Anion exchanger1 (AE1/SLC4A1) function is impaired in red blood cells from prediabetic subjects: Potential benefits of finger lime (Citrus australasica, Faustrime cultivar) juice extract.

Prediabetes is a risk state that defines a high chance of developing diabetes and cardiovascular disease. Oxidative stress mediated by hyperglycemia-induced production of reactive species could play a crucial role in this context. In the present study, we investigated whether the anion exchange capability mediated by AE1 (SLC4A1), which is sensitive to oxidative stress, was altered in human red blood cells (RBCs) obtained from prediabetic volunteers. In addition, we assessed the precise composition of bioactive compounds and the potential benefits of finger lime juice extract (Citrus australasica, Faustrime cultivar) in counteracting oxidative stress-related functional alterations. Human RBCs from normal and prediabetic volunteers were incubated with 50 µg/mL juice extract for 2 h at 25°C. Juice extract restored alterations of the anion exchange capability mediated by AE1 and prevented the structural rearrangements of AE1 and α/ß-spectrin in prediabetic RBCs. AE1 functional and structural alterations were not associated with an increase in lipid peroxidation or protein oxidation at the level of the plasma membrane. An increased production of intracellular ROS, which provoked the oxidation of hemoglobin to methemoglobin, both reverted by juice extract, was instead observed. Importantly, juice extract also induced a reduction in glycated hemoglobin levels in prediabetic RBCs. Finally, juice extract blunted the overactivation of the endogenous antioxidant enzymes catalase and superoxide dismutase and prevented glutathione depletion in prediabetic RBCs. These findings contribute to clarifying cellular and molecular mechanisms related to oxidative stress and glycation events that may influence RBC and systemic homeostasis in prediabetes, identify AE1 as a sensitive biomarker of RBC structural and function alterations in prediabetes and propose finger lime juice extract as a natural antioxidant for the treatment and/or prevention of the complications associated with the prediabetic condition.

Cellular and Molecular Mechanisms in Oxidative Stress-Related Diseases 2.0/3.0.

Oxidative stress is frequently described as the balance between the production of reactive species (including oxygen and nitrogen) in biological systems and the ability of the latter to defend itself through the sophisticated antioxidant machinery [...].

Oxidative Stress and Immune Response in Melanoma: Ion Channels as Targets of Therapy.

Oxidative stress and immune response play an important role in the development of several cancers, including melanoma. Ion channels are aberrantly expressed in tumour cells and regulate neoplastic transformation, malignant progression, and resistance to therapy. Ion channels are localized in the plasma membrane or other cellular membranes and are targets of oxidative stress, which is particularly elevated in melanoma. At the same time, ion channels are crucial for normal and cancer cell physiology and are subject to multiple layers of regulation, and therefore represent promising targets for therapeutic intervention. In this review, we analyzed the effects of oxidative stress on ion channels on a molecular and cellular level and in the context of melanoma progression and immune evasion. The possible role of ion channels as targets of alternative therapeutic strategies in melanoma was discussed.

d-Galactose induced early aging in human erythrocytes: Role of band 3 protein.

Aging, a time-dependent multifaceted process, affects both cell structure and function and involves oxidative stress as well as glycation. The present investigation focuses on the role of the band 3 protein (B3p), an anion exchanger essential to red cells homeostasis, in a d-galactose ( d-Gal)-induced aging model. Anion exchange capability, measured by the rate constant of SO4²- uptake through B3p, levels of lipid peroxidation, oxidation of membrane sulfhydryl groups, B3p expression, methemoglobin, glycated hemoglobin (Hb), and the reduced glutathione/oxidized glutathione ratio were determined after exposure of human erythrocytes to 25, 35, 50, and 100 mmol/L d-Gal for 24 h. Our results show that: (i) in vitro application of d-Gal is useful to model early aging in human erythrocytes; (ii) assessment of B3p ion transport function is a sensitive tool to monitor aging development; (iii) d-Gal leads to Hb glycation and produces substantial changes on the endogenous antioxidant system; (iv) the impact of aging on B3p function proceeds through steps, first involving Hb glycation and then oxidative events at the membrane level. These findings offer a useful tool to understand the mechanisms of aging in human erythrocytes and propose B3p as a possible target for new therapeutic strategies to counteract age-related disturbances.

Cellular and Molecular Mechanisms in Oxidative Stress-Related Diseases.

The redox equilibrium is important in preserving the correct functionality of vital cellular functions [...].

Oxidation Stress as a Mechanism of Aging in Human Erythrocytes: Protective Effect of Quercetin.

Aging is a multi-factorial process developing through a complex net of interactions between biological and cellular mechanisms and it involves oxidative stress (OS) as well as protein glycation. The aim of the present work was to verify the protective role of Quercetin (Q), a polyphenolic flavonoid compound, in a d-Galactose (d-Gal)-induced model of aging in human erythrocytes. The anion-exchange capability through the Band 3 protein (B3p) measured by the rate constant of the SO42- uptake, thiobarbituric acid reactive substances (TBARS) levels-a marker of lipid peroxidation-total sulfhydryl (-SH) groups, glycated hemoglobin (A1c), and a reduced glutathione/oxidized glutathione (GSH-GSSG) ratio were determined following the exposure of erythrocytes to 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µM Q. The results confirmed that d-Gal activated OS pathways in human erythrocytes, affecting both membrane lipids and proteins, as denoted by increased TBARS levels and decreased total sulfhydryl groups, respectively. In addition, d-Gal led to an acceleration of the rate constant of the SO42- uptake through the B3p. Both the alteration of the B3p function and oxidative damage have been improved by pre-treatment with Q, which preferentially ameliorated lipid peroxidation rather than protein oxidation. Moreover, Q prevented glycated A1c formation, while no protective effect on the endogenous antioxidant system (GSH-GSSG) was observed. These findings suggest that the B3p could be a novel potential target of antioxidant treatments to counteract aging-related disturbances. Further studies are needed to confirm the possible role of Q in pharmacological strategies against aging.

Antioxidant Activity of Quercetin in a H2O2-Induced Oxidative Stress Model in Red Blood Cells: Functional Role of Band 3 Protein.

During their lifespan, red blood cells (RBCs) are exposed to a large number of stressors and are therefore considered as a suitable model to investigate cell response to oxidative stress (OS). This study was conducted to evaluate the potential beneficial effects of the natural antioxidant quercetin (Q) on an OS model represented by human RBCs treated with H2O2. Markers of OS, including % hemolysis, reactive oxygen species (ROS) production, thiobarbituric acid reactive substances (TBARS) levels, oxidation of protein sulfhydryl groups, CD47 and B3p expression, methemoglobin formation (% MetHb), as well as the anion exchange capability through Band 3 protein (B3p) have been analyzed in RBCs treated for 1 h with 20 mM H2O2 with or without pre-treatment for 1 h with 10 µM Q, or in RBCs pre-treated with 20 mM H2O2 and then exposed to 10 µM Q. The results show that pre-treatment with Q is more effective than post-treatment to counteract OS in RBCs. In particular, pre-exposure to Q avoided morphological alterations (formation of acanthocytes), prevented H2O2-induced OS damage, and restored the abnormal distribution of B3p and CD47 expression. Moreover, H2O2 exposure was associated with a decreased rate constant of SO42- uptake via B3p, as well as an increased MetHb formation. Both alterations have been attenuated by pre-treatment with 10 µM Q. These results contribute (1) to elucidate OS-related events in human RBCs, (2) propose Q as natural antioxidant to counteract OS-related alterations, and (3) identify B3p as a possible target for the treatment and prevention of OS-related disease conditions or aging-related complications impacting on RBCs physiology.

Band 3 protein function and oxidative stress in erythrocytes.

Band 3 protein (B3p), anion transporter, allows the HCO3- /Cl- exchange across plasma membrane and plays an important role for erythrocytes homeostasis. In addition, B3p is linked to proteins cytoskeleton, thus contributing to cell shape and deformability, essential to erythrocytes adjustment within narrowest capillaries. Taking into account that erythrocytes are a suitable cell model to investigate the response of the oxidative stress effects, B3p functions, and specifically anion exchange capability, determining the rate constant for SO42- uptake, has been considered. As, in the latter years, rising attention has been addressed to membrane transport system, and particularly to this protein, the present mini-review has been conceived to report the most recent knowledge about B3p, with specific regard to its functions in oxidative stress conditions, including oxidative stress-related diseases.

Mechanisms of Activation of LRRC8 Volume Regulated Anion Channels.

Volume regulated anion channels (VRACs) are ubiquitously expressed in all vertebrate cells. Despite many years of research, the fundamental mechanisms underlying VRAC activation are not understood. The recent molecular identification of the LRRC8 genes underlying VRAC revealed that VRACs are formed by a hexameric assembly of members of the LRRC8 gene family. Knowing the genes underlying VRACs allowed the discovery of novel VRAC functions into cell volume regulation, and first structure function studies revealed important insight in channel activation mechanisms. The determination of cryo-EM structures of homomeric LRRC8A and LRRC8D complexes provide a framework for a rational approach to investigate biophysical mechanisms. We discuss several recent advances within the structural framework, and we critically review the literature on the main mechanisms proposed to be involved in VRAC activation, including low intracellular ionic strength, membrane unfolding, oxidation, phosphorylation and G-protein coupling.

TRPM2 Oxidation Activates Two Distinct Potassium Channels in Melanoma Cells through Intracellular Calcium Increase.

Tumor microenvironments are often characterized by an increase in oxidative stress levels. We studied the response to oxidative stimulation in human primary (IGR39) or metastatic (IGR37) cell lines obtained from the same patient, performing patch-clamp recordings, intracellular calcium ([Ca2+]i) imaging, and RT-qPCR gene expression analysis. In IGR39 cells, chloramine-T (Chl-T) activated large K+ currents (KROS) that were partially sensitive to tetraethylammonium (TEA). A large fraction of KROS was inhibited by paxilline-a specific inhibitor of large-conductance Ca2+-activated BK channels. The TEA-insensitive component was inhibited by senicapoc-a specific inhibitor of the Ca2+-activated KCa3.1 channel. Both BK and KCa3.1 activation were mediated by an increase in [Ca2+]i induced by Chl-T. Both KROS and [Ca2+]i increase were inhibited by ACA and clotrimazole-two different inhibitors of the calcium-permeable TRPM2 channel. Surprisingly, IGR37 cells did not exhibit current increase upon the application of Chl-T. Expression analysis confirmed that the genes encoding BK, KCa3.1, and TRPM2 are much more expressed in IGR39 than in IGR37. The potassium currents and [Ca2+]i increase observed in response to the oxidizing agent strongly suggest that these three molecular entities play a major role in the progression of melanoma. Pharmacological targeting of either of these ion channels could be a new strategy to reduce the metastatic potential of melanoma cells, and could complement classical radio- or chemotherapeutic treatments.

Protective Role of Magnesium against Oxidative Stress on SO4= Uptake through Band 3 Protein in Human Erythrocytes.

BACKGROUND/AIMS: Magnesium, whose supplementation provides beneficial effects against oxidative stress-related conditions, has been here used to possibly protect Band 3 protein anion exchange capability and underlying signaling in an in vitro model of oxidative stress. METHODS: Whole blood samples pre-exposed to 10 mM MgCl2, were treated for 30 min with H2O2 (300 µM, 600 µM and 1 mM) chosen as oxidant molecule. In a separate protocol, NEM (0.5,1 and 2 mM), a phosphatase inhibitor and thiol-alkilant agent, has been also applied. The rate constant for SO4= uptake, accounting for Band 3 protein anion exchange capability, has been measured by a turbidimetric method, while intracellular reduced glutathione (GSH) levels and membrane -SH groups mostly belonging to Band 3 protein were spectrophotometrically quantified after reaction with DTNB (5,5'-dithiobis-(2-nitrobenzoic acid). Expression levels of Band 3 protein, phosporylated Tyrosine (P-Tyr) and tyrosine kinase (Syk) involved in signaling have been also measured. RESULTS: Our results show that Mg2+ prevented the reduction in the rate constant for SO4= uptake on H2O2-treated erythrocytes, not involving GSH levels and membrane -SH groups, unlike NEM, remaining both P-Tyr and Syk expression levels high. CONCLUSION: Hence, i) the measurement of the rate constant for SO4= uptake is a useful tool to evaluate Mg2+ protective effect; ii) the use of two different oxidant molecules shed light on Mg2+ effect which seems not to modulate phosphorylative pathways but would putatively stabilize membrane organization; iii) the use of Mg2+ in food supplementation can be reasonably supported to protect erythrocytes homeostasis in case of oxidative stress-related diseases.

Melatonin Protects Band 3 Protein in Human Erythrocytes against H2O2-Induced Oxidative Stress.

The beneficial effect of Melatonin (Mel), recognized as an anti-inflammatory and antioxidant compound, has been already proven to prevent oxidative stress-induced damage associated to lipid peroxidation. As previous studies modeled the impact of oxidative stress on Band 3 protein, an anion exchanger that is essential to erythrocytes homeostasis, by applying H2O2 at not hemolytic concentrations and not producing lipid peroxidation, the aim of the present work was to evaluate the possible antioxidant effect of pharmacological doses of Mel on Band 3 protein anion exchange capability. The experiments have been performed on human erythrocytes exposed to 300 µM H2O2-induced oxidative stress. To this end, oxidative damage has been verified by monitoring the rate constant for SO4= uptake through Band 3 protein. Expression levels of this protein Mel doses lower than 100 µM have also been excluded due to lipid peroxidation, Band 3 protein expression levels, and cell shape alterations, confirming a pro-oxidant action of Mel at certain doses. On the other hand, 100 µM Mel, not provoking lipid peroxidation, restored the rate constant for SO4= uptake, Band 3 protein expression levels, and H2O2-induced cell shape alterations. Such an effect was confirmed by abolishing the endogenous erythrocytes antioxidant system. Therefore, the present findings show the antioxidant power of Mel at pharmacological concentrations in an in vitro model of oxidative stress not associated to lipid peroxidation, thereby confirming Band 3 protein anion exchange capability measurement as a suitable model to prove the beneficial effect of Mel and support the use of this compound in oxidative stress-related diseases affecting Band 3 protein.

Neuronal-like differentiated SH-SY5Y cells adaptation to a mild and transient H2 O2 -induced oxidative stress.

Preconditioning (PC) is a cell adaptive response to oxidative stress and, with regard to neurons, can be considered as a neuroprotective strategy. The aim of the present study was to verify how neuronal-like differentiated SH-SY5Y cells adapt to a mild and transient H2 O2 -induced oxidative stress and, hence, whether may be considered as more sensitive cell model to study PC pathways. A first screening allowed to define H2 O2 concentrations for PC (10µM-50µM), applied before damage(100µM H2 O2 ). Cell viability measured 24 hours after 100µM H2 O2 -induced damage was ameliorated by 24-hour pre-exposure to low-concentration H2 O2 (10µM-30µM) with cell size as well restored. Markers for apoptosis (Bcl-2 and Bad), inflammation (iNOS), and redox system (MnSOD) were also determined, showing that, in cells pre-exposed to 10µM H2 O2 and then submitted to 100µM H2 O2 , Bcl-2 levels were higher, Bad and iNOS levels were lower than those observed in damaged cells, and MnSOD levels were unchanged. Such findings show that (1) neuronal-like differentiated SH-SY5Y cells are a suitable model to investigate PC response and more sensitive to the effect of a mild and transient H2 O2 -induced oxidative stress with respect to other neuronal cells; (2) 10µM H2 O2 -induced PC is mediated by apoptotic and inflammatory pathways, unlike antioxidant system; (3) such neuroprotective strategy and underlying signals proven in neuronal-like differentiated SH-SY5Y cells may contribute to understand in vivo PC mechanisms and to define a window for pharmacological intervention, namely, related to ischemic brain damage. SIGNIFICANCE OF THE STUDY: Neuronal-like differentiated SH-SY5Y cells are a suitable model to investigate PC, an endogenous neuroprotective response to a mild and transient H2 O2 -induced oxidative stress, elicited by 24-hour exposure to very low H2 O2 concentrations and mediated by both apoptotic and inflammatory pathways. This model reflects in vivo PC mechanisms occurring after brain trauma and provides novel information about pathways and time of protection useful for an appropriate pharmacological intervention.

Anion exchange through band 3 protein in canine leishmaniasis at different stages of disease.

Band 3 protein efficiency in mediating Cl-/HCO3- exchange through erythrocytes membrane is reduced by oxidative stress. The aim of the present study was to verify whether and how anion transport through band 3 protein may be useful in monitoring canine leishmaniasis (Leishmania infantum) development, a disease associated to membrane protein degradation and oxidative stress. To accomplish this aim, serological analysis to determine IFAT (immunofluorescence antibody test) titers against leishmaniasis has been performed and 1:160 and 1:540 titers, determined at diagnosis and after 6 months, were considered for experiments. Oxidative conditions have been assessed by estimating MDA (malondialdehyde) plasma levels, intracellular GSH (reduced glutathione) content, and membrane -SH groups. Band 3 protein anion exchange capability was evaluated by measuring the rate constant for SO4= uptake, and its expression levels, along with those of P-Tyr (phosphorylated tyrosine), involved in pathways underlying band 3 protein function, have been also determined. Our results show that, in infected dogs with 1:160 IFAT titer, high MDA plasma levels and oxidation of -SH groups are associated to increased P-Tyr expression levels, leading to a reduction in anion exchange capability throughout 6 months of diagnosis. On the other hand, infected dogs with 1:540 IFAT titer, exhibited oxidative conditions associated to an impaired anion exchange capability at diagnosis, were ameliorated after 6 months. Such findings suggest that (1) band 3 protein-mediated anion transport is reduced by oxidative conditions associated to leishmaniasis, putatively via phosphorylative pathways; (2) band 3 protein efficiency may account for canine leishmaniasis development; and (3) the assessment of band 3 protein function may represent an additional tool for canine leishmaniasis diagnosis and monitoring of its development, with potential application to humans, either in case of leishmaniasis or other oxidative-related pathologies.

SO4= uptake and catalase role in preconditioning after H2O2-induced oxidative stress in human erythrocytes.

Preconditioning (PC) is an adaptive response to a mild and transient oxidative stress, shown for the first time in myocardial cells and not described in erythrocytes so far. The possible adaptation of human erythrocytes to hydrogen peroxide (H2O2)-induced oxidative stress has been here verified by monitoring one of band 3 protein functions, i.e., Cl-/HCO3- exchange, through rate constant for SO4= uptake measurement. With this aim, erythrocytes were exposed to a mild and transient oxidative stress (30 min to either 10 or 100 µM H2O2), followed by a stronger oxidant condition (300- or, alternatively, 600-µM H2O2 treatment). SO4= uptake was measured by a turbidimetric method, and the possible role of catalase (CAT, significantly contributing to the anti-oxidant system in erythrocytes) in PC response has been verified by measuring the rate of H2O2 degradation. The preventive exposure of erythrocytes to 10 µM H2O2, and then to 300 µM H2O2, significantly ameliorated the rate constant for SO4= uptake with respect to 300 µM H2O2 alone, showing thus an adaptive response to oxidative stress. Our results show that (i) SO4= uptake measurement is a suitable model to monitor the effects of a mild and transient oxidative stress in human erythrocytes, (ii) band 3 protein anion exchange capability is retained after 10 µM H2O2 treatment, (iii) PC response induced by the 10 µM H2O2 pretreatment is clearly detected, and (iv) PC response, elicited by low-concentrated H2O2, is mediated by CAT enzyme and does not involve band 3 protein tyrosine phosphorylation pathways. Erythrocyte adaptation to a short-term oxidative stress may serve as a basis for future studies about the impact of more prolonged oxidative events, often associated to aging, drug consumption, chronic alcoholism, hyperglycemia, or neurodegenerative diseases.

Redox Homeostasis and Antioxidant Strategies in the Pathophysiology.

The molecular mechanisms underlying oxidative stress, and pathophysiological consequences in cell and tissue function, are frequently described as the imbalance between the production of reactive species and the ability to defend through sophisticated antioxidant machinery (Contributions 1-3) [...].

Oxidative stress-related cellular aging causes dysfunction of the Kv3.1/KCNC1 channel reverted by melatonin.

The voltage-gated Kv3.1/KCNC1 channel is abundantly expressed in fast-spiking principal neurons and GABAergic inhibitory interneurons throughout the ascending auditory pathway and in various brain regions. Inactivating mutations in the KCNC1 gene lead to forms of epilepsy and a decline in the expression of the Kv3.1 channel is involved in age-related hearing loss. As oxidative stress plays a fundamental role in the pathogenesis of epilepsy and age-related hearing loss, we hypothesized that an oxidative insult might affect the function of this channel. To verify this hypothesis, the activity and expression of endogenous and ectopic Kv3.1 were measured in models of oxidative stress-related aging represented by cell lines exposed to 100 mM d-galactose. In these models, intracellular reactive oxygen species, thiobarbituric acid reactive substances, sulfhydryl groups of cellular proteins, and the activity of catalase and superoxide dismutase were dysregulated, while the current density of Kv3.1 was significantly reduced. Importantly, the antioxidant melatonin reverted all these effects. The reduction of function of Kv3.1 was not determined by direct oxidation of amino acid side chains of the protein channel or reduction of transcript or total protein levels but was linked to reduced trafficking to the cell surface associated with Src phosphorylation as well as metabolic and endoplasmic reticulum stress. The data presented here specify Kv3.1 as a novel target of oxidative stress and suggest that Kv3.1 dysfunction might contribute to age-related hearing loss and increased prevalence of epilepsy during aging. The pharmacological use of the antioxidant melatonin can be protective in this setting.

Internalization of nano- and micro-plastics in human erythrocytes leads to oxidative stress and estrogen receptor-mediated cellular responses.

Plastic material versatility has resulted in a substantial increase in its use in several sectors of our everyday lives. Consequently, concern regarding human exposure to nano-plastics (NPs) and micro-plastics (MPs) has recently increased. It has been shown that plastic particles entering the bloodstream may adhere to the erythrocyte surface and exert adverse effects following erythrocyte aggregation and adhesion to blood vessels. Here, we explored the effects of polystyrene nano-plastics (PS-NPs) and micro-plastics (PS-MPs) on human erythrocytes. Cellular morphology, binding/internalization of PS-NPs and PS-MPs, oxidative stress parameters, as well as the distribution and anion exchange capability of band 3 (anion exchanger 1; SLC4A1) have been analyzed in human erythrocytes exposed to 1 µg/mL PS-NPs or PS-MPs for 3 and 24 h, respectively. The data obtained showed significant modifications of the cellular shape after exposure to PS-NPs or PS-MPs. In particular, a significantly increased number of acanthocytes, echinocytes and leptocytes were detected. However, the percentage of eryptotic cells (<1 %) was comparable to physiological conditions. Analytical cytology and confocal microscopy showed that PS-NPs and PS-MPs bound to the erythrocyte plasma membrane, co-localized with estrogen receptors (Erα/ERß), and were internalized. An increased trafficking from the cytosol to the erythrocyte plasma membrane and abnormal distribution of ERs were also observed, consistent with ERα-mediated binding and internalization of PS-NPs. An increased phosphorylation of ERK1/2 and AKT kinases indicated that an activation of the ER-modulated non-genomic pathway occurred following exposure to PS-NPs and PS-MPs. Interestingly, PS-NPs or PS-MPs caused a significant production of reactive oxygen species, resulting in an increased lipid peroxidation and protein sulfhydryl group oxidation. Oxidative stress was also associated with an altered band 3 ion transport activity and increased oxidized haemoglobin, which led to abnormal clustering of band 3 on the plasma membrane. Taken together, these findings identify cellular events following the internalization of PS-NPs or PS-MPs in human erythrocytes and contribute to elucidating potential oxidative stress-related harmful effects, which may affect erythrocyte and systemic homeostasis.

Melatonin protects Kir2.1 function in an oxidative stress-related model of aging neuroglia.

Melatonin is a pleiotropic biofactor and an effective antioxidant and free radical scavenger and, as such, can be protective in oxidative stress-related brain conditions including epilepsy and aging. To test the potential protective effect of melatonin on brain homeostasis and identify the corresponding molecular targets, we established a new model of oxidative stress-related aging neuroglia represented by U-87 MG cells exposed to D-galactose (D-Gal). This model was characterized by a substantial elevation of markers of oxidative stress, lipid peroxidation, and protein oxidation. The function of the inward rectifying K+ channel Kir2.1, which was identified as the main Kir channel endogenously expressed in these cells, was dramatically impaired. Kir2.1 was unlikely a direct target of oxidative stress, but the loss of function resulted from a reduction of protein abundance, with no alterations in transcript levels and trafficking to the cell surface. Importantly, melatonin reverted these changes. All findings, including the melatonin antioxidant effect, were reproduced in heterologous expression systems. We conclude that the glial Kir2.1 can be a target of oxidative stress and further suggest that inhibition of its function might alter the extracellular K+ buffering in the brain, therefore contributing to neuronal hyperexcitability and epileptogenesis during aging. Melatonin can play a protective role in this context.

Aging Injury Impairs Structural Properties and Cell Signaling in Human Red Blood Cells; Açaì Berry Is a Keystone.

Red blood cell (RBC) deformability is the ability of cells to modulate their shape to ensure transit through narrow capillaries of the microcirculation. A loss of deformability can occur in several pathological conditions, during natural RBC aging through an increase in membrane protein phosphorylation, and/or through the structural rearrangements of cytoskeletal proteins due to oxidative conditions, with a key role played by band 3. Due to the close relationship between aging and oxidative stress, flavonoid-rich foods are good candidates to counteract age-related alterations. This study aims to verify the beneficial role of Açaì extract in a d-Galactose (d-Gal)-induced model of aging in human RBCs. To this end, band 3 phosphorylation and structural rearrangements in membrane cytoskeleton-associated proteins, namely spectrin, ankyrin, and/or protein 4.1, are analyzed in RBCs treated with 100 mM d-Gal for 24 h, with or without pre-incubation with 10 µg/mL Açaì extract for 1 h. Furthermore, RBC deformability is also measured. Tyrosine phosphorylation of band 3, membrane cytoskeleton-associated proteins, and RBC deformability (elongation index) are analyzed using western blotting analysis, FACScan flow cytometry, and ektacytometry, respectively. The present data show that: (i) Açaì berry extract restores the increase in band 3 tyrosine phosphorylation and Syk kinase levels after exposure to 100 mM d-Gal treatment; and (ii) Açaì berry extract partially restores alterations in the distribution of spectrin, ankyrin, and protein 4.1. Interestingly, the significant decrease in membrane RBC deformability associated with d-Gal treatment is alleviated by pre-treatment with Açaì extract. These findings further contribute to clarify mechanisms of natural aging in human RBCs, and propose flavonoid substances as potential natural antioxidants for the treatment and/or prevention of oxidative-stress-related disease risk.