Abstract
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.
Highlights
IntroductionSO4 = uptake measurement has been previously confirmed as a suitable tool to verify the impact of oxidative conditions on erythrocytes homeostasis [4]
The efficiency of Band 3 protein, the anion exchanger present in million copies on erythrocytes membrane and accounting for ion balance, erythrocytes deformability and tissue oxygenation [1], can be monitored by determining the rate constant for SO4 = uptake, which is slower and more detectable than Cl- or HCO3 - uptake [2,3]
After treatment with either 100 μM or 250 μM or 500 μM Mel, Thiobarbituric-Acid-Reactive Substances (TBARS) levels were significantly lower (p < 0.001) than those determined after exposure to either 1 μM or 10 μM or 50 μM Mel, while remaining unchanged with respect to control
Summary
SO4 = uptake measurement has been previously confirmed as a suitable tool to verify the impact of oxidative conditions on erythrocytes homeostasis [4]. In this regard, H2 O2 , used to produce oxidative stress at not hemolytic concentrations [5], impacts on the phospholipid bilayer arrangement and the integral proteins associated to cytoskeleton, including Band protein [6]. Our previous investigations demonstrated a reduction in Band 3 protein anion exchange capability and protein expression levels after a short-term period of exposure (30 min) to 300 μM H2 O2 associated neither to lipid peroxidation, nor to membrane –SH groups oxidation The role of antioxidants in preventing oxidative damage at Band 3 levels has been investigated [11]
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