rats. METHODS: IRI was achieved via tourniquet application in Wistar-albino rats. Experimental groups were chosen as (i) sham control, (ii) IRI (3+2 h), (iii) IRI and Legalon (r) SIL-50 (50 mg/kg/i.p.), (iv) IRI and Legalon (r) SIL-100 (100 mg/kg/i.p.), and (v) IRI and Legalon (r) SIL-200 (200 mg/kg/ i.p.). Muscle viability (evaluated by triphenyltetrazolium chloride dye method), malondialdehyde, superoxide dismutase, catalase, and glutathione peroxidase were assessed in muscle samples using a spectrophotometer. RESULTS: Although viability of the injured limb non-significantly declined in the IRI group, administration of Legalon (r) SIL did not prevent injury. However, dramatic increase observed in malondialdehyde levels in the IRI group was prohibited by Legalon (r) SIL in a statistically significant manner. In comparison with the sham-control group, IRI and Legalon (r) SIL administration did not cause any significant alterations in the levels of superoxide dismutase, catalase, and glutathione peroxidase. CONCLUSION: Although Legalon (r) SIL was not sufficient to prevent muscle injury in terms of viability, it is found to be an effective option to reduce reactive oxygen species-induced cell injury.

Avaliação da peroxidação lipídica no plasma de ratos submetidos à lesão tecidual e tratados com hidrogel de poliamido de mandioca / Evaluación de la peroxidación de lípidos en plasma de ratas sometidas a lesión tisular y tratadas con hidrogel de poliamida de yuca / Evaluación de la peroxidación de lípidos en plasma de ratas sometidas a lesión tisular y tratadas con hidrogel de poliamida de yuca

As espécies reativas ao oxigênio (EROS) são produzidas como mecanismo de defesa celular, participando dos processos de cicatrização celular. Entretanto, altos níveis de EROS podem causar danos como a peroxidação lipídica (PL). O presente estudo teve como objetivo, verificar os níveis de PL por meio da determinação das substâncias reativas ao ácido tiobarbitúrico (TBARS) no plasma de ratos com lesão tecidual induzida. Foram utilizados 32 ratos machos, Rattus norvegicus albinus da linhagem Wistar, os quais foram pesados e da média ± 10% do peso foram distribuídos em quatro grupos: A ­ controle negativo; B - Vetaglós®; C ­ hidrogel de poliamido de mandioca+ Vetaglós®; D ­ Hidrogel de poliamido de mandioca. Após 21 dias, todos os animais foram anestesiados com isoflurano e foi feita a coleta de sangue por punção cardíaca, e os plasmas foram obtidos após centrifugação, na sequência por superdosagem do anestésico foi realizada a eutanásia. Os níveis de PL nos plasmas dos ratos foram determinados pelo método do TBARS. Não houve diferença significativa entre os grupos em relação à PL, indicando um equilíbrio entre as defesas antioxidantes celulares e os níveis de EROS produzidos durante o processo de cicatrização celular. Essa ausência nos diferentes grupos experimentais, em relação à PL, deixa claro a importância de se contemplar estudos de parâmetros de bioindicadores de estresse oxidativo em protocolos experimentais.

Reactive oxygen species (ROS) are produced as a cellular defense mechanism, participating in the processes of cellular healing. However, high levels of ROS can cause damages such as lipid peroxidation (LPO). This study aimed to verify the levels of LPO through the determination of reactive substances to thiobarbituric acid (TBARS) in rat plasma with induced tissue injury. A total of 32 Rattus norvegicus albinus Wistar were used, with a mean weight ± 10%. They were divided into four groups: A ­ negative control; B - Vetaglós®; C - Polyamide cassava; D - Polyamide cassava + Vetaglós®. After 21 days, all animals were anesthetized with isoflurane and blood was collected by cardiac puncture. Plasma was obtained after centrifugation. Euthanasia was performed with administration of an overdose of inhalational anesthetic previously used. The LPO levels in rat plasma were determined using the TBARS method. There was no significant difference between the groups in relation to LPO, indicating a balance between antioxidant defenses and cellular levels of ROS produced during the cellular healing process. This absence in the different experimental groups in relation to LPO emphasizes the importance of further studies related to the bio-indicator parameters for oxidative stress in experimental protocols.

Las especies reactivas al oxígeno (EROS) se producen como mecanismo de defensa celular, que participan en los procesos de curación celulares. Sin embargo, los altos niveles de EROS pueden causar daños como la peroxidación lipídica (PL). Este estudio tuvo como objetivo verificar los niveles de peroxidación lipídica por sustancias reactivas al ácido tiobarbitúrico (TBARS) en el plasma de ratas con lesión tisular inducida. Se han utilizado 32 ratas machos, Rattus norvegicus albinus de linaje Wistar, que se pesaron y la media ± 10% en peso, y se dividieron en cuatro grupos: A ­ control negativo; B - Vetaglós®; C ­ hidrogel de poliamida de yuca + Vetaglós®; D ­ Hidrogel de poliamida de yuca. Después de 21 días, todos los animales fueron anestesiados con isoflurano y se hizo la extracción de sangre por punción cardiaca, y se obtuvieron los plasmas después de la centrifugación, enseguida con sobredosis de anestésico se realizó la eutanasia. Los niveles de PL en los plasmas de las ratas se determinaron por el método de TBARS. No hubo diferencia significativa entre los grupos en relación a la peroxidación lipídica, lo que indica un equilibrio entre las defensas antioxidantes celulares y los niveles de EROS producidos durante el proceso de curación celular. Esa ausencia en los diferentes grupos experimentales, en relación a la PL, pone de manifiesto la importancia de contemplarse estudios de parámetros de bioindicadores de estrés oxidativo en los protocolos experimentales.

Cadmium-induced apoptosis of Siberian tiger fibroblasts via disrupted intracellular homeostasis

BACKGROUND: Heavy metals can cause great harm to Siberian tigers in the natural environment. Cadmium (Cd2+) is an environmental contaminant that affects multiple cellular processes, including cell proliferation, differentiation, and survival. It has been shown to induce apoptosis in a variety of cell types and tissues. RESULTS: We investigated the apoptotic effects of Cd2+ on Siberian tiger fibroblasts in vitro. Our research revealed the typical signs of apoptosis after Cd²+ exposure. Apoptosis was dose- (0-4.8 µM) and duration-dependent (12-48 h), and proliferation was strongly inhibited. Cd²+ increased the activity of caspase-3, -8, and -9 and disrupted calcium homeostasis by causing oxidative stress and mitochondrial dysfunction. It also increased K+ efflux and altered the mRNA levels of Bax, Bcl-2, caspase-3, caspase-8, Fas, and p53. CONCLUSIONS: Our results suggest that Cd2+ triggers the apoptosis of Siberian tiger fibroblasts by disturbing intracellular homeostasis. These results will aid in our understanding of the effects of Cd2+ on Siberian tigers and in developing interventions to treat and prevent cadmium poisoning.

The antioxidative defense system is involved in the premature senescence in transgenic tobacco (Nicotiana tabacum NC89)

BACKGROUND: α-Farnesene is a volatile sesquiterpene synthesized by the plant mevalonate (MVA) pathway through the action of α-farnesene synthase. The α-farnesene synthase 1 (MdAFS1) gene was isolated from apple peel (var. white winterpearmain), and transformed into tobacco (Nicotiana tabacum NC89). The transgenic plants had faster stem elongation during vegetative growth and earlier flowering than wild type (WT). Our studies focused on the transgenic tobacco phenotype. RESULTS: The levels of chlorophyll and soluble protein decreased and a lower seed biomass and reduced net photosynthetic rate (Pn) in transgenic plants. Reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) and superoxide radicals (O2._) had higher levels in transgenics compared to controls. Transgenic plants also had enhanced sensitivity to oxidative stress. The transcriptome of 8-week-old plants was studied to detect molecular changes. Differentially expressed unigene analysis showed that ubiquitin-mediated proteolysis, cell growth, and death unigenes were upregulated. Unigenes related to photosynthesis, antioxidant activity, and nitrogen metabolism were downregulated. Combined with the expression analysis of senescence marker genes, these results indicate that senescence started in the leaves of the transgenic plants at the vegetative growth stage. CONCLUSIONS: The antioxidative defense system was compromised and the accumulation of reactive oxygen species (ROS) played an important role in the premature aging of transgenic plants.