Upper graphs: stimulation with DDS-NHOH
Upper graphs: stimulation with DDS-NHOH. acid (GA) concentrations, measured by HPLCCMS. Results showed that licorice intake decreased the level of DDS-NHOH-related oxidative alterations in RBCs, and the reduction was directly correlated with plasma GA concentration. In conclusion, in PG, the inability to counteract oxidative stress is a serious concern in the evaluation of therapeutic approaches. GA, by protecting RBC from oxidative assault, as in dapsone therapy, might be considered as a new potential tool for preventing further switching into severe endometriosis. contamination, and a variety of immuno-related conditions [18,19]. Unfortunately, DDS shares a well-documented toxicity, related to its routes of biotransformation [20,21] leading to the formation of dapsone hydroxylamine (DDS-NHOH), the powerful oxidizing dapsone metabolite [20,22]. In in vitro studies, DDS-NHOH has been demonstrated to shorten RBC lifespan through the progressive oxidative alteration pathway starting from methemoglobin formation, glutathione oxidation, [22,23,24,25], and band 3 high molecular weight aggregates (HMWA) [26], which leads to autologous antibody recognition [24]. Among the antioxidants assumed with the diet, such as vitamins, carotenoids, and minerals, which have been shown to contribute to maintaining the redox homeostasis, glycyrrhizin, the glycoside extracted from roots of Rabbit Polyclonal to A20A1 the liquorice, known for its Hordenine characteristic sweetness (about 30C50 occasions sweeter than sucrose), is usually widely used in the treatment of many diseases, such as chronic hepatitis [27], erythrodermic psoriasis [28], a variety of human viruses such as avian infectious bronchitis computer virus [29], HIV [30,31], and SARS-CoV-2 [32,33], as a few examples. Orally administered glycyrrhizin is usually Hordenine metabolized by intestinal bacteria into 18-glycyrrhetinic acid (GA) [34], a pentacyclic triterpenoid, whose structure is similar to those of the mineralocorticoid and glucocorticoid hormones secreted by the adrenal cortex. In in vitro experiments in human RBCs, GA prevented oxidative-induced alterations, greatly reducing both band 3 Tyr-P and band 3 high molecular weight aggregate (HMWA) formation [35]. The aim of this study was to evaluate the effect of the licorice intake around the oxidative stress generated by DDS-NHOH in RBCs from PG by monitoring band 3 Tyr-P levels and HMWA as parameters for the detection of RBC membrane denaturation. In addition, we analyzed the state and the activity of cytosolic carbonic anhydrase (CA), to investigate if GA could mitigate DDS-NHOH side-effects involving Hordenine this enzyme, a useful parameter of the potential worsening of RBC oxidative status [36]. 2. Results 2.1. Evaluation of the RBC Oxidative Status (Diamide) and Response to DDS-NHOH before Licorice Intake Cytosolic compartments of PG RBC showed much higher monomeric CAII isoform (30 kDa) than CG RBC (35 3.7% compared to 12 3%, respectively, 0.001). Being the activity of CA strictly depending on its monomerization [36], a CA Hordenine activity assay was performed, with PG RBC exhibiting almost six occasions higher values compared to CG RBC (Physique 1). When RBCs were treated with diamide, the amount of the 30 kDa band of CA increased to 24% and 49%, in CG and PG RBC, respectively, ( 0.001), with a parallel activity increase (5.2 2 in CG and 32.7 8.0 in PG, 0.001). In addition, in the presence of DDS-NHOH, if the process of formation of the 30 kDa isoform was more evident (26% in CG, 68% in PG), the correspondent values of CA activity were not so drastically increased as expected (3.3 0.6 and 12.2 3.1 in CG and PG, respectively). Open in a separate window Physique 1 Fresh blood was collected from CG and PG RBCs (isolated as described in the Methods section) and was incubated with and without 1.5 mM diamide or 0.3 mM DDS-NHOH. (a) Diluted cytosol from 1 L of packed cells, underwent Western blotting in non-reducing conditions. Bands immunostained with anti-CA antibodies were densitometrically analyzed, and the sum of the 30 and 60 kDa bands was arbitrarily calculated as 100%, taking into account that amount of proteolytic 30 kDa bands accounts for half the larger bands. Values were expressed as the means SD of = 12 CG and = 18 PG patients. * 0.001, comparison of CA 30 kDa isoform before and after treatments within groups, Students 0.001, comparison of the 30 kDa band between CG and PG groups, in both experimental conditions (diamide and DDS-NHOH treatments), Students = 12 CG and = 18 PG patients. * 0.001, comparison of CA activity to CG, before and after diamide treatment, Students = 12 CG and = 18 PG patients. Comparison from respective baseline values: * 0.005)..