Eritrocitos humanos como biomodelo farmacológico de citoprotección antioxidante para la evaluación de nuevas alternativas terapéuticas en pacientes con COVID-19 / Human erythrocytes as pharmacological biomodel of antioxidant cytoprotection for the evaluation of new therapeutic alternatives in patients with COVID-19

Introducción: El estrés oxidativo puede afectar las membranas biológicas de diferentes tipos celulares en el organismo, lo cual se ha evidenciado en los daños a los tejidos y órganos de pacientes con COVID-19, por lo cual las investigaciones recientes están relacionadas con la búsqueda de fármacos citoprotectores y antioxidantes que minimicen estos daños. Objetivo: Evaluar los eritrocitos humanos como biomodelo farmacológico de citoprotección antioxidante. Métodos: Se evaluó el modelo de citotoxicidad en eritrocitos inducido por peróxido de hidrógeno y se valoró el sistema de diagnóstico propuesto en un ensayo de citoprotección en eritrocitos, con el empleo del ácido ascórbico como sustancia de referencia. Resultados: Para la concentración de eritrocitos utilizada se logró un modelo de citotoxicidad a la concentración de 10 mM de peróxido a los 30 minutos de incubación. La sustancia de referencia empleada no mostró signos de citotoxicidad en el test de hemólisis. En el ensayo de citoprotección se evidenció un efecto farmacológico del referente, con un valor del índice de citoprotección de 12,71 µg/mL. El estudio de microscopía óptica mostró daños morfológicos severos en los eritrocitos tratados con peróxido de tipo esferocitos, equinocitos y esferoequinocitos, que disminuyeron significativamente en presencia de dicha sustancia de referencia. Conclusiones: El biomodelo farmacológico propuesto puede ser empleado en la evaluación de nuevas alternativas terapéuticas con propiedades citoprotectoras antioxidantes para el tratamiento de pacientes con COVID-19.

Introduction: The oxidative stress can affect the biological membranes of different cellular types in the organism, which has been evidenced in the damages to the tissues and organs of patients with COVID-19, reason why the recent investigations are related to the search of cytoprotector and antioxidant drugs that minimize these damages. Objective: To evaluate the human erythrocytes as pharmacological biomodel of antioxidant cytoprotection. Methods: The cytotoxicity pattern was evaluated in erythrocytes induced by peroxide of hydrogen and the system of diagnosis proposed was valued in a cytoprotection assay in erythrocytes, with the use of ascorbic acid as reference substance. Results: For the concentration of erythrocytes used a cytotoxicity model was achieved to the concentration of 10 mM of peroxide at 30 minutes of incubation. The substance of reference used didn't show cytotoxicity signs in the hemolysis test. In the cytoprotection assay a pharmacological effect of the referent was evidenced, with a value of the cytoprotection index of 12.71 µg/mL. The study of optic microscopy showed severe morphological damages in the erythrocytes treated with peroxide of spherocytes, echinocytes and spheroechinocytes type that significantly diminished in presence of this reference substance. Conclusions: The proposed pharmacological biomodel can be used in the evaluation of new therapeutic alternatives with antioxidant cytoprotector properties for the treatment of patients with COVID-19.

The role of seasonality on the chemical composition, antioxidant activity and cytotoxicity of Polish propolis in human erythrocytes

ABSTRACT Propolis extracts possess beneficial biological effects, such as antioxidant activity. However, the composition of propolis and biological properties of its extracts depend on many factor, including time of harvesting. The main purpose of this study was to evaluate the seasonal effect on the phenolic profile of Polish propolis extracts and their antioxidant activity. Propolis samples were collected from the same apiary during three seasons of the year. The chemical composition (contents of phenolic acids and flavonoids) of ethanolic propolis extracts was determined by ultra-performance liquid chromatography equipped with a photodiode detector and a triple quadrupole mass spectrometer. The antioxidant potential of propolis extracts was evaluated. Additionally, in vitro effects of propolis extracts on the morphology of human red blood cells and the selective permeability of their membrane were determined. The capacity of propolis extracts to protect human red blood cells against free radical-induced hemolysis was also studied. The analyze of the chemical composition of propolis extracts collected in three season of the year indicated that the sum of determined flavonoids and phenolic acids was the highest in the sample harvested in the spring (125.14 mg/g) and it was the lowest in the extract of material collected in the fall (110.09 mg/g), but the differences were slightly. The concentration of examined phenols in propolis samples collected in different seasons was similar and only content of seven among fifteen determined compound was significantly different in extracts according to statistical analysis. The propolis extracts possess high antioxidant potential and significantly protect human red blood cells from oxidative damage. There was no significant differences with regard to the seasonal effect on the chemical profile and antioxidant potential of Polish propolis extracts. These results indicate that the time of Polish propolis harvesting have no influence on phenolic profile and antioxidant activity of its extract.

Estudio de la desialización producida por larvas musculares de Trichinella spiralis / Desialylation study produced by muscle larvae of Trichinella spiralis

Trichinella spiralis es agente causal de una zoonosis endémica en Argentina. El objetivo fue estudiar la desialización eritrocitaria producida por larvas musculares (LM) de T. spiralis. Se trabajó con concentrados de LM, incubados en partes iguales con glóbulos rojos (GR) Grupo O (37 °C) durante 3 horas (con y sin agitación controlada) durante 150 minutos, a intervalos de 30 minutos, para estudiar el curso de la desialización en el tiempo. Los GR controles fueron incubados de la misma manera, con igual volumen de solución fisiológica. Se aplicó el método de titulación de la agregación, calculando título y coeficiente de puntuación total (CexpST). Se encontró que los eritrocitos incubados con LM presentaron mayor agregación que los controles. El valor medio de CexpST con agitación (0,43) fue significativamente menor que cuando los GR no se agitaron (0,72). El estudio de la desialización eritrocitaria en el tiempo mostró que el título de los GR control disminuyó significativamente a los 90 minutos en 5/10 repeticiones y a los 150 minutos en 9/10. El aumento del tiempo de incubación produjo el incremento de la desialización excepto a los 120 y 150 minutos donde no existieron diferencias significativas en el valor de CexpST. La experiencia realizada in vitro, sugeriría que en la infección in vivo, las LM podrían captar el ácido siálico a partir de los residuos sializados presentes en la célula muscular.

Trichinella spiralis is the cause of an endemic zoonosis in Argentina. The objective was to study the erythrocyte desialylation by T. spiralis muscle larvae (ML) applying an aggregation titulation method. We worked with ML concentrates, which were incubated with an equal volume of O Group erythrocytes (RBCs) at 37° C for 3 hours, (with and without controlled agitation) and for 150 minutes, taking samples at 30 minutes intervals to study the course in time of the desialylation. RBCs control were incubated with an equal volume of physiological saline solution. Aggregation titulation method was applied and the title and total score coefficient (TSexpC) were calculated. The results showed that erythrocytes incubated with ML showed more aggregation than controls. The average TSexpC with agitation (0.43) was significantly lower than when erythrocytes were not stirred (0.72). The course in time of the erythrocyte desialylation showed that the RBCs contol title decreased significantly at 90 minutes in 5/10 repetitions and at 150 minutes in 9/10. Increasing the incubation time produced an increase in erythrocyte desialylation, except at the 120 and 150 minutes interval where no significant differences in TSexpC values were found. The in vitro experience would suggest that in cases of in vivo infection, ML could capture sialic acid from sialylate residues present in the muscle cell.

Acción biorreológica de extractos de Ascaris lumbricoides en eritrocitos humanos / Biorheological activity of Ascaris lumbricoides extracts in human erythrocytes

Experiencias previas han reportado que los extractos de A. lumbricoides captan ácido siálico del eritrocito humano modificando su carga. El objetivo fue evaluar in vitro las posibles alteraciones biorreológicas debidas al efecto de extractos del parásito adulto. Se analizó la acción de 8 extractos sobre eritrocitos Grupo O, incubando el sedimento globular con igual volumen de extracto y con PBS. Se determinó la distribución y morfología de los agregados (Análisis Digital de Imágenes), carga porcentual (método Azul de Alcian), cinética de agregación (eritroagregámetro) y viscoelasticidad (eritrodefórmetro). Los resultados mostraron que todos los extractos causaron una disminución del porcentaje de células aisladas y un aumento en el tamaño de los agregados, sin alterar su morfología. Los glóbulos tratados con 6 extractos mediaron una significativa alteración de la carga superficial y con 5 de ellos la velocidad de agregación fue menor que en el control. Se observó una correlación lineal entre los parámetros de cinética y la concentración proteica de los extractos. Uno de los tratamientos modificó la fluidez de membrana y dos disminuyeron la deformabilidad eritrocitaria, pero ninguno afectó la elasticidad. Estas experiencias in vitro permiten concluir que el contacto de los extractos con los eritrocitos produce alteraciones hemorreológicas.

Previous studies have reported that A. lumbricoides extracts capture sialic acid from human erythrocytes, modifying their charge. The purpose was to evaluate in vitro the possible biorheological alterations due to the effect of extracts from adult parasites. The activity of 8 extracts over Group O erythrocytes was analyzed incubating the globular sediment with an equal volume of extract and with PBS. The distribution and morphology of the aggregates was determined (Digital Image Analysis), percent of charge (Alcian Blue method), aggregation kinetics (erythroaggregameter), and visco-elasticity (erythroderformeter). The results showed that all the extracts produce a decrease of the percentage of isolated cells and an increase of the size of aggregates, without altering their morphology. Red cells treated with 6 extracts mediated a significant alteration of their superficial charge and with 5 of them the aggregation velocity was less than that of the control. A lineal correlation between the kinetic parameters and the protein concentration of the extracts occurred. One of the treatments modified the fluidity of the membrane and two decreased erythrocyte deformability, but none of them affected elasticity. These in vitro experiences allowed concluding that contact of the extracts with the erythrocytes produces hemorheological alterations.

Citotoxicidad de extracto acuoso de corteza de Manguifera indica L (Vimang®) en Escherichia coli y linfocitos humanos / Cytotoxicity of the aqueous extract from the Manguifera indica L (Vimang®) in Escherichia coli and human lymphocytes

Vimang® es un producto de origen natural que se obtiene del árbol del mango (Manguifera indica L. familia Anacardiaceae). Este compuesto ha sido clasificado como antioxidante, inmunomodulador, etc. Por ello, resulta importante conocer su potencial citotóxico. OBJETIVOS: evaluar la citotoxicidad de un extracto acuoso del Vimang®. MÉTODOS: se emplearon los modelos procariótico (Escherichia coli, cepa PQ37) y eucariótico (eritrocitos humanos), se realizaron curvas de supervivencia celular con la cepa PQ37 (con activación metabólica y sin esta); así como se cuantificaron los niveles de la actividad fosfatasa alcalina (mediante la realización del SOS Chromotest). Posteriormente, se desarrolló el ensayo de inhibición de la actividad mitocondrial en eritrocitos. Las concentraciones de Vimang® estudiadas fueron: 50, 250, 500 y 1 000 mg de extracto liofilizado/mL. RESULTADOS: el ensayo procariótico indicó que, en ausencia de fracción S9, el Vimang® diminuye significativamente la viabilidad celular cuando se aplica a concentración igual o superior que 500 mg/mL. Sin embargo, la presencia de activación metabólica podría ocasionar una biotransformación de los componentes del Vimang® que conduce a la no citotoxicidad del producto en el rango de concentraciones analizado. El análisis de los niveles de fosfatasa alcalina cuantificados en presencia del Vimang®; sugirió que la citotoxicidad detectada en E. coli PQ37 no parece estar relacionada con la inhibición de la síntesis proteica. En el caso del ensayo eucariótico empleado y las concentraciones ensayadas, la supervivencia celular de los eritrocitos (en presencia de Vimang®)no disminuyó de forma significativa en relación con los controles correspondientes. CONCLUSIONES: el Vimang® es citotóxico para la cepa PQ37 de Escherichia coli...

Vimang® is a product of natural origin obtained from the mango tree (Manguifera indica L. Anacardiaceae family. This compound has been classified as antioxidant, immunomodulation agent, etc. Thus, it is important to know its cytotoxic potential. OBJECTIVES: to assess the cytotoxicity of a aqueous extract of Vimang®. METHODS: the prokaryotic (PQ37 strain-Escherichia coli and eukaryotic (human erutjrocytes) models and cellular survival curves with PQ37 strain (with and without metabolic activation) were made and the levels of alkaline phosphatase were quantified (by Chromotest SOS test). Later, a trial of mitochondria activity was developed in erythrocytes. The concentrations of study Vimang® were: 50, 250, 500 and 1 000 µg of lyophilized/mL extract. RESULTS: the prokaryotic trial indicated that, in absence of S9, Vimang® decrease significantly the cell viability when it is applied at a concentration similar o higher than 500 µg/mL. However, the presence of a metabolic activation could to cause a biotransformation of the Vimang's® components leading to the no-cytotoxicity of product within the study concentration rank. Analysis of alkaline phosphatase levels quantified in presence of Vimang® suggested that the cytotoxicity detected in PQ37 Escherichia coli apparently isn't related to protein synthesis inhibition. In the case of the eukaryotic trial used and the assayed concentrations, the cell survival of erythrocytes (in presence of Vimang® not decreased significantly in relation to the corresponding controls. CONCLUSIONS: Vimang® is cytotoxic for the PQ37 strain of E.coli when it is applied at a concentration similar or higher than 500 µg/mL. This effect is not observed in these cells neither when a metabolic activation is applied nor in the human erythrocytes for the conditions reported in present paper

Developmental Changes of Membrane Proteins from Human Erythrocytes

To investigate the developmental changes of human erythrocytes, adult and fetal erythrocyte membranes prepared from adult and cord blood were analyzed in regard to their proteins, glycoproteins, enzyme activities and glucose transport systems. 1. Adult and fetal erythrocyte membranes appeared to have the same major proteins and gl-ycoproteins in the electrophoretic pattern. 2. ATPase activity in adult erythrocyte membranes was lower than that of fetal erythrocyte membranes, whereas adult cholinesterase was higher. No significant change was observed in the activities of alkaline and acid phosphatases during development. 3. Glucose uptake was higher in fetal erythrocytes than in adult erythrocytes, suggesting some loss of glucose carrier system during devlopment. The results suggest that the major structural proteins in human erythrocyte membrane relatively unchanged while some functional proteins such as membrane enzymes and carrier proteins in transport systems are markedly changed during development.