Effect from dinoponeratoxin M-PONTXDq3a arginine and lysine substituted analogues against Staphylococcus aureus strains.

The growing incidence of methicillin-resistant Staphylococcus aureus (MRSA) infections is associated with increased mortality rates, which has generated interest in the development of antimicrobial peptides (AMP), such as those found in the giant ant Dinoponera quadríceps. In order to improve the net positive charge and the antibacterial activity of the AMP, amino acids with positive side chain single substituted analogues have been proposed, mainly arginine or lysine. The present work aims to study the antimicrobial activity of the analogues of M-PONTX-Dq3a, a 23 amino acid AMP identified in the D. quadriceps venom. M-PONTX-Dq3a[1-15], a fragment containing the 15 central amino acids, and eight derivatives of single arginine or lysine substituted analogues were proposed. The antimicrobial activity of peptides was evaluated against Staphylococcus aureus ATCC 6538 P (MSSA) and ATCC 33591 (MRSA) strains, followed by minimum inhibitory concentration (MIC), minimum lethal concentration (MLC), and minimum biofilm inhibitory concentration (MBIC) measurement. The membrane permeability was then assessed via crystal violet assay and flow cytometry analysis. The effect of exposure time on microbial viability (Time-Kill) was evaluated. Finally, ultrastructural alterations were evaluated through scanning electron microscopy (SEM). Both arginine-substituted peptides [Arg]3M-PONTX-Dq3a[1-15] and [Arg]4M-PONTX-Dq3a[1-15], showed lowest MIC and MLC values (each 0.78 µM). In the biofilm formation assays, the peptide [Arg]3M-PONTX-Dq3a [1-15] showed MBIC of 3.12 µM against the two tested strains. Both peptides were able to alter the membrane permeability approximately by 80%. The treatment with MIC was able to eliminate bacteria after 2 h of contact on the other hand, treatment with half of the MIC, the population of both bacterial strains remained constant for up to 12 h, indicating a possible bacteriostatic effect. The SEM results showed that the treatment with the lowest concentration (0.78 µM) of both peptides caused disruption of the cell membrane, destabilization of the intercellular interaction and the CLM of [Arg]4M-PONTX-Dq3a [1-15] resulted in the complete eradication of the bacteria. Thus, this study describes two AMPs active against MSSA and MRSA and also inhibits the biofilm formation of these stains. This study finds [Arg]3M-PONTX-Dq3a[1-15] and [Arg]4M-PONTX-Dq3a[1-15] as alternative substances to treat resistant and/or biofilm-forming strains.

Resveratrol-Loaded Polymeric Nanoparticles: The Effects of D-α-Tocopheryl Polyethylene Glycol 1000 Succinate (TPGS) on Physicochemical and Biological Properties against Breast Cancer In Vitro and In Vivo.

Resveratrol (RSV), a phytoalexin from grapes and peanuts, has been reported to exhibit antiproliferative effects on various cancer cell lines. In breast cancer, RSV has been demonstrated to exert an antiproliferative effect on both hormone-dependent and hormone-independent breast cancer cell lines. However, RSV is a lipophilic drug, and its therapeutic effect could be improved through nanoencapsulation. Functionalizing polymeric nanoparticles based on polycaprolactone (PCL) with polyethylene glycol 1000 tocopheryl succinate (TPGS) has been reported to prolong drug circulation and reduce drug resistance. However, the effect of TPGS on the physicochemical properties and biological effects of breast cancer cells remains unclear. Therefore, this study aimed to develop RSV-loaded PCL nanoparticles using nanoprecipitation and investigate the effect of TPGS on the nanoparticles' physicochemical characteristics (particle size, zeta potential, encapsulation efficiency, morphology, and release rate) and biological effects on the 4T1 breast cancer cell line (cytotoxicity and cell uptake), in vitro and in vivo. The optimized nanoparticles without TPGS had a size of 138.1 ± 1.8 nm, a polydispersity index (PDI) of 0.182 ± 0.01, a zeta potential of -2.42 ± 0.56 mV, and an encapsulation efficiency of 98.2 ± 0.87%, while nanoparticles with TPGS had a size of 127.5 ± 3.11 nm, PDI of 0.186 ± 0.01, zeta potential of -2.91 ± 0.90 mV, and an encapsulation efficiency of 98.40 ± 0.004%. Scanning electron microscopy revealed spherical nanoparticles with low aggregation tendency. Differential Scanning Calorimetry (DSC) and Fourier Transform Infrared Spectroscopy (FTIR) identified the constituents of the nanoparticles and the presence of drug encapsulation in an amorphous state. In vitro release studies showed that both formulations followed the same dissolution profiles, with no statistical differences. In cytotoxicity tests, IC50 values of 0.12 µM, 0.73 µM, and 4.06 µM were found for the formulation without TPGS, with TPGS, and pure drug, respectively, indicating the potentiation of the cytotoxic effect of resveratrol when encapsulated. Flow cytometry and confocal microscopy tests indicated excellent cellular uptake dependent on the concentration of nanoparticles, with a significant difference between the two formulations, suggesting that TPGS may pose a problem in the endocytosis of nanoparticles. The in vivo study evaluating the antitumor activity of the nanoparticles confirmed the data obtained in the in vitro tests, demonstrating that the nanoparticle without TPGS significantly reduced tumor volume, tumor mass, maintained body weight, and improved survival in mice. Moreover, the biochemical evaluation evidenced possible hepatotoxicity for formulation with TPGS.

Níveis séricos de vitamina D e urinários de KIM-1 e MCP-1 em pacientes com anemia falciforme / Serum levels of vitamin D, and urinary levels of KIM-1 and MCP-1 in patients with sickle cell anemia

Objetivo: realizar dosagens de biomarcadores de função renal não convencionais em pacientes com anemia falciforme e associar com os níveis séricos de vitamina D. Métodos: trata-se de um estudo observacional, analítico de corte transversal. Participaram do estudo 51 pacientes adultos com anemia falciforme, e o grupo controle foi composto por 17 adultos saudáveis doadores de sangue. Os níveis séricos de 25- hidroxi-vitamina D foram determinados por imunoensaio quimioluminecente de micropartículas (CMIA), e a função renal foi avaliada pelas dosagens de molécula-1 de lesão renal (KIM-1) e proteína-1 quimiotática de monócitos (MCP-1). Os resultados foram expressos como mediana (intervalo interquartil). Os testes t-Student de amostras independentes, análise de variância de Welch e teste não paramétrico de Kruskal-Wallis foram realizados para comparar as diferenças entre os grupos. Resultados: os pacientes apresentaram níveis séricos de vitamina D superiores ao grupo controle, além de uma maior prevalência de suficiência de vitamina D. Os níveis urinários de KIM-1 e MCP-1 estavam aumentados nos pacientes em relação ao grupo controle. Não houve relação entre baixos níveis séricos de vitamina D e a probabilidade de desenvolvimento de doença renal. Conclusões: este estudo fornece dados importantes sobre a prevalência da deficiência de vitamina D em pacientes com anemia falciforme e demonstra não haver relação entre baixos níveis de vitamina D e desenvolvimento de doença renal.

Objective: to measure non-conventional renal function biomarkers in patients with sickle cell anemia and associate them with serum levels of vitamin D. Method: this is an observational, analytical, cross-sectional study. Fifty-one adult patients with sickle cell anemia participated in the study, and the control group consisted of 17 healthy adult blood donors. Serum levels of 25-hydroxyvitamin D were determined by chemiluminescent microparticle immunoassay (CMIA), and renal function was assessed by measuring urinary Kidney Injury Molecule-1 (KIM-1) and Monocyte Chemoattractant Protein-1 (MCP-1). Results were expressed as median (interquartile range). Student's t-test, Welch analysis of variance, and non-parametric Kruskal-Wallis test were performed to compare differences between groups. Results: patients had higher serum levels of vitamin D than the control group, besides a higher prevalence of vitamin D sufficiency. Urinary levels of KIM-1 and MCP-1 were increased in patients compared to the control group. There was no relationship between low serum vitamin D levels and the likelihood of developing kidney disease. Conclusions: this study provides important data on the prevalence of vitamin D deficiency in patients with sickle cell anemia and demonstrates that there is no relationship between low levels of vitamin D and the development of kidney disease.

1α,25-Dihydroxyvitamin D3 (VD3) Shows a Neuroprotective Action Against Rotenone Toxicity on PC12 Cells: An In Vitro Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by dopaminergic cell loss in the substantia nigra, and PD brains show neuroinflammation, oxidative stress, and mitochondrial dysfunction. The study evaluated the neuroprotective activity of 1α,25-dihydroxy vitamin D3 (VD3), on the rotenone (ROT)-induced cytotoxicity in PC12 cells. The viability parameters were assessed by the MTT and flow cytometry, on cells treated or not with VD3 and/or ROT. Besides, ROS production, cell death, mitochondrial transmembrane potential, reduced GSH, superoxide accumulation, molecular docking (TH and Keap1-Nrf2), and TH, Nrf2, NF-kB, and VD3 receptor protein contents by western blot were evaluated. VD3 was shown to improve the viability of ROT-exposed cells. Cells exposed to ROT showed increased production of ROS and superoxide, which decreased after VD3. ROT decrease in the mitochondrial transmembrane potential was prevented, after VD3 treatment and, VD3 was shown to interact with tyrosine hydroxylase (TH) and Nrf2. While ROT decreased TH, Nrf2, and NF-kB expressions, these effects were reversed by VD3. In addition, VD3 also increased VD3 receptor protein contents and values went back to those of controls after ROT exposure. VD3 protects PC12 cells against ROT damage, by decreasing oxidative stress and improving mitochondrial function. One target seems to be the TH molecule and possibly an indirect Nrf2 activation could also justify its neuroprotective actions on this PC12 cell model of PD.

Nephroprotective effects of Lippia sidoides ethanolic extract against ischemia/reperfusion-induced acute kidney injury

Abstract Ischemia/reperfusion injury (I/R) is commonly related to acute kidney injury (AKI) and oxidative stress. Antioxidant agents are used to treat this condition. Lippia sidoides is a brazillian shrub with anti-inflammatory and anti-oxidative properties. Thus, the aim of this study is to evaluate the effect of Lippia sidoides ethanolic extract (LSEE) on in vivo and in vitro models of AKI induced by I/R. Male Wistar rats were submitted to unilateral nephrectomy and ischemia on contralateral kidney for 60 min via clamping followed by reperfusion for 48 h. They were divided into four groups: Sham, LSEE (sham-operated rats pre-treated with LSEE), I/R (rats submitted to ischemia) and I/R-LSEE (rats treated with LSEE before ischemia). Kidney tissues homogenates were used to determine stress parameters and nephrin expression. Plasma and urine samples were collected for biochemical analysis. I/R in vitro assays were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and flow cytometry assays in Rhesus Monkey Kidney Epithelial Cells (LLC-MK2). The LSEE treatment prevented biochemical and nephrin expression alterations, as well as oxidative stress parameters. In the in vitro assay, LSEE protected against cell death, reduced the reactive oxygen species and increased mitochondrial transmembrane potential. LSEE showed biotechnological potential for a new phytomedicine as a nephroprotective agent.

Association of serum lipoproteins and inflammatory parameters derived from the blood test with renal function in COVID-19 outpatients

Abstract Changes in lipoprotein metabolism are among the main causes of hemodynamic impairment in renal function. COVID-19 is an multisystemic inflammatory disease, aggravating this situation. This cross-sectional study investigated the relationship of serum lipoprotein profile with inflammatory parameters and renal function in 95 COVID-19 outpatients in comparison with 173 with flu-like symptoms. Serum samples were collected for the determination of total cholesterol and fractions, apolipoproteins (Apo A-I and Apo B), urea (sUr) and creatinine (sCr). The glomerular filtration rate (eGFR) was calculated. Neutrophil/lymphocyte (NLR) and platelet/lymphocyte (PLR) ratios were calculated as inflammatory parameters derived from the blood tests. COVID-19 patients presented lower high-density lipoprotein cholesterol (HDL-c) (47.90 ± 1.543 vs. 51.40 ± 0.992) and higher PLR (190.9 ± 9.410 vs. 137.6 ± 5.534) and NLR (3.40 ± 0.22 vs. 2.80 ± 0.15). Both NLR and PLR correlated with each other (r = 0.639). Furthermore, the Apo B/Apo A-I ratio was correlated with PLR (r = 0.5818) and eGFR (r = -0.2630). COVID-19 patients classified as at high risk of developing acute myocardial infarction based on the Apo B/ Apo A-I ratio had higher values for sUr/sCr. Thus, serum apolipoproteins, PLR, and NLR could be related to renal dysfunction in COVID-19.

Hypertriglyceridemia promotes dysfunctions in high-density lipoprotein increasing the cardiovascular risk

Abstract Hypertriglyceridemia is associated with several metabolic diseases. The triglycerides (TG) disrupt the cholesterol reverse transport and contribute to increased levels of low-density lipoprotein (LDL). High-density lipoprotein (HDL) acts in cholesterol reverse transport as an anti-inflammatory and antioxidant. This study aims to investigate the role of hypertriglyceridemia in the functionality of HDL. Individuals were divided into 4 groups based on high or low HDL-c and triglycerides levels. Biochemical and anthropometric analysis were performed. This study demonstrated that triglycerides promote dysfunctions on HDL, increasing the cardiovascular risk. Blood pressure was higher in subjects with low HDL. Women presented higher levels of HDL-c and low percentage of fat mass. The highest levels of triglycerides were observed in older age. In addition, high levels of triglycerides were associated with higher total cholesterol and LDL-c levels, non-HDL-c, non-esterified fatty acids, and blood glucose, increasing in the ratio of non-HDL-c/HDL-c and ApoB/ApoA-I. The increase of triglycerides levels progressively impairs the antioxidant capacity of HDL, probably due to a higher occurrence of fatty acid peroxidation in individuals with hypertriglyceridemia. Patients with high HDL and low TG levels increased the Lag Time. Furthermore, a positive correlation was found between TG versus HDL particle size, variables that depend on age and anthropometric parameters.

Tyramine exerts hypolipidemic and anti-obesity effects in vivo

Abstract Obesity and dyslipidemia are conditions often associated with cardiovascular risk, inflammation, oxidative stress, and death. Thus, a new approach has been highlighted to promote research and development of pharmacological tools derived from natural sources. Among the most widely studied groups of substances, polyphenols such as tyramine stand out. This study investigated hypolipidemic and anti-obesity properties of tyramine. Oral toxicity evaluation, models of dyslipidemia and obesity were used. To induce dyslipidemia, Poloxamer-407 (P-407) was administered intraperitoneally. In the hypercholesterolemic and obesity model, specific diet and oral tyramine were provided. After 24h of P-407 administration, tyramine 2 mg/kg (T2) decreased triglycerides (TG) (2057.0 ± 158.5 mg/dL vs. 2838 ± 168.3 mg/dL). After 48h, TG were decreased by T2 (453.0 ± 35.47 vs. 760.2 ± 41.86 mg/dL) and 4 mg/kg (T4) (605.8 ± 26.61 760.2 ± 41.86 mg/dL). T2 reduced total cholesterol (TC) after 24h (309.0 ± 11.17 mg/dL vs. 399.7 ± 15.7 mg/dL); After 48h, 1 mg/kg (T1) (220.5 ± 12.78 mg/dL), T2 (205.8 ± 7.1 mg/dL) and T4 (216.8 ± 12.79 mg/dL), compared to P-407 (275.5 ± 12.1 mg/dL). The treatment decreased thiobarbituric acid reactive substances and nitrite in liver, increased superoxide dismutase, reduced the diet-induced dyslipidemia, decreasing TC around 15%. Tyramine reduced body mass, glucose, and TC after hypercaloric feed. Treatment with 5 mg/L (0.46 ± 0.04 ng/dL) and 10 mg/L (0.44 ± 0.02 ng/dL) reduced plasma insulin (1.18 ± 0.23 ng/dL). Tyramine increased adiponectin at 5 mg/L (1.02 ± 0.02 vs. 0.83 ± 0.02 ng/mL) and 10mg/L (0.96 ± 0.04 ng/mL). In conclusion, tyramine has low toxicity in rodents, has antioxidant effect, reduces plasma triglycerides and cholesterol levels. However, further studies should be conducted in rodents and non-rodents to better understand the pharmacodynamic and pharmacokinetic properties of tyramine

Linfoma de Hodgkin em Pacientes HIV Positivo em Uso de Antirretrovirais de Alta Efetividade / Hodgkin Lymphoma in HIV Positive Patients in The Use of High-Effective Antiretroviral / Linfoma de Hodgkin en Pacientes VIH Positivos en el Uso de Antiretrovirales de Alta Efectividad

Introdução: A capacidade do vírus da imunodeficiência humana (HIV) de invadir células do sistema imunológico, principalmente células T CD4+, para se multiplicar e manter-se vivo, quando não revertido, possui, como desfecho inevitável, a síndrome da imunodeficiência adquirida (SIDA), evento no qual os pacientes começam a apresentar doenças secundárias como infecções oportunistas e câncer. Objetivo:Identificar casos de linfoma de Hodgkin em pacientes HIV+ em uso dos antirretrovirais de alta efetividade. Método: Estudo do tipo observacional transversal com delineamento exploratório e descritivo e abordagem quali-quantitativa, realizado durante o mês de outubro de 2018, a partir da análise de 57prontuários de pacientes HIV+ diagnosticados com câncer e internados em um hospital localizado em Fortaleza, CE. Resultados: Foi identificado um total de 21 cânceres não definidores de SIDA. Destes, os mais comuns foram o câncer de pele com 14,3% (3); seguido do câncer de mama com 9,5% (2); linfoma de Hodgkin com 9,5% (2); e o câncer de estômago com 9,5% (2) dos casos. Conclusão: Os dados obtidos no presente estudo colocam o linfoma de Hodgkin em segundo lugar entre os cânceres não definidores de SIDA encontrados. Contudo, ao considerar o baixo número de casos, resultante das limitações da pesquisa, essas informações não permitem concluir sobre a real quantidade de linfomas de Hodgkin entre os demais cânceres não definidores de SIDA ocorridos em pacientes HIV+ na localidade, tampouco estimar a participação do HIV, carga viral, condição imunológica e coinfecções como fatores de risco.

Introduction: The ability of the human immunodeficiency virus (HIV) of invading immune system cells, especially CD4+ to multiply and stay alive, when not reversed, has as inevitable outcome the acquired immunodeficiency syndrome (AIDS), an event in which patients start to develop secondary diseases such as opportunistic infections and cancer. Objective: To identify cases of Hodgkin's lymphoma in HIV+ patients using highly effective antiretrovirals. Method: Cross-sectional observational study with exploratory and descriptive design and qualitative and quantitative approach, carried out during the month of October 2018 based on the analysis of 57 HIV-infected patients' charts diagnosed with cancer and admitted to a hospital located in Fortaleza, CE. Results: A total of 21 non-AIDS-defining cancers were detected. Of these, skin cancer, with 14.3% (3) followed by breast cancer, with 9.5% (2), Hodgkin's lymphoma, 9.5% (2) and stomach cancer, with 9.5% (2) were the most common cases. Conclusion: The data obtained in the present study rank Hodgkin's lymphoma in second place among the non-AIDS-defining cancers encountered. However, while considering the small number of cases, due to the study limitations, these data are scanty to conclude the actual quantity of Hodgkin's lymphoma among the non-AIDS-defining cancers occurred in HIV-positive patients locally or to estimate the participation of HIV, viral load, immune condition and co-infection as risk factors.

Introducción: La capacidad del virus de inmunodeficiencia humana (VIH) para invadir las células del sistema inmunitario, especialmente las células T CD4 + para multiplicarse y mantenerse con vida, cuando no se revierte, tiene el resultado inevitable del síndrome de inmunodeficiencia adquirida (SIDA), evento en el que los pacientes comienzan a presentar enfermedades secundarias como infecciones oportunistas y cáncer. Objetivo: Identificar los casos de linfoma de Hodgkin en pacientes VIH+ utilizando los antirretrovirales de alta eficacia. Método: Este es un estudio observacional transversal con diseño exploratorio y descriptivo y enfoque cualitativo, realizado durante octubre de 2018 a partir del análisis de 57 registros médicos de pacientes VIH + diagnosticados con cáncer y hospitalizados en un hospital ubicado en Fortaleza, CE. Resultados: Se identificaron un total de 21 cánceres que no definen el SIDA. De estos, los más comunes fueron cáncer de piel con 14,3% (3), seguido de cáncer de seno con 9,5% (2), linfoma de Hodgkin 9,5% (2) y cáncer de estómago con 9,5% (2) de los casos. Conclusión: Los datos obtenidos en el presente estudio colocan al linfoma de Hodgkin en segundo lugar entre los cánceres no definitorios de SIDA encontrados. Sin embargo, considerando el bajo número de casos resultantes de las limitaciones de la investigación, esta información no nos permite concluir acerca de la cantidad real de linfoma de Hodgkin entre los otros cánceres no definitorios de SIDA en pacientes VIH + en la localidad, ni estimar la participación del VIH, la carga viral, la condición inmune y las coinfecciones como factores de riesgo.

Protective Effect of Quercetin on Renal Tubular Cells and the Involvement with the Renin-Angiotensin-Aldosterone Axis

Abstract Ischemia-reperfusion (I/R) plays an important role in the process of acute kidney injury (AKI) due to the generation of reactive oxygen species (ROS). Substances of natural origin have been studied in the prevention of oxidative damage related to I/R. Quercetin is a flavonoid with antioxidant potential and modulate enzymes, such the inhibition of the Rennin-Angiotensin System (RAS). The aim of this study is to evaluate the nephroprotective effect of quercetin against the I/R and analyze the inhibition of RAS. Rhesus monkey Kidney Epithelial Cells (LLC-MK2 line) were submitted to an in vitro ischemia/reperfusion model. After the reperfusion cells were treated with quercetin, the cell viability was accessed by the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) assay. Tubular cell damage was assessed by the Kidney Injury Molecule-1 (KIM-1) measurement. Oxidative stress was evaluated through Thiobarbituric Acid Reactive Substances (TBARS) and reduced glutathione (GSH). The evaluation of cell death and the mitochondrial depolarization were analyzed by flow cytometry. Quercetin prevents cell death reducing oxidative stress and preventing mitochondrial membrane depolarization. Molecular docking showed that quercetin prevents cell damage better than losartan and lisinopril, inhibitors of RAS. Quercetin has a potential to interact with type 1 angiotensin II receptor (AT1) with greater affinity through the formation of five hydrogen bonds of strong intensity.

Involvement of NADPH-oxidase enzyme in the nephroprotective effect of (-)-α-bisabolol on HK2 cells exposed to ischemia - Reoxygenation.

Acute Kidney Injury (AKI) is associated with high morbidity and mortality. Ischemia and reperfusion (I/R) are events that lead to AKI through hypoxia, reactive oxygen species (ROS) production, oxidative stress and apoptosis. We aimed to evaluate the mechanism of nephroprotection mediated by Bisabolol in human tubular kidney cells after injury by I/R in vitro. HK2 cells were exposed to I/R and treated with Bisabolol. Cell viability was accessed by MTT assay. Cells were submitted to flow cytometry to evaluate necrotic/apoptotic cells, reactive oxygen species production and mitochondrial transmembrane depolarization. TBARS and GSH were used as parameters of redox balance. Also, KIM-1 supernatant levels were measured. In order to identify an interaction between bisabolol and NOX4, molecular docking and enzymatic assays were performed. Expression of isoform NOX4 on treated cells was examined by western-blot. Finally, cells were visualized by scanning electron microscopy. Bisabolol improved cell viability and prevented cell death by apoptosis, indicated also by the decreased levels of KIM-1. It was observed a decrease on reactive oxygen species production and mitochondrial depolarization, with antioxidant regulation by increased GSH and decreased lipid peroxidation. It was also demonstrated that bisabolol treatment can inhibit NOX4. Finally, SEM images showed that bisabolol reduced I/R-induced cell damage. Bisabolol treatment protects HK2 cells against oxidative damage occasioned by I/R. This effect is related to inhibition of apoptosis, decrease on KIM-1 release, reactive oxygen species accumulation and mitochondrial dysfunction. Bisabolol inhibited NOX4 activity in the tubular cells, impairing reactive oxygen species synthesis.

Evaluation of KIM-1 as an early biomarker of snakebite-induced AKI in mice.

Acute kidney injury (AKI) is one of the most important complications of bothropic poisoning and its early identification remains challenging. The nephrotoxicity of Bothrops insularis venom (BinsV) was previously described by our research group. In this study, we continued to evaluate the effect of BinsV on kidney function in mice and LLC-MK2 proximal tubule cells, evaluating KIM-1 protein as an early AKI biomarker. Male Swiss mice were inoculated with BinsV intramuscularly and observed for 24 h in a metabolic cage model. Urine and blood were collected for biochemical analyses and the kidneys were examined for oxide-reducing balance and submitted to histological analysis. LLC-MK2 cells incubated with BinsV were assessed for cell viability and cell death mechanism by flow cytometry. Histological analysis of the kidneys indicated AKI and the oxide-reducing analyses demonstrated a decreasing in reduced glutathione (GSH) levels and an increasing on Malondialdehyde (MDA) levels. BinsV was cytotoxic to LLC-MK2 and the cytometry analyses suggested necrosis. Within 24 h after the envenomation, urinary creatinine did not increase, but the urinary levels of KIM-1 increased. In conclusion, we found AKI evidence in the kidney tissue and the increase in the KIM-1 levels suggest it can be used as an early AKI biomarker.

The dinoponeratoxin peptides from the giant ant Dinoponera quadriceps display in vitro antitrypanosomal activity.

The crude venom of the giant ant Dinoponera quadriceps is a cocktail of polypeptides and organic compounds that shows antiparasitic effects against Trypanosoma cruzi, the causative agent of Chagas disease. In order to investigate the venom-derived components responsible for such antitrypanosomal activity, four dinoponeratoxins (DnTxs) were identified, namely M-PONTX-Dq3a, -Dq3b, -Dq3c and -Dq4e, that are diverse in size, net charge, hydrophobicity and propensity to interact with eukaryote cell membranes. These peptides were tested against epimastigote, trypomastigote and amastigote forms of benznidazole (Bz)-resistant Y strain of T. cruzi and in mammalian host cells. The M-PONTX-Dq3a and -Dq4e inhibited all developmental forms of T. cruzi, including amastigotes, the responsible form for the maintenance of infection on chronic phase of the disease. The M-PONTX-Dq3a showed the highest selectivity index (SI) (80) and caused morphological alterations in T. cruzi, as observed by scanning electron microscopy (SEM), and induced cell death through necrosis, as seen by multiparametric flow cytometry analysis with specific biochemical markers. Altogether, the D. quadriceps venom appears as a source for the prospection of trypanocidal peptides and the M-PONTX-Dq3a arises as a candidate among the dinoponeratoxin-related peptides in the development of compounds against Chagas disease.

Nephroprotective effects of (-)-α-bisabolol against ischemic-reperfusion acute kidney injury.

BACKGROUND: Ischemia/reperfusion (I/R) in kidney is commonly related to acute kidney injury (AKI), essentially through oxidative stress. (-)-α-Bisabolol is a sesquiterpene isolated from the essential oil of a variety of plants, including chamomile, which has important antioxidant activity. STUDY DESIGN: This study intends to evaluate the nephroprotective activity of (-)-α-bisabolol (Bis) in both in vivo and in vitro models of kidney I/R. METHODS: Male Wistar rats were submitted to right nephrectomy, followed by ischemia by clamping of the renal artery in the left kidney for 60min. and 48h of reperfusion. The animals were treated orally with Bis (100mg/kg) or vehicle for 24h after reperfusion, and placed in metabolic cages, to evaluate water consumption, diuresis, urinary osmolality, classic biochemical markers and urinary KIM-1 (kidney injury molecule-1). Additionally, the left kidney was collected for histological evaluation and determination of glutathione (GSH) and Thiobarbituric Acid Reactive Substances (TBARS) levels. Tubular epithelial cells LLC-MK2 were used to assess Bis effect on in vitro I/R, by MTT assay. It was performed the cellular respiration tests by flow cytometry: evaluation of the production of cytoplasmic reactive oxygen species by DCFH-DA assay and mitochondrial transmembrane potential analysis with the dye rhodamine 123. RESULTS: I/R caused alterations in diuresis, water intake, urinary osmolality, plasmatic creatinine, urea and uric acid, creatinine clearance, proteinuria and microalbuminuria. Treatment with Bis ameliorated all of these parameters. Also, KIM-1 level enhanced by I/R was also diminished in groups treated with Bis. The histological examination showed that Bis attenuated the morphological changes caused by I/R, markedly vascular congestion and intratubular deposits of proteinaceous material. Additionally, Bis was able to reduce the changes observed in TBARS and GSH levels in kidney tissue. In in vitro assay, Bis was capable to partially protect the cell lineage against cell damage induced by I/R. CONCLUSION: (-)-α-Bisabolol has a nephroprotective effect in kidney I/R, with antioxidant effect. Moreover, this result seems to be associated to a direct protective effect on tubular epithelia.

Involvement of Nitric Oxide on Bothropoides insularis Venom Biological Effects on Murine Macrophages In Vitro.

Viperidae venom has several local and systemic effects, such as pain, edema, inflammation, kidney failure and coagulopathy. Additionally, bothropic venom and its isolated components directly interfere on cellular metabolism, causing alterations such as cell death and proliferation. Inflammatory cells are particularly involved in pathological envenomation mechanisms due to their capacity of releasing many mediators, such as nitric oxide (NO). NO has many effects on cell viability and it is associated to the development of inflammation and tissue damage caused by Bothrops and Bothropoides venom. Bothropoides insularis is a snake found only in Queimada Grande Island, which has markedly toxic venom. Thus, the aim of this work was to evaluate the biological effects of Bothropoides insularis venom (BiV) on RAW 264.7 cells and assess NO involvement. The venom was submitted to colorimetric assays to identify the presence of some enzymatic components. We observed that BiV induced H2O2 production and showed proteolytic and phospholipasic activities. RAW 264.7 murine macrophages were incubated with different concentrations of BiV and then cell viability was assessed by MTT reduction assay after 2, 6, 12 and 24 hours of incubation. A time- and concentration-dependent effect was observed, with a tendency to cell proliferation at lower BiV concentrations and cell death at higher concentrations. The cytotoxic effect was confirmed after lactate dehydrogenase (LDH) measurement in the supernatant from the experimental groups. Flow cytometry analyses revealed that necrosis is the main cell death pathway caused by BiV. Also, BiV induced NO release. The inhibition of both proliferative and cytotoxic effects with L-NAME were demonstrated, indicating that NO is important for these effects. Finally, BiV induced an increase in iNOS expression. Altogether, these results demonstrate that B. insularis venom have proliferative and cytotoxic effects on macrophages, with necrosis participation. We also suggest that BiV acts by inducing iNOS expression and causing NO release.

The acute inflammatory response induced in mice by the venom of the giant ant Dinoponera quadriceps involves macrophage and interleukin-1ß.

Dinoponera quadriceps (Hymenoptera, Formicidae, Ponerinae) is a primitive and endemic ant of Northeastern Brazil, that uses its sting and associated venom gland to capture preys and for defense. Venom of Dinoponera is of potential clinical importance, since it causes intense local pain, accompanied by erythema and edema, when injected by the sting. With other hymenopteran venoms, inflammatory effects are also reported. The aim of this study was to evaluate the inflammatory activity of D. quadriceps venom (DqV) in mice. Acrylamide electrophoresis of DqV revealed five main protein bands varying between 15 and 100 kDa, confirming the proteinous nature of DqV. DqV subplantar injection elicited edema at 5 µg/kg (3 fold), 50 µg/kg (4 fold) or 500 µg/kg (7 fold) from zero to 360 min compared to saline. DqV (50 µg/kg) increased vascular permeability (4 fold) in the first hour after induction. The paw tissue histology showed moderate inflammatory focus caused by DqV (50 µg/kg) in the first hour of paw edema, but severe tissue changes (edema, inflammatory infiltrate and focal areas of hemorrhage) in the third hour. Intraperitoneal injection of DqV (50 µg/kg) stimulated neutrophil (7 fold) and mononuclear (1.4 fold) migration vs saline. DqV edematogenic effect was inhibited by dexamethasone (92%), thalidomide (82%), cyproheptadine (62%), AA861 (58%), celecoxib (34%) or l-NAME (34%), but the neutrophil migration was only by dexamethasone (57%). DqV-elicited neutrophil migration at 50 µg/kg was potentiated 1.7 fold by the animals pre-treatment with 3% thioglycolate. DqV injection increased the levels of interleukin-1 beta (IL-1ß) in peritoneal cavities. DqV (50, 100 and 200 µg/mL) increased phospholipase activity (A425nm) from 10 min to 40 min. Raw 267 macrophages incubated with DqV (from 3.12 to 50 mg/mL) showed no significant decrease in cell viability or LDH measurements and at 35 µg/mL induced increase in IL-1ß (from 3 to 6 h). This study demonstrated, in mice, the inflammatory effect of D. quadriceps venom, characterized by edema, increase in vascular permeability and neutrophil migration, implying the participation of resident macrophages and IL-1ß, among other inflammatory mediators.

Efeito nefroprotetor do α bisabolol na lesão renal aguda em modelos de isquemia reperfusão

A Isquemia/Reperfusão (I/R)é um fenômeno complexo, que contribui para a mortalidade e morbidade e é um fator predisponente para o estabelecimento da Lesão Renal Aguda (LRA). Várias estratégias e recursos são utilizados pelascélulas para prevenir ou diminuir a injúria celular causada pelo estresse oxidativo. Dessa forma, ressalta-se a importância do estudo utilizando substâncias como o (-)-α-Bisabolol, o qual apresenta um potencial antioxidante. Ademais, o estudo de ferramentaspara diagnóstico precoce do desenvolvimento da lesão renal utilizando biomarcadores cada vez mais sensíveis e específicos, como ressaltado neste trabalho o KIM-1, pode auxiliar na detecção da injúria e acompanhamento da progressão do processo de melhorae cura. Logo, o presente trabalho tem como objetivo estudar os possíveis efeitos nefroprotetores do (-)-α-Bisabolol na Lesão Renal Aguda (LRA) em modelos de Isquemia/Reperfusão (I/R), bem como estudar obiomarcador KIM-1 como preditor precoce da injúria renal. Ratos Wistar machos foram submetidos aoprocedimento cirúrgico de nefrectomia direita e clampeamento da artéria renal esquerda. Amostras de urina, sangue e tecido renal foram coletadas para avaliações adicionais. Um modelo in vitro de I/R foi realizado em cultura de linhagens de células tubulares renais LLC-MK2 para avaliar a viabilidade celular pelo ensaio de redução do MTT...

Ischemia / reperfusion (IR) isa complex phenomenon that contributes to mortality and morbidity and is a predisposing factor for the establishment of acute kidney injury (AKI). Various strategies and resources are used by cells to prevent or decrease the cellular injury caused by oxidative stress. Thus, it emphasizes the importance of studying using substances such as (-)-α-Bisabolol, which has an antioxidant potential. Furthermore, the study of strategies for early detection of developing kidney damage using increasingly sensitive and specific biomarkers, as pointed out in this paper the KIM-1, can aid in the detection of injury and follow the progression of improvement and healing process. Thus, this paper aims to study the possible nephroprotectiveseffects of (-)-α-Bisabolol in AcuteKidney Injury (AKI) in models of ischemia/reperfusion (I/R), as well as study the KIM-1 biomarker as a predictor early renal injury. Male Wistar rats underwent surgical procedure right nephrectomy and clamping of the left renal artery. Samples of urine, blood and kidney tissue were collected for further evaluation. An in vitro model of I/R was performed in a culture of renal tubular cell lines, LLC-MK2 to assess cell viability by the MTT reduction assay...